StepOne™ and StepOnePlus™ Real-Time PCR

Systems

Installation, Networking, and Maintenance

for use with:

Applied Biosystems™StepOne™ Real-Time PCR System Software

Catalog Number 4376357, 4376374, 4376373, 4379216, 4376600, 4376598, and 4376599
Publication Number 4376782
Revision H

For Research Use Only. Not for use in diagnostic procedures.

Installation, Networking, and Maintenance Guide

Get Started

Applied Biosystems StepOne™

and StepOnePlus™
Real-Time PCR Systems Set Up the
Instrument
Installation, Networking, and Maintenance Guide

Install the
Colocated Layout

Install the
Standalone Layout

Connect the
System
to a Network

Maintain the
System
The information in this guide is subject to change without notice.
DISCLAIMER
TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT,
PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

Revision history

Table 1 Revision history of Pub. no. 4376782

Revision Date Description

H February 2016 Minor update in materials required for replacing

external fuses. Update in logos and covers.

G November 2011 Added information about excitation wavelength

for custom dyes. Cleaned up website URLs (and
some website-related instructions) in boilerplate
areas so they now are LT, and changed SDS to
MSDS. Replaced logos and back cover with
current versions.

Limited Use Label License No. 474: Real-Time PCR System for Research Use Only
Notice to Purchaser: The purchase of this instrument conveys to the purchaser the limited, non-transferable right to use the purchased instrument
only, under intellectual property rights that are owned and/or controlled by Life Technologies and relate specifically to the instrument. Purchase of the
instrument includes the right to use the instrument for internal research and to perform services (including the right to report the results of services
for a fee) by the purchaser only, but does not convey rights to use any other products, reagents, assays or methods such as the 5' nuclease assay
process. The sale of this instrument is expressly conditioned on the purchaser not reselling, repackaging, or distributing this instrument, or any of its
components, and no such rights are conveyed expressly, by implication, or by estoppel. For information on obtaining additional rights, please contact
outlicensing@lifetech.com or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008.

Corporate entity
Life Technologies Corporation | Carlsbad, CA 92008 USA | Toll Free in USA 1 800 955 6288

TRADEMARKS
All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
©2016 Thermo Fisher Scientific Inc. All rights reserved.
 Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Purpose of This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
How to Obtain More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
How to Obtain Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Safety Conventions Used in This Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Symbols on the Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Safety Labels on Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
General Instrument Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Chemical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Chemical Waste Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Electrical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
LED Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Biological Hazard Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Workstation Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Safety and Electromagnetic Compatibility (EMC) Standards . . . . . . . . . . . . . . . . . . . . 24

Chapter 1 Get Started. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

About the StepOne™ and StepOnePlus™ Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Select Protective Hardware and Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Select an Installation Layout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Plan the Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Chapter 2 Set Up the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . 37

About the Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Unpack the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Check the Shipped Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Set Up the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Complete the Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Chapter 3 Install the Colocated Layout . . . . . . . . . . . . . . . . . . . . 49

About the Colocated Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Set Up the Computer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Install the Computer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

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Installation, Networking, and Maintenance Guide
 Install the StepOne™ Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Check for StepOne™ Software Updates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Connect the System Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Perform the RNase P Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Set Up the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Run the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Analyze the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
After the Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

Chapter 4 Install the Standalone Layout. . . . . . . . . . . . . . . . . . . . 75

About the Standalone Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Set Up the Computer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Install the Computer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Install the StepOne™ Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Check for StepOne™ Software Updates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Prepare the StepOne™ Software for Standalone Use . . . . . . . . . . . . . . . . . . . . . . . . . 86
Perform the RNase P Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
Set Up the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Run the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Analyze the RNase P Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Unload the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Transfer the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Analyze the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
After the Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

Chapter 5 Connect the System to a Network . . . . . . . . . . . . . . . 101

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Connect the Instrument to a Network . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Set Up a Computer for Remote Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
Monitor the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

Chapter 6 Maintain the System . . . . . . . . . . . . . . . . . . . . . . . . . . 115

Regular Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
Perform a Spatial Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
Perform a Background Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
Perform a Dye Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
Archive and Back Up Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
Infrequent Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
Decontaminate the Sample Block(s) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Move the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

8 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems

Installation, Networking, and Maintenance Guide
 Replace the External Fuses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
Ship the Instrument for Service . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
Update the StepOne™ Software or the Operating System . . . . . . . . . . . . . . . . . 154

Appendix A How to Troubleshoot the Installation . . . . . . . . . . . . . . . 159

StepOne™ Software Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
Network Connection Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
Background Calibration Failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
Dye Calibration Failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166

Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems 9

Installation, Networking, and Maintenance Guide
10 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems
Installation, Networking, and Maintenance Guide
 Preface

Purpose of This Guide

About the System The guides listed below are shipped with the Applied Biosystems StepOne™ and
Documentation StepOnePlus™ Real-Time PCR Systems (StepOne™ and StepOnePlus™ systems).

Guide Purpose and Audience PN

Applied Biosystems StepOne™/StepOnePlus™ Real- Explains how to perform experiments on the 4376786
Time PCR System Getting Started Guide for StepOne™ and StepOnePlus™ systems. Each Getting
Genotyping Experiments Started Guide functions as both:

Applied Biosystems StepOne™/StepOnePlus™ Real- • A tutorial, using example experiment data provided 4376787
Time PCR System Getting Started Guide for with the Applied Biosystems StepOne™ Real-Time
Presence/Absence Experiments PCR System Software (StepOne™ software).
• A guide for your own experiments.
Applied Biosystems StepOne™/StepOnePlus™ Real- 4376785
Intended for laboratory staff and principal
Time PCR System Getting Started Guide for Relative
investigators who perform experiments using the
Standard Curve and Comparative CT Experiments
StepOne™ or StepOnePlus™ system.
Applied Biosystems StepOne™/StepOnePlus™ Real- 4376784
Time PCR System Getting Started Guide for Standard
Curve Experiments

Applied Biosystems 7500/7500Fast, StepOne™, and 4411937

StepOnePlus™ Real-Time PCR Systems Quick
Reference Card for Comparative CT Experiments and
Studies

Applied Biosystems StepOne™/StepOnePlus™ Real- Explains how to install and maintain the StepOne™ 4376782
Time PCR System Installation, Networking, and and StepOnePlus™ systems.
Maintenance Guide
Intended for laboratory staff responsible for the
Applied Biosystems StepOne™/StepOnePlus™ Real- installation and maintenance of the StepOne™ or 4376783
Time PCR System Installation Quick Reference Card StepOnePlus™ system.

Applied Biosystems StepOne™/StepOnePlus™ Real- Provides information about the reagents you can use 4379704
Time PCR System Reagent Guide on the StepOne™ and StepOnePlus™ systems,
including:
• An introduction to TaqMan® and SYBR® Green
reagents
• Descriptions and design guidelines for the
following experiment types:
– Quantitation experiments
– Genotyping experiments
– Presence/absence experiments
Intended for laboratory staff and principal
investigators who perform experiments using the
StepOne™ or StepOnePlus™ system.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems 11

Installation, Networking, and Maintenance Guide
Preface
Purpose of This Guide

Guide Purpose and Audience PN

Applied Biosystems StepOne™/StepOnePlus™ Real- Explains how to prepare your site to receive and install 4376768
Time PCR System Site Preparation Guide the StepOne™ or StepOnePlus™ systems.
Intended for personnel who schedule, manage, and
perform the tasks required to prepare your site for
installation of the StepOne™ or StepOnePlus™ system.

Applied Biosystems StepOne™ Real-Time PCR Explains how to use the StepOne™ software to: NA
System Software Help
• Set up, run, and analyze experiments using the
StepOne™ and StepOnePlus™ systems.
• Monitor networked StepOne™ and StepOnePlus™
instruments.
• Calibrate StepOne™ and StepOnePlus™
instruments.
• Verify the performance of StepOne™ and
StepOnePlus™ instruments with an RNase P run.
Intended for:
• Laboratory staff and principal investigators who
perform experiments using the StepOne™ or
StepOnePlus™ system.
• Laboratory staff responsible for the installation and
maintenance of the StepOne™ or StepOnePlus™
system.

Text Conventions This guide uses the following conventions:

• Bold text indicates user action. For example:
Type 0, then press Enter for each of the remaining fields.
• Italic text indicates new or important words and is also used for emphasis. For example:
Before analyzing, always prepare fresh matrix.
• A right arrow symbol ( ) separates successive commands you select from a drop-down
or shortcut menu. For example:
Select File Open.

User Attention Two user attention words appear in the user documentation. Each word implies a particular
Words level of observation or action as described below:

Note: – Provides information that may be of interest or help but is not critical to the use of
the product.

IMPORTANT! – Provides information that is necessary for proper instrument operation,

accurate chemistry kit use, or safe use of a chemical.

Examples of the user attention words appear below:

Note: The Calibrate function is also available in the Control Console.

IMPORTANT! To verify your client connection, you need a valid user ID.

12 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems

Installation, Networking, and Maintenance Guide
 Preface
How to Obtain More Information

Safety Alert Words Safety alert words also appear in user documentation. For more information, see “Safety
Alert Words” on page 15.

How to Obtain More Information

Related StepOne™ and StepOnePlus™ System Documents Available for Sale
Documentation
Document PN

Applied Biosystems StepOne™/StepOnePlus™ Real-Time PCR System Installation 4376791

Performance Verification Protocol

Applied Biosystems StepOne™/StepOnePlus™ Real-Time PCR System Installation 4376790

Qualification-Operation Qualification Protocol

Applied Biosystems StepOne™/StepOnePlus™ Real-Time PCR System Planned 4376788

Maintenance Protocol

Note: For more documentation, see “How to Obtain Support” on page 14.

Obtaining The StepOne™ Software Help describes how to use each feature of the user interface. Access
Information from the Help from within the StepOne™ software by doing one of the following:
the Software Help • Press F1.
• Click in the toolbar.
• Select Help StepOne Help.
To find topics of interest in the Help:
• Review the table of contents.
• Search for a specific topic.
• Search an alphabetized index.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems 13

Installation, Networking, and Maintenance Guide
Preface
How to Obtain Support

How to Obtain Support

For the latest services and support information for all locations, go to
www.thermofisher.com, then click the link for Support.
At the Support page, you can:
• Search through frequently asked questions (FAQs)
• Submit a question directly to Technical Support
• Order user documents, SDSs, certificates of analysis, and other related documents
• Download PDF documents
• Obtain information about customer training
• Download software updates and patches
In addition, the Support page provides access to worldwide telephone and fax numbers to
contact Technical Support and Sales facilities.

Customer Care Center

Contact the Customer Care Center when directed by this guide, or to schedule maintenance
for your instrument (such as, annual planned maintenance or temperature
verification/calibration). To contact the Customer Care Center call 1-800-762-4001, Option 1
(U.S.A. only). For all other countries, please contact your local support service
representative.

14 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems

Installation, Networking, and Maintenance Guide
 Preface
Safety Conventions Used in This Document

Safety Conventions Used in This Document

Safety Alert Words Four safety alert words appear in the user documentation at points in the document where you
need to be aware of relevant hazards. Each alert word—IMPORTANT, CAUTION,
WARNING, DANGER—implies a particular level of observation or action, as defined
below.

Definitions

IMPORTANT! – Indicates information that is necessary for proper instrument operation,

accurate chemistry kit use, or safe use of a chemical.

– Indicates a potentially hazardous situation that, if not avoided, may

result in minor or moderate injury. It may also be used to alert against unsafe practices.

– Indicates a potentially hazardous situation that, if not avoided, could

result in death or serious injury.

– Indicates an imminently hazardous situation that, if not avoided, will

result in death or serious injury. This signal word is limited to the most extreme situations.

Except for IMPORTANTs, each safety alert word in the document appears with an open
triangle figure that contains a hazard symbol. These hazard symbols are identical to the
hazard symbols that are affixed to the instruments (see “Safety Symbols” on page 16).

Examples

IMPORTANT! You must create a separate spreadsheet for each 96-well plate.

CHEMICAL HAZARD. TaqMan Universal PCR Master Mix may

cause eye and skin irritation. Exposure may cause discomfort if swallowed or inhaled. Read
the SDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing,
and gloves.

PHYSICAL INJURY HAZARD. During instrument operation, the

heated cover and sample block can reach temperatures in excess of 100°C.

ELECTRICAL HAZARD. Grounding circuit continuity is vital for the

safe operation. Never operate the system with the grounding conductor disconnected.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems 15

Installation, Networking, and Maintenance Guide
Preface
Symbols on the Instruments

Symbols on the Instruments

Electrical The following electrical symbols may be displayed on the instruments.
Symbols
Symbol Description Symbol Description

Indicates the On position of the Indicates a terminal that may be

main power switch. connected to the signal ground
reference of another instrument.
This is not a protected ground
Indicates the Off position of the terminal.
main power switch.
Indicates a protective grounding
terminal that must be connected to
Indicates a standby switch by which earth ground before any other
the instrument is switched on to the electrical connections are made to
Standby condition. Hazardous the instrument.
voltage may be present if this
switch is on standby. Indicates a terminal that can receive
or supply alternating current or
voltage.

Indicates the On/Off position of a Indicates a terminal that can receive

push-push main power switch. or supply alternating or direct
current or voltage.

Safety Symbols The following safety symbols may be displayed on the instruments. Each symbol may appear
by itself or with text that explains the relevant hazard (see “Safety Labels on Instruments” on
page 17). These safety symbols may also appear next to DANGERS, WARNINGS, and
CAUTIONS that occur in this and other documents.

Symbol Description Symbol Description

Indicates that you should consult Indicates the presence of moving

the manual for further information parts and to proceed with
and to proceed with appropriate appropriate caution.
caution.
Indicates the presence of an
electrical shock hazard and to
Indicates the presence of a hot proceed with appropriate caution.
surface or other high-temperature
hazard and to proceed with Indicates the presence of a laser
appropriate caution. inside the instrument and to
proceed with appropriate caution.

Environmental The following symbol applies to all all electrical and electronic products placed on the
Symbols European market after August 13, 2005.

Symbol Description

Do not dispose of this product as unsorted municipal waste.

Follow local municipal waste ordinances for proper disposal provisions to reduce
the environmental impact of waste electrical and electronic equipment (WEEE).
European Union customers:
Call your local Customer Service office for equipment pick-up and recycling. See
www.lifetechnologies.com for a list of customer service offices in the European
Union.

16 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems

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 Preface
Safety Labels on Instruments

Safety Labels on Instruments

The following CAUTION, WARNING, and DANGER statements may be displayed on the
instruments in combination with the safety symbols described in the preceding section.

English Francais

CAUTION Hazardous chemicals. Read the ATTENTION Produits chimiques dangeureux.

Safety Data Sheets (SDSs) before handling. Lire les fiches techniques de sûreté de
matériels avant la manipulation des produits.

CAUTION Hazardous waste. Refer to SDS(s) ATTENTION Déchets dangereux. Lire les
and local regulations for handling and fiches techniques de sûreté de matériels et la
disposal. régulation locale associées à la manipulation
et l'élimination des déchets.

CAUTION Hot surface. ATTENTION Surface brûlante.

DANGER High voltage. DANGER Haute tension.

WARNING To reduce the chance of electrical AVERTISSEMENT Pour éviter les risques
shock, do not remove covers that require tool d'électrocution, ne pas retirer les capots dont
access. No user-serviceable parts are inside. l'ouverture nécessite l'utilisation d'outils.
Refer servicing to qualified service personnel. L’instrument ne contient aucune pièce
réparable par l’utilisateur. Toute intervention
doit être effectuée par le personnel de service
qualifié de Thermo Fisher Scientific.

CAUTION Moving parts. ATTENTION Parties mobiles.

DANGER Class 3B (III) visible and/or invisible DANGER Rayonnement visible ou invisible
LED radiation present when open and d’un faisceau LED de Classe 3B, (III) en cas
interlocks defeated. Avoid exposure to beam. d’ouverture et de neutralisation des
dispositifs de sécurité. Evitez toute
exposition au faisceau.

Locations of The system contains a warning at the location shown below:

Warnings

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems 17

Installation, Networking, and Maintenance Guide
Preface
General Instrument Safety

General Instrument Safety

PHYSICAL INJURY HAZARD. Using the instrument in a manner not

specified by us may result in personal injury or damage to the instrument.

Moving and Lifting PHYSICAL INJURY HAZARD. The instrument is to be moved and
the Instrument positioned only by the personnel or vendor specified in the applicable site preparation guide.
If you decide to lift or move the instrument after it has been installed, do not attempt to lift or
move the instrument without the assistance of others, the use of appropriate moving
equipment, and proper lifting techniques. Improper lifting can cause painful and permanent
back injury. Depending on the weight, moving or lifting an instrument may require two or
more persons.

Moving and Lifting Do not attempt to lift or move the computer or the monitor without the
Computers and assistance of others. Depending on the weight of the computer and/or the monitor, moving
Monitors them may require two or more people.

Things to consider before lifting the computer and/or the monitor:

• Make sure that you have a secure, comfortable grip on the computer or the monitor
when lifting.
• Make sure that the path from where the object is to where it is being moved is clear of
obstructions.
• Do not lift an object and twist your torso at the same time.
• Keep your spine in a good neutral position while lifting with your legs.
• Participants should coordinate lift and move intentions with each other before lifting and
carrying.
• Instead of lifting the object from the packing box, carefully tilt the box on its side and
hold it stationary while someone slides the contents out of the box.

Operating the Ensure that anyone who operates the instrument has:
Instrument • Received instructions in both general safety practices for laboratories and specific safety
practices for the instrument.
• Read and understood all applicable Safety Data Sheets (SDSs). See “About SDSs” on
page 19.

Cleaning or Before using a cleaning or decontamination method other than those

Decontaminating recommended by the manufacturer, verify with the manufacturer that the proposed method
the Instrument will not damage the equipment.

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 Preface
Chemical Safety

Chemical Safety
Chemical Hazard CHEMICAL HAZARD. Before handling any chemicals, refer to the
Warning Safety Data Sheet (SDS) provided by the manufacturer, and observe all relevant precautions.

CHEMICAL STORAGE HAZARD. Never collect or store waste in a

glass container because of the risk of breaking or shattering. Reagent and waste bottles can
crack and leak. Each waste bottle should be secured in a low-density polyethylene safety
container with the cover fastened and the handles locked in the upright position. Wear
appropriate eyewear, clothing, and gloves when handling reagent and waste bottles.

Chemical Safety To minimize the hazards of chemicals:

Guidelines • Read and understand the Safety Data Sheets (SDSs) provided by the chemical
manufacturer before you store, handle, or work with any chemicals or hazardous
materials. (See “About SDSs” on page 19.)
• Minimize contact with chemicals. Wear appropriate personal protective equipment
when handling chemicals (for example, safety glasses, gloves, or protective clothing).
For additional safety guidelines, consult the SDS.
• Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only
with adequate ventilation (for example, fume hood). For additional safety guidelines,
consult the SDS.
• Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the
manufacturer’s cleanup procedures as recommended in the SDS.
• Comply with all local, state/provincial, or national laws and regulations related to
chemical storage, handling, and disposal.

About SDSs Chemical manufacturers supply current Safety Data Sheets (SDSs) with shipments of
hazardous chemicals to new customers. They also provide SDSs with the first shipment of a
hazardous chemical to a customer after an SDS has been updated. SDSs provide the safety
information you need to store, handle, transport, and dispose of the chemicals safely.
Each time you receive a new SDS packaged with a hazardous chemical, be sure to replace the
appropriate SDS in your files.

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Preface
Chemical Safety

Obtaining The SDS for any chemical supplied by us is available to you free 24 hours a day. To
SDSs obtain SDSs:

1. Go to www.lifetchnologies.com/support

2. Click the MSDS, COA & other Support Documents link.

3. In the Redifine your search field:

a. Type the chemical name, part number, or other information that you expect to
appear in the SDS of interest.
b. Select Material Safety Data Sheets in the drop-down list.

c. Click Search.

4. To view, download, or print the document of interest:

a. Click the link below MSDS Files.

b. Click the link for the language of choice.

The SDS opens in the web broswer. You can print or save the SDS.

Note: For the SDSs of chemicals not distributed by us, contact the chemical
manufacturer.

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 Preface
Chemical Waste Safety

Chemical Waste Safety

Chemical Waste HAZARDOUS WASTE. Refer to Safety Data Sheets and local
Hazard regulations for handling and disposal.

CHEMICAL WASTE HAZARD. Wastes produced by instruments are

potentially hazardous and can cause injury, illness, or death.

CHEMICAL STORAGE HAZARD. Never collect or store waste in a

glass container because of the risk of breaking or shattering. Reagent and waste bottles can
crack and leak. Each waste bottle should be secured in a low-density polyethylene safety
container with the cover fastened and the handles locked in the upright position. Wear
appropriate eyewear, clothing, and gloves when handling reagent and waste bottles.

Chemical Waste To minimize the hazards of chemical waste:

Safety Guidelines • Read and understand the Safety Data Sheets (SDSs) provided by the manufacturers of
the chemicals in the waste container before you store, handle, or dispose of chemical
waste.
• Provide primary and secondary waste containers. (A primary waste container holds the
immediate waste. A secondary container contains spills or leaks from the primary
container. Both containers must be compatible with the waste material and meet federal,
state, and local requirements for container storage.)
• Minimize contact with chemicals. Wear appropriate personal protective equipment
when handling chemicals (for example, safety glasses, gloves, or protective clothing).
For additional safety guidelines, consult the SDS.
• Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only
with adequate ventilation (for example, fume hood). For additional safety guidelines,
consult the SDS.
• Handle chemical wastes in a fume hood.
• After emptying a waste container, seal it with the cap provided.
• Dispose of the contents of the waste tray and waste bottle in accordance with good
laboratory practices and local, state/provincial, or national environmental and health
regulations.

Waste Disposal If potentially hazardous waste is generated when you operate the instrument, you must:
• Characterize (by analysis if necessary) the waste generated by the particular
applications, reagents, and substrates used in your laboratory.
• Ensure the health and safety of all personnel in your laboratory.
• Ensure that the instrument waste is stored, transferred, transported, and disposed of
according to all local, state/provincial, and/or national regulations.

IMPORTANT! Radioactive or biohazardous materials may require special handling, and

disposal limitations may apply.

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Preface
Electrical Safety

Electrical Safety

ELECTRICAL SHOCK HAZARD. Severe electrical shock can result

from operating the system without its instrument panels in place. Do not remove instrument
panels. High-voltage contacts are exposed when instrument panels are removed from the
instrument.

Fuses FIRE HAZARD. Improper fuses or high-voltage supply can damage

the instrument wiring system and cause a fire. Before turning on the instrument, verify that
the fuses are properly installed and that the instrument voltage matches the power supply in
your laboratory.

FIRE HAZARD. For continued protection against the risk of fire,

replace fuses only with fuses of the type and rating specified for the instrument.

Power ELECTRICAL HAZARD. Grounding circuit continuity is vital for the

safe operation of equipment. Never operate equipment with the grounding conductor
disconnected.

ELECTRICAL HAZARD. Use properly configured and approved line

cords for the voltage supply in your facility.

ELECTRICAL HAZARD. Plug the system into a properly grounded

receptacle with adequate current capacity.

Overvoltage Rating The StepOne™ and StepOnePlus™ systems have an installation (overvoltage) category of II,
and are classified as portable equipment.

LED Safety
To ensure safe LED operation:
• The system must be maintained by a Technical Representative from the company.
• All instrument panels must be in place on the instrument while the instrument is
operating. When all panels are installed, there is no detectable radiation present. If any
panel is removed when the LED is operating (during service with safety interlocks
disabled), you may be exposed to LED emissions in excess of the Class 3B rating.
• Do not remove safety labels or disable safety interlocks.

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 Preface
Biological Hazard Safety

Biological Hazard Safety

General Biohazard BIOHAZARD. Biological samples such as tissues, body fluids,
infectious agents, and blood of humans and other animals have the potential to transmit
infectious diseases. Follow all applicable local, state/provincial, and/or national regulations.
Wear appropriate protective equipment, which includes but is not limited to: protective
eyewear, face shield, clothing/lab coat, and gloves. All work should be conducted in properly
equipped facilities using the appropriate safety equipment (for example, physical
containment devices). Individuals should be trained according to applicable regulatory and
company/institution requirements before working with potentially infectious materials. Read
and follow the applicable guidelines and/or regulatory requirements in the following:
• U.S. Department of Health and Human Services guidelines published in Biosafety in
Microbiological and Biomedical Laboratories (stock no. 017-040-00547-4;
bmbl.od.nih.gov)
• Occupational Safety and Health Standards, Bloodborne Pathogens (29 CFR§1910.1030;
www.access.gpo.gov/ nara/cfr/waisidx_01/ 29cfr1910a_01.html).
• Your company’s/institution’s Biosafety Program protocols for working with/handling
potentially infectious materials.
Additional information about biohazard guidelines is available at:
www.cdc.gov

Workstation Safety
Correct ergonomic configuration of your workstation can reduce or prevent effects such as
fatigue, pain, and strain. Minimize or eliminate these effects by configuring your workstation
to promote neutral or relaxed working positions.

MUSCULOSKELETAL AND REPETITIVE MOTION HAZARD.

These hazards are caused by potential risk factors that include but are not limited to repetitive
motion, awkward posture, forceful exertion, holding static unhealthy positions, contact
pressure, and other workstation environmental factors.

To minimize musculoskeletal and repetitive motion risks:

• Use equipment that comfortably supports you in neutral working positions and allows
adequate accessibility to the keyboard, monitor, and mouse.
• Position the keyboard, mouse, and monitor to promote relaxed body and head postures.

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Preface
Safety and Electromagnetic Compatibility (EMC) Standards

Safety and Electromagnetic Compatibility (EMC) Standards

U.S. and Canadian The system has been tested to and complies with standard:
Safety
UL 61010-1 / CAN/CSA C22.2 No. 61010-1, “Safety Requirements for Electrical
Standards
Equipment for Measurement, Control, and Laboratory Use, Part 1: General
Requirements.”

Canadian EMC The instrument has been tested to and complies with ICES-001, Issue 4: “Industrial,
Standard Scientific, and Medical Radio Frequency Generators.”

European Safety, Safety

RoHS Directive, This instrument meets European requirements for safety (Low Voltage Directive
and EMC 2014/35/EU). This instrument has been tested to and complies with standards EN 61010-1,
Standards “Safety Requirements for Electrical Equipment for Measurement, Control and Laboratory
Use, Part 1: General Requirements.”
EN 61010-2-010, “Particular Requirements for Laboratory Equipment for the Heating of
Materials.”
EN 61010-2-081, “Particular Requirements for Automatic and Semi-Automatic Laboratory
Equipment for Analysis and Other Purposes.”

EMC
This instrument meets European requirements for emission and immunity (EMC Directive
2014/30/EU). This instrument has been tested to and complies with standard EN 61326
(Group 1, Class B), “Electrical Equipment for Measurement, Control and Laboratory Use –
EMC Requirements.”
This instrument meets European RoHS Directive 2011/65/EU.

Australian EMC This instrument has been tested to and complies with standard AS/NZS CISPR 11, “Limits
Standards and Methods Measurement of Electromagnetic Disturbance Characteristics of Industrial,
Scientific, and Medical (ISM) Radio-frequency Equipment.”

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 Chapter 1

Get Started

This chapter covers:

■ About the StepOne™ and StepOnePlus™ Systems . . . . . . . . . . . . . . . . . . . . . . . . 26
■ Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
■ Select Protective Hardware and Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
■ Select an Installation Layout. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
■ Plan the Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Note: For more information about any of the topics discussed in this guide, access the
Help from within the Applied Biosystems StepOne™ Real-Time PCR System Software
by pressing F1, or clicking in the toolbar, or selecting Help StepOne Software
Help.

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 Chapter 1 Get Started
About the StepOne™ and StepOnePlus™ Systems

About the StepOne™ and StepOnePlus™ Systems

There are two models available for this Real-Time PCR System:

System Features

Applied Biosystems StepOne™ Real-Time • 48-well platform

PCR System (StepOne™ system) • Three-color system

Applied Biosystems StepOnePlus™ Real- • 96-well platform

Time PCR System (StepOnePlus™ system) • Four-color system
• VeriFlex™ sample blocks

The Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems use
fluorescent-based polymerase chain reaction (PCR) reagents to provide:
• Quantitative detection of target nucleic acid sequences (targets) using real-time
analysis.
• Qualitative detection of targets using post-PCR (endpoint) analysis.
• Qualitative analysis of the PCR product (achieved by melt curve analysis that occurs
post-PCR).

Notes

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About the StepOne™ and StepOnePlus™ Systems

About Data The StepOne™ and StepOnePlus™ systems collect raw fluorescence data at different
Collection points during a PCR, depending on the type of run that the instruments perform:

Run Type Data Collection Point

Real-time runs Standard curve The instrument collects data following each
extension step of the PCR.
Relative standard curve

Comparative CT (ΔΔCT)

Post-PCR Genotyping The instrument collects data:

(endpoint) runs
Presence/absence • Before the PCR (For presence/absence
experiments, data collection before the PCR is
optional, but recommended.)
• (Optional) During the PCR. The instrument can
collect data during the run (real-time);
collecting data during the run can be helpful
for troubleshooting.
• After the PCR

Regardless of the run type, a data collection point or read on the StepOne™ or
StepOnePlus™ instrument consists of three phases:

1. Excitation – The instrument illuminates all wells of the reaction plate within the
instrument, exciting the fluorophores in each reaction.

2. Emission – The instrument optics collect the residual fluorescence emitted from the
wells of the reaction plate. The resulting image collected by the device consists only
of light that corresponds to the range of emission wavelengths.

3. Collection – The instrument assembles a digital representation of the residual

fluorescence collected over a fixed time interval. The StepOne™ software stores the
raw fluorescent image for analysis.
After a run, the StepOne™ software uses calibration data (spatial, dye, and background)
to determine the location and intensity of the fluorescent signals in each read, the dye
associated with each fluorescent signal, and the significance of the signal.

Notes

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About the StepOne™ and StepOnePlus™ Systems

Filters and StepOnePlus™ System Filters and Dyes

Supported Dyes The StepOnePlus™ instrument features a four-color filter set that supports the following
dyes: FAM™ dye, JOE™ dye, NED® dye, ROX™ dye, TAMRA® dye, VIC® dye, and
SYBR® Green dye. The following figure shows the emission spectrum for each dye, and
the filter at which each dye is read.

Filters 1 2 3 4
Wavelength 500 600 700
(nm)
Emission
Spectra

Dyes ~520 nm ~550 nm ~580 nm ~610 nm

• FAM • JOE • NED • ROX
• SYBR Green • VIC • TAMRA

StepOne™ System Filters and Dyes

The StepOne™ instrument features a three-color filter set that supports the following
dyes: FAM™ dye, JOE™ dye, ROX™ dye, VIC® dye, and SYBR® Green dye. The
following figure shows the emission spectrum for each dye, and the filter at which each
dye is read.

Filters 1 2 3
Wavelength 500 600 700
(nm)
Emission
Spectra

Dyes ~520 nm ~550 nm ~610 nm

• FAM • JOE • ROX
• SYBR Green • VIC

About the The StepOnePlus instrument contains six independently thermally regulated VeriFlex™
VeriFlex™ blocks to help you optimize your thermal cycling conditions. You can set a different
Technology temperature for one or more of the VeriFlex blocks, creating up to six different zones for
samples, or you can set the same temperature for each of the VeriFlex blocks.

Notes

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 Chapter 1 Get Started
Before You Begin

Before You Begin

Before you install the Applied Biosystems StepOne™/StepOnePlus™ Real-Time PCR
System, familiarize yourself with this guide, read the supporting site preparation
documentation, and obtain the materials required for the installation.

Review You can use this guide to:

This Guide • Install the system.
Chapters 2 through 5 of this guide contain all the information necessary for you to
perform a complete installation of the system. You can install the system within
4 hours; however, the installation may require more time if you plan to connect the
system to a network.
• Maintain the system.
Chapter 6 contains all the information necessary to perform routine maintenance of
the system.
Go to Chapter 6 to learn how to:
– Archive and back up data
– Calibrate the instrument
– Decontaminate the instrument
– Move the instrument
– Replace the instrument fuses
– Ship the instrument for service

Prepare for the • Read the Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Site
Installation Preparation Guide and complete the preinstallation checklist contained in the guide.
• Obtain the materials for installation (see “Obtain the Required Materials” on page 30).
• Read “Select Protective Hardware and Software” on page 31 and obtain any protective
hardware and software that you want to install to the system.

IMPORTANT! You can use this guide to reinstall the StepOne™ or StepOnePlus™ system
after transportation, but you must have the necessary installation reagents. See “Check
the Shipped Materials” on page 41 for a list of materials that are required for the
installation and to order more materials as needed.

Notes

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Before You Begin

Obtain the • Tools and equipment

Required – Centrifuge with a reaction plate adapter of the appropriate size
Materials – Powder-free gloves
– Safety glasses
– Scissors or box cutters
– Screwdriver, flathead
• Computer (see below)
• (Optional) Protective hardware and software to install to the instrument and the
computer (see “Select Protective Hardware and Software” on page 31).

Minimum If you did not order a computer with your system, supply one that meets the following
Computer requirements:
Requirements

Minimum Requirements ‡
Component
For operating the StepOne or StepOnePlus For analyzing experiments in comparative CT
instrument studies

Computer • Intel processor, 1.0 GHz • Intel Core Duo T5500E processor, 1.66 GHz
• 512 MB of RAM • 1 GB of RAM #
• One 60-GB hard drive • One 60-GB hard drive
• 20/48X IDE CD-ROM • 20/48X IDE CD-ROM
• Ethernet network interface adapter (10BASE-T) § • Ethernet network interface adapter (10BASE-T)§
• UL-listed • UL-listed
• CE-marked • CE-marked
• FCC-labeled • FCC-labeled

Monitor • 1280 × 1024 pixel resolution for full-screen • 1280 × 1024 pixel resolution for full-screen
display display
• 32-bit color • 32-bit color
• UL-listed • UL-listed

Operating Microsoft Windows® XP Professional Operating Microsoft Windows® XP Professional Operating

System ‡‡ System, Service Pack 2 or later System, Service Pack 2 or later

‡ A computer that meets the minimum requirement provides optimal software performance and is supported by Thermo Fisher
Scientific.
§ Necessary only if you plan to connect the computer to the instrument or to a local area network.
# The number of reaction plates (experiments) that can be analyzed in a comparative CT study depends on the processor speed
and the amount of RAM.
‡‡We support the Windows® Vista Business Operating System, Service Pack 1, for performing data analysis (that is, analyzing
your experiments with the computer). We do not support the Vista operating system for instrument control purposes.

Notes

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 Chapter 1 Get Started
Select Protective Hardware and Software

Select Protective Hardware and Software

Before installing the system, obtain any of the following optional hardware and/or
software.

Electrical We recommend several protective devices to prevent loss of data and to protect the
Protective system from damage resulting from electrical hazards.
Devices
Power Line Regulator
We recommend the use of a 1.5-kVA power line regulator in areas where the supplied
power fluctuates in excess of ±10% of the normal voltage. Power fluctuations can
adversely affect the function of the system.

Note: A power line regulator monitors the input current and adjusts the power supplied to the
instrument or computer. It does not protect against a power surge or failure.

Uninterruptible Power Supply (UPS)

We recommend the use of a 1.5-kVA uninterruptible power supply (UPS), especially in
areas prone to power failure. Power failures and other events that abruptly terminate the
function of the system can corrupt data and possibly damage the system.

IMPORTANT! UPSs have finite battery lives and they provide power for a limited time.
They are meant to delay the effects of a power outage, not to serve as replacement power
sources. In the event of a power loss, power off the system unless you expect to regain
power within the battery life of the UPS.

Surge Protector
We recommend the use of a 10-kVA surge protector (line conditioner) in areas with
frequent electrical storms or near devices that are electrically noisy, such as refrigerators,
air conditioners, or centrifuges. Short-duration, high-voltage power fluctuations can
abruptly terminate the function of, and thereby damage the components of, the computer
and the system.

Note: A dedicated line and ground between the instrument/computer and the building’s
main electrical service can also prevent problems caused by power fluctuations.

Data Backup/ We recommend the use of one or more backup storage devices to prevent potential loss
Storage Devices of data caused by unforeseen failures of the instrument or the computer. If your system
includes a computer, then the CD/DVD drive of the computer can serve as the backup
storage device for your system. By saving your experiment files (EDSs) to one or more
writable CD or DVDs on a weekly basis, you can back up the data generated by your
system. Before installing the system, decide on a method for backing up your data
(“Archive and Back Up Data” on page 142).

Notes

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 Chapter 1 Get Started
Select Protective Hardware and Software

Third-Party We recommend the use of several types of commercial software to ensure optimal
Software performance of the system and software, including:
• File compression software for archiving data generated by the system
• System optimization software for defragmenting the computer hard drive(s)
• Antivirus and firewall software for protecting the system if you plan to connect it to
a network
Before you install third-party software to the computer running the StepOne™ software,
confirm that the software will not:
• Restrict Ethernet communication
• Interfere with StepOne™ software operation (see below)

To confirm that third-party software does not interfere with the StepOne™ software:

1. Install the software to the computer that contains the StepOne™ software.

2. Perform several test experiments using “dummy” plates (plates that do not contain
reagents).

Note: The goal of the test experiments is to run plates under conditions that match
normal instrument operation. Therefore, the characteristics of the test experiments
(plate layout and run method) must closely resemble your actual experiments.

3. Confirm that the system performs each test experiment without producing errors.
If the system performs the tests successfully, perform experiments normally. If the
system encounters errors during the test runs, the software may not be compatible
with the StepOne™ software.

Notes

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 Chapter 1 Get Started
Select an Installation Layout

Select an Installation Layout

Before you install your StepOne™ or StepOnePlus™ system, select the installation layout
that best suits your laboratory environment. The layout that you select affects the flow of
the installation, so note your choice for later reference. The StepOne™ and StepOnePlus™
instruments can be installed in the two layouts described below (standalone or
colocated).

Colocated In the colocated layout, the instrument is directly connected to the computer by the
Layout yellow StepOne system cable. In this configuration, you can set up, run, and analyze
experiments from the colocated computer. The following figure shows an instrument in a
colocated layout.

caps scroll
Scroll Pause num lock lock
Print Break lock
Screen Lock
F12 SysRq

F8 F9 F10 F11
Page
Up
Num
Lock
* 9 +
F6 F7 Home 8
F5 Insert Page 7
F3 F4 _ += Backspace End Down 6
F1 F2 - Delete 4 5
Esc 9 0 ] Enter
8 [ 2 3
6 7 O P 1
4 5 I " Enter
2 3 Y U L ' 0
~ 1 R T J K ? /
` E H < > Shift
Q W F G Ctrl
Tab S D N M
A V B Alt
Caps Lock Z X C
Shift Alt
Ctrl

Note: You can control a colocated instrument from the instrument touchscreen, but only
when the StepOne™ software is not performing a run.

Note: Experiment data can be transferred between the standalone instrument and
computer over an Ethernet network ( ) connection. See “Networked Colocated and
Standalone Layouts” on page 34 for more information.

The following steps summarize the experiment workflow when using a colocated
system:

1. Create the experiment on the colocated computer using the StepOne™ software.

2. Start the run from the colocated computer.

During the run, use the colocated computer to:
• Monitor the status of a run in progress.
• View temperature and amplification data as they are collected.

3. Analyze the experiment on the colocated computer using the StepOne™ software.

Notes

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Select an Installation Layout

Standalone In the standalone layout, the instrument is not connected to the computer. Instead, a USB
Layout drive ( ) is used to transfer data between the instrument and the computer. In this
configuration, you set up and analyze experiments from the computer but run them from
the instrument touchscreen. The following figure shows an instrument in a standalone
layout.

Note: Experiment data can be transferred between the standalone instrument and
computer over an Ethernet network ( ) connection. See “Networked Colocated and
Standalone Layouts” below for more information.

The workflow for the standalone layout is more complicated than for the colocated
layout because the system components do not share a direct connection. The following
steps summarize the experiment workflow for a standalone system:

1. Create the experiment on the standalone computer using the StepOne™ software.

2. Transfer the experiment to the standalone instrument on a USB drive.

3. Start the experiment from the instrument touchscreen.

During the run, monitor the run from the instrument touchscreen.

4. Transfer the experiment to the standalone computer on a USB drive.

5. Analyze the experiment on the standalone computer using the StepOne™ software.

Networked You can expand the functionality of a colocated or standalone instrument by connecting
Colocated and it to an Ethernet network. When an instrument is part of a network, other computers on
Standalone the network that are running the StepOne™ software can:
Layouts • Monitor the status of the instrument
• Send and download experiments to and from the instrument

IMPORTANT! Computers on the network cannot control the instrument, only monitor it.

Note: Chapter 5, “Connect the System to a Network,” on page 101 contains a detailed
explanation of how to install the instrument to a network.

Notes

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 Chapter 1 Get Started
Plan the Installation

Plan the Installation

You can install the system within a 4-hour period. The installation does not require your
constant attention; however, plan to spend most of your time working with the system.

Recommended Unpack and Set Up the

Workflow instrument (Chapter 2)

caps scroll
Scroll Pause num lock lock
Print Break lock
Screen Lock
F12 SysRq

F8 F9 F10 F11
Page
Up
Num
Lock
* 9 +
F6 F7 Home 8
F5 Insert Page 7
F3 F4 _ += Backspace End Down 6
F1 F2 - Delete 4 5
Esc 9 0 ] Enter
8 [ 2 3
6 7 O P 1
4 5 I " Enter
2 3 Y U L ' 0
~ 1 R T J K ? /
` E H < > Shift
Q W F G Ctrl
Tab S D N M
A V B Alt
Caps Lock Z X C
Shift Alt
Ctrl

Colocated Installation Standalone Installation

(Chapter 3) (Chapter 4)

Install the Computer

Set Up the Computer

Connect the system

Perform the RNase P

Experiment
Perform the RNase P
Experiment

(Optional) Install the system to a

Network (Chapter 5)
Note: Consult your IT department
to network a colocated computer.
To enable the full functionality of the
StepOne™ software, the colocated
computer requires a network
connection.

Notes

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Plan the Installation

Notes

36 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems

Installation, Networking, and Maintenance Guide
 Chapter 2

Set Up the Instrument

This chapter covers:

■ About the Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
■ Unpack the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
■ Check the Shipped Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
■ Set Up the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
■ Complete the Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Note: For more information about any of the topics discussed in this guide, access the
Help from within the Applied Biosystems StepOne™ Real-Time PCR System Software
by pressing F1, or clicking in the toolbar, or selecting Help StepOne Software
Help.

Notes

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems 37

Installation, Networking, and Maintenance Guide
 Chapter 2 Set Up the Instrument
About the Installation

About the Installation

This chapter explains how to install the Applied Biosystems StepOne™/StepOnePlus™
Real-Time PCR Instrument for both colocated and standalone layouts.

Installation Chapter 1,
Workflow Get Started

Unpack and Set Up the Instrument

1. Unpack and place the instrument.
2. Check the shipped materials.
3. Set up the instrument.
a. Prepare the instrument.
b. Set the instrument settings.

Chapter 3, Chapter 4,
Install the Colocated Layout Install the Standalone Layout

caps scroll
Scroll Pause num lock lock
Print Break lock
Screen Lock
F12 SysRq

F8 F9 F10 F11
Page
Up
Num
Lock
* 9 +
F6 F7 Home 8
F5 Insert Page 7
F3 F4 _ += Backspace End Down 6
F1 F2 - Delete 4 5
Esc 9 0 ] Enter
8 [ 2 3
6 7 O P 1
4 5 I " Enter
2 3 Y U L ' 0
~ 1 R T J K ? /
` E H < > Shift
Q W F G Ctrl
Tab S D N M
A V B Alt
Caps Lock Z X C
Shift Alt
Ctrl

Notes

38 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems

Installation, Networking, and Maintenance Guide
 Chapter 2 Set Up the Instrument
Unpack the Instrument

Unpack the Instrument

Prepare the installation site and unpack the instrument.

Before • Review and complete the preinstallation checklists in the Applied Biosystems
Unpacking StepOne™ and StepOnePlus™ Real-Time PCR Systems Site Preparation Guide. The
guide is shipped to you before the instrument arrives, and it contains important
environmental and electrical requirements for the system.
• Prepare the installation site as described in the Applied Biosystems StepOne™ and
StepOnePlus™ Real-Time PCR Systems Site Preparation Guide.

Confirm the Site IMPORTANT! Avoid placing the system on surfaces that are subject to constant or
Requirements intermittent vibration. Tabletop centrifuges, vortex mixers, and other laboratory equipment
can vibrate the instrument during a run and produce data collection errors.

Confirm that the installation site meets the physical and environmental requirements for
the instrument listed in the table below. Refer to the Applied Biosystems StepOne™ and
StepOnePlus™ Real-Time PCR Systems Site Preparation Guide for a complete summary.

Requirement Instrument Additional Total

Length 48.5 cm (19.1 in) • Front ‡ – 18.8 cm (7.4 in) 82.5 cm (32.5 in)
(drawer closed) • Rear § – 15.2 cm (6.0 in)

Width 24.6 cm (9.7 in) 15.2 cm (6.0 in) # 39.8 cm (15.7 in)

Height 51.8 cm (20.4 in) 30.5 cm (12.0 in) 81.7 cm (32.4 in)

• StepOne™ Instrument – 23.59 ± 0.45 kg (52 ± 1 lbs) ‡‡

Weight
• StepOnePlus™ Instrument – 24.04 ± 0.45 kg (53 ± 1 lbs)

Temperature 15 to 30°C (59 to 86°F) / Maximum change < 15°C every 24 hrs

Humidity 15 to 80% relative humidity, noncondensing

Power 100 to 240 VAC (50 to 60 Hz)

‡ Clearance required to provide adequate space for the instrument drawer to open.
§ Clearance required to ensure adequate airflow and cooling.
# Clearance at the left and right sides to ensure adequate airflow and cooling.
‡‡ Or at least 38.32 kg (84.5 lbs) if you plan to place a computer with the instrument. Actual weights
depend on the computer model you use.

1 square. = (10 cm)2 (3.94 in)2

Notes

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems 39

Installation, Networking, and Maintenance Guide
 Chapter 2 Set Up the Instrument
Unpack the Instrument

Place the PHYSICAL INJURY HAZARD. Do not attempt to lift the

Instrument instrument or any other heavy objects unless you have received related training. Incorrect
lifting can cause painful and sometimes permanent back injury. Use proper lifting
techniques when lifting or moving the instrument. At least two people are required to lift
the instrument.

1. Cut the tape securing the top flaps of the crate and open them.

2. Remove the system packing kit from the instrument and set it aside.

3. Remove the packing material from the crate.

4. Remove the protective cover from the instrument.

2
3

5. Position yourselves on either side of the instrument and grasp it firmly at the
corners.

6. Lift and move the instrument to the installation location.

Guidelines for lifting and moving the instrument:
• Keep your spine in a neutral position.
• Bend at the knees and lift with your legs.
• Do not lift an object and twist your torso at the same time.
• Coordinate your intentions with your assistant before lifting and carrying.

Do not carry the instrument by the instrument drawer. Carrying

the instrument by the drawer can damage the instrument optics.

Yes No

Notes

40 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems

Installation, Networking, and Maintenance Guide
 Chapter 2 Set Up the Instrument
Check the Shipped Materials

Check the Shipped Materials

After unpacking the instrument, confirm that you have received all of the components
shipped with the Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR
Systems.

Instrument and
Computer 9 Component

StepOne™ and StepOnePlus™ instrument

(Optional) Dell® computer:

• Dell® tower computer with a Dell® flat screen monitor, or
• Dell® laptop computer with a PCMCIA Network Card
IMPORTANT! If you did not order a computer from us, provide one that meets the
requirements listed in “Minimum Computer Requirements” on page 30.

Packing Kit StepOnePlus™ System Packing Kit

9 Component PN

Business card holder 4333831

Cable, Ethernet (blue) 4376698

Cable, StepOne™ system (yellow) 4376700

Cables, universal voltage kit ‡ 4377117

MicroAmp® 96-Well Support Base 4379590

MicroAmp® 96-Well Tray for VeriFlex Blocks 4379983

MicroAmp® Adhesive Film Applicator 4333183

MicroAmp® Cap Installing Tool (Handle) 4330015

MicroAmp® Fast 8-Tube Strip (0.1 mL) 4358293

MicroAmp® Fast Optical 96-Well Reaction Plate with Barcode (0.1-mL) 4346906

MicroAmp® Fast Reaction Tube with Cap (0.1-mL) 4358297

MicroAmp® Optical 8-Cap Strip 4323032

MicroAmp® Optical Adhesive Film 4360954

StepOnePlus™ System USB Drive 4333831

Warranty card, release notes —

‡ Contains Australian, British, European, North American, and Japanese power cords.

Notes

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems 41

Installation, Networking, and Maintenance Guide
 Chapter 2 Set Up the Instrument
Check the Shipped Materials

StepOne™ System Packing Kit

9 Component PN

Business card holder 4333831

Cable, Ethernet (blue) 4376698

Cable, StepOne™ system (yellow) 4376700

Cables, universal voltage kit ‡ 4377117

MicroAmp® Fast Reaction Tube with Cap (0.1-mL) 4358297

®
MicroAmp 48-Well Base Adapter 4375284

MicroAmp® 48-Well Optical Adhesive Film 4375928

MicroAmp® 96-Well Support Base 4379590

MicroAmp® Fast 48-Well Tray 4375282

MicroAmp® Fast 8-Tube Strip (0.1-mL) 4358293

MicroAmp® Fast Optical 48-Well Reaction Plate 4375816

MicroAmp® Optical 8-Cap Strip 4323032

StepOne™ System USB Drive 4333831

Warranty card, release notes —

‡ Contains Australian, British, European, North American, and Japanese power cords.

Chemistry StepOnePlus™ System Chemistry Installation Kit

Installation Kit
9 Component PN

TaqMan® RNase P Fast 96-Well Instrument Verification Plate 4351979

StepOne™ System Chemistry Installation Kit

9 Component PN

TaqMan® RNase P Fast 48-Well Instrument Verification Plate 4371439

Notes

42 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems

Installation, Networking, and Maintenance Guide
 Chapter 2 Set Up the Instrument
Check the Shipped Materials

Software and
Documentation 9 Component PN
Kit Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Site 4376768
Preparation Guide

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems 4376783

Quick Reference Card

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems 4376782

Installation, Networking, and Maintenance Guide (this document)

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems 4376786

Getting Started Guide for Genotyping Experiments

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems 4376787

Getting Started Guide for Presence/Absence Experiments

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems 4376784

Getting Started Guide for Standard Curve Experiments

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems 4376785

Getting Started Guide for Relative Standard Curve and Comparative CT
Experiments

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems 4379740

Reagent Guide

Applied Biosystems StepOne™ Real-Time PCR System Software 4379099

Primer Express® Software v3.0 4363991

Miscellaneous items (mouse pad, registration card, and others) —

Notes

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems 43

Installation, Networking, and Maintenance Guide
 Chapter 2 Set Up the Instrument
Set Up the Instrument

Set Up the Instrument

After confirming that you have received all system components, perform an initial
system test of the instrument, then define the instrument settings.

Prepare the 1. Connect the power cord to the system.

Instrument

2. (Optional) Install electrical protective devices to the power cord.

We recommend that you install at least one of the electrical protective devices
(power line regulator, uninterruptible power supply, and/or surge protector) to
prevent loss of data and to protect the instrument from damage resulting from
electrical hazards.

Note: For more information on selecting electrical protective devices for the
instrument, see “Select Protective Hardware and Software” on page 31.

3. Connect the power cord to the power receptacle.

4. Power on the instrument, then wait for it to perform a diagnostic of the system
components.
When you power on the instrument for the first time, it may require more than 5 min
to boot. Proceed with the installation when the touchscreen displays the Main Menu,
indicating that the instrument has completed the boot.

Note: If you are updating the instrument firmware, the instrument may pause for up
to 20 min before completing the boot.

If the instrument displays an error, power off the instrument, wait 30 sec, then power
on the instrument. If the instrument displays the error again, contact Support as
explained in “How to Obtain Support” on page 14.

Notes

44 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems

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 Chapter 2 Set Up the Instrument
Set Up the Instrument

5. Remove the packing material from the sample block(s):

a. Open the instrument drawer.

b. Remove the packing material from the sample block(s).

c. Close the instrument drawer.

When removing the packing material from the sample block(s), you must power on
the instrument to lower the instrument drawer. The drawer is in the “raised” position
during transportation and it will not open until the instrument is powered on.

Define the 1. Set the instrument name:

Settings
a. In the Main Menu, touch Settings Menu, then touch Admin Menu, then
touch Set Instrument Name.
b. Touch the Instrument Name field, enter a name for the instrument, then touch
Done.
The instrument name identifies your computer on the network. If you want to
connect the instrument to a network, the name must be unique.

Note: The instrument does not restrict the length or content of the instrument
name. However, if you plan to connect the instrument to a network, limit the
instrument name to fifteen characters and do not include spaces or special
characters (such as ; : " < > * + = \ | ? , ).

c. Touch Done to save the settings, then touch OK when prompted.

1b

1c

Notes

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems 45

Installation, Networking, and Maintenance Guide
 Chapter 2 Set Up the Instrument
Set Up the Instrument

2. Define the date and time settings:

a. In the Admin Menu, touch Set Date & Time.

b. Touch the Date field, then enter the current date (in year/month/day format),
then touch Done.
c. Touch the Time field, then enter the time in hour:minute format, then
touch Done.
d. Touch AM/PM to display the correct time period.

e. Touch Done to save the settings, then touch OK when prompted.

2b

2c/2d

2e

3. Touch , then touch Yes in the Confirmation Screen to enter standby mode.

Notes

46 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems

Installation, Networking, and Maintenance Guide
 Chapter 2 Set Up the Instrument
Complete the Installation

Complete the Installation

After setting up the instrument, install the remaining system components according to the
layout you selected (standalone or colocated), then perform the RNase P verification
experiment.

Perform the As explained in “Select an Installation Layout” on page 33, the system supports two
Appropriate basic installation layouts: colocated and standalone. Perform the installation according to
Installation the layout you selected.

If you want to install the… Go to…

instrument and the computer to the same location, connected by the Chapter 3, “Install
yellow StepOne system cable. the Colocated
Layout,” on page 49.

caps scroll
Scroll Pause num lock lock
Print Break lock
Screen Lock
F12 SysRq

F8 F9 F10 F11
Page
Up
Num
Lock
* 9 +
F6 F7 Home 8
F5 Insert Page 7
F3 F4 _ += Backspace End Down 6
F2 - Delete 4 5
F1 0 Enter
Esc 8 9 [ ] 2 3
6 7 O P 1
4 5 I " Enter
2 3 Y U L ' 0
~ 1 R T J K ? /
` E H < > Shift
Q W F G Ctrl
Tab S D N M
A V B Alt
Caps Lock Z X C
Shift Alt
Ctrl

instrument and the computer to different locations. Chapter 4, “Install

the Standalone
Layout,” on page 75.

IMPORTANT! Do not install the instrument to an Ethernet network until you complete
the installation by performing an RNase P experiment that passes. See Chapter 5,
“Connect the System to a Network,” on page 101 to connect the instrument to a network
after the installation.

Notes

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems 47

Installation, Networking, and Maintenance Guide
 Chapter 2 Set Up the Instrument
Complete the Installation

Notes

48 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems

Installation, Networking, and Maintenance Guide
 Chapter 3

Install the Colocated Layout

This chapter covers:

■ About the Colocated Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
■ Set Up the Computer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Install the Computer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Install the StepOne™ Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
■ Check for StepOne™ Software Updates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
■ Connect the System Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
■ Perform the RNase P Experiment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Set Up the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Run the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Analyze the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
■ After the Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

Note: For more information about any of the topics discussed in this guide, access the
Help from within the Applied Biosystems StepOne™ Real-Time PCR System Software
by pressing F1, or clicking in the toolbar, or selecting Help StepOne Software
Help.

Notes

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems 49

Installation, Networking, and Maintenance Guide
 Chapter 3 Install the Colocated Layout
About the Colocated Installation

About the Colocated Installation

This chapter explains how to install the Applied Biosystems StepOne™ or StepOnePlus™
Real-Time PCR System for colocated operation.

When to Perform In this layout, the yellow StepOne system cable connects the instrument to the colocated
a Colocated computer. Install the system in the colocated layout when both the computer and the
Installation instrument will be placed together in the same location. See “Colocated Layout” on
page 33 for a complete description of the layout.

caps scroll
Scroll Pause num lock lock
Print Break lock
Screen Lock
F12 SysRq

F8 F9 F10 F11
Page
Up
Num
Lock
* 9 +
F6 F7 Home 8
F5 Insert Page 7
F3 F4 _ += Backspace End Down 6
F2 - Delete 4 5
F1 0 Enter
Esc 8 9 [ ] 2 3
6 7 O P 1
4 5 I " Enter
2 3 Y U L ' 0
~ 1 R T J K ? /
` E H < > Shift
Q W F G Ctrl
Tab S D N M
A V B Alt
Caps Lock Z X C
Shift Alt
Ctrl

Note: If you do not want to connect the computer to the instrument, perform the
standalone installation as described in Chapter 4, “Install the Standalone Layout.”

Notes

50 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems

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 Chapter 3 Install the Colocated Layout
About the Colocated Installation

Colocated Chapter 2, Set Up the Instrument

Installation
Workflow
Set Up the Computer
1. Install the computer.
2. Install the StepOne™ software.
IMPORTANT! Perform step 2 only if you did not
order a computer from us.

Check for Software Updates

Connect the System

1. Connect the instrument to the computer.
2. Confirm the connection.

Perform the RNase P Experiment

1. Set up the experiment.
2. Run the experiment.
3. Analyze the experiment.

Chapter 5, Connect the System

to a Network

Notes

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems 51

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 Chapter 3 Install the Colocated Layout
Set Up the Computer

Set Up the Computer

After you install the StepOne™ or StepOnePlus™ instrument, set up the colocated
computer.

IMPORTANT! If you ordered a computer from us, you need only to unpack the computer
as explained on page 53. Computers shipped by us are ready for use with the instrument
and already contain the StepOne™ software.

Materials • CD, Applied Biosystems StepOne™ Real-Time PCR System Software

Required • Computer

IMPORTANT! If you did not order a computer from us, provide a computer that
satisfies the requirements listed on “Minimum Computer Requirements” on
page 30.

• Screwdrivers, flathead and Phillips

• StepOne system cable, yellow (from the system packing kit)
• (Optional) Protective hardware to install to the computer
We recommend that you install one or more of the following electrical devices to the
computer to prevent loss of data and to protect the computer from damage resulting
from electrical hazards:
• Power line regulator
• Uninterruptible power supply (UPS)
• Surge protector
• Backup storage device
For more information, see “Select Protective Hardware and Software” on page 31.

Notes

52 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems

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 Chapter 3 Install the Colocated Layout
Set Up the Computer

Install the Computer

After obtaining the required materials, install the computer.

Confirm the Site Confirm that the installation site meets the physical and environmental requirements for
Requirements the computer. See the documentation for your computer for the site requirements.

Place the PHYSICAL INJURY HAZARD. Improper lifting can cause painful
Computer and sometimes permanent back injury. Use proper lifting techniques when lifting or moving
the computer. We recommend safety training for proper lifting techniques. Do not attempt to
lift or move the computer or the monitor without the assistance of others. Depending on the
weight of the computer and the monitor, this may require two or more people.

1. If you have not done so already, unpack the monitor, computer, keyboard, and
mouse, and assemble the computer components as described in the computer
installation guide.

caps scroll
Pause num lock lock
Print Scroll Break lock
Screen Lock
F12 SysRq

F8 F9 F10 F11
Page
Up
Num
Lock
* 9 +
F6 F7 Home 8
F5 Insert Page 7
F3 F4 _ += Backspace End Down 6
F2 - Delete 4 5
F1 0 Enter
Esc 8 9 [ ] 2 3
6 7 O P 1
4 5 I " Enter
2 3 Y U L ' 0
~ 1 R T J K ? /
` E H < > Shift
Q W F G Ctrl
Tab S D N M
A V B Alt
Caps Lock Z X C
Shift Alt
Ctrl

Guidelines for lifting and moving the computer:

• Make sure that you have a secure grip on the computer when lifting.
• Make sure that the pathway from the beginning of the lift to the final location is
clear of obstructions.
• Do not lift an object and twist your torso at the same time.
• Keep your spine in a neutral position while lifting with your legs.
• Coordinate lift and movement with all participants before lifting and carrying
the computer.
• Instead of lifting the computer/monitor from the packing box, tilt the box on its
side, then slide the contents out of the box.

2. Make the following connections:

Connect Do Not Connect

Power Mouse Keyboard Monitor Ethernet

or or or

Notes

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 Chapter 3 Install the Colocated Layout
Set Up the Computer

3. If you ordered a laptop computer from us, install the PCMCIA Network Card as
described in the documentation accompanying the card.
After installing the PCMCIA Network Card, wait several minutes to allow the
computer to enable the device. The computer may require time to register the
network card with the operating system before it is available for use.

Note: If you are installing your system in a colocated setup, do not connect the
StepOne system cable to the Ethernet port of the PCMCIA card. If you have already
installed the instrument connection through the card, unplug and connect the
StepOne system cable to the appropriate port.

4. If you ordered a tower computer from us, set the voltage appropriately for your
region as described in the computer installation guide.

5. Get the yellow StepOne system cable from the system packing kit (see “Check the
Shipped Materials” on page 41), then connect the:
• Yellow Ethernet port ( ) of the instrument, to the
• Ethernet port ( ) of the computer

IMPORTANT! Do not connect the StepOne system cable to the blue LAN Port ( );
this port is reserved for a network connection.

6. (Optional) Install electrical protective devices to the computer.

Note: For more information, see “Select Protective Hardware and Software” on
page 31.

Notes

54 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems

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 Chapter 3 Install the Colocated Layout
Set Up the Computer

Power On the 1. Power on the computer and monitor.

Computer
2. Log onto the operating system:
• If you are using a computer that you provided, log on to the operating system as
a member of the Administrators user group.
• If you are using a computer from us, enter Administrator in the User field, then
click Login. Leave the Password field blank.

IMPORTANT! If you are installing a computer that you provided, you must log onto
the Windows® operating system using a user account that belongs to the
Administrators user group.

3. In the Getting Started with Windows® XP window, deselect Show this screen at
startup, then click Exit.

4. Determine the next step:

• If you are installing a computer that you provided, go to “Install the StepOne™
Software” on page 56.
• If you are installing a computer from us, go to “Check for StepOne™ Software
Updates” on page 60.

Notes

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 Chapter 3 Install the Colocated Layout
Set Up the Computer

Install the StepOne™ Software

After setting up the computer, define the display properties, computer name, and the date
and time settings, then install the StepOne™ software.

IMPORTANT! If you ordered a computer from us, skip “Install the StepOne™ Software”
and go to “Check for StepOne™ Software Updates” on page 60. Computers that are
supplied by us already contain the StepOne™ software, and are ready for use with the
instrument.

Prepare the 1. Change the screen saver and power options settings:
Operating
a. Right-click the desktop, then select Properties.
System
b. In the Display Properties dialog box, select the Screen Saver tab.

c. In the Screen saver group box, select (None) in the dropdown menu.

d. Click Power.

e. In the Power Options Properties dialog box, select Home\Office Desk from the
Power schemes dropdown list.
f. Set the Plugged in power scheme settings:
• Monitor – Select After 20 mins from the Plugged in dropdown menu.
• Hard Disks – Select Never from the Plugged in dropdown menu.
• System standby – Select Never from the Plugged in dropdown menu.
g. Click OK to save the settings.

1b

1e

1c
1f

1d

1g

Notes

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 Chapter 3 Install the Colocated Layout
Set Up the Computer

2. Change the screen resolution:

a. In the Display Properties dialog box, select the Settings tab.

b. In the Screen Area group box, use the slider to select 1280 by 1024 pixels.

c. In the Colors group box, select Highest (32 bit) in the dropdown menu.

d. Click OK twice, then click Yes to accept the new resolution.

2a

2b
2c

2d

3. Define the date and time settings:

a. Right-click the time readout in the toolbar, then select Adjust Date/Time.

b. In the Date & Time tab, define the date and time settings.

c. Select the Time Zone tab.

d. Select a time zone from the dropdown menu, then select Automatically adjust
clock for daylight saving changes if necessary.
e. Click OK.

Note: Computers on a network synchronize their date/time settings with the server.

3c
3d

3b

3d

3e

Notes

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 Chapter 3 Install the Colocated Layout
Set Up the Computer

4. Change the computer name:

a. In the desktop, right-click My Computer, then select Properties.
b. In the System Properties dialog box, select the Computer Name tab.

c. Click Change.

d. In the Computer Name field, enter the computer name.

Do not give the computer the same name as the instrument.

A shared name could lead to network conflicts if you install both devices to an
Ethernet network.

e. If necessary, define the Domain or Workgroup settings if you are going to

connect the computer to a network.
f. Click OK twice.

g. In the Network Identification dialog box, click OK.

Note: The computer name identifies the computer on the network and must be
different than that of the instrument. After you install the system, you can change
the computer name without disrupting the function of the StepOne™ software.

4b

4d

4e

4c

4f

4g

5. In the Systems Settings Change dialog box, click Yes to restart the computer.

6. Log onto the operating system as a member of the Administrators user group.

Notes

58 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems

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 Chapter 3 Install the Colocated Layout
Set Up the Computer

Install the IMPORTANT! You must be logged into the Windows® operating system as an
Software administrator to install the StepOne™ software.

Note: If you encounter errors during the installation of the StepOne™ software, reinstall
the software as described in “Uninstall the StepOne™ Software” on page 160.

1. Insert the Applied Biosystems StepOne™ Real-Time PCR System Software CD into
the CD drive of the computer and wait for the installer to start.
If the installer does not start automatically, double-click (My Computer),
navigate to the CD drive, then double-click Setup.exe.

2. In the Welcome page of the InstallShield Wizard, click Next.

3. In the License Agreement page, click Yes to accept the agreement.

4. In the Choose Destination Location page, click Next to accept the default location.

Note: We recommend installing the StepOne™ software to the D drive; however,

you can install the software to another location.

5. In the Default Instrument Type page, select the type of instrument that you are
installing (StepOnePlus™ Instrument or StepOne™ Instrument), then click Next.

6. In the Start Copying Files page, confirm that the installation location displayed in
the Current Settings field is correct, then click Next to begin installing the
StepOne™ software.

7. When the StepOne™ software completes the installation, click Finish.

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Check for StepOne™ Software Updates

Check for StepOne™ Software Updates

Updates and/or patches may be available for the StepOne software. Applied Biosystems
recommends that you check for software updates before you proceed with the
installation.

Note: To periodically check for software updates after the StepOne or StepOnePlus
system is installed (as part of routine maintenance), see “Update the StepOne™ Software
or the Operating System” on page 154.

To check for software updates:

1. Go to: http://www.lifetechnologies.com/support and click the Instrument

Software, Patches & Updates link.

2. In the Instrument Software, Patches & Updates page, click the link for the
appropriate software.

3. Under Software Download, view the available downloads.

4. If there are updates and/or patches for StepOne software v2.1 or later, select the
appropriate option, then follow the prompts to begin the download.
You may be asked for your instrument serial number. The serial number is on the
back panel of your instrument, or can be obtained from the instrument touchscreen.

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Connect the System Components

Connect the System Components

After preparing the computer, connect the instrument to the computer, then confirm that
the StepOne™ software can access the instrument by retrieving the maintenance data.

Establish the 1. If necessary, power on the instrument.

Connection

2. Power on the computer and monitor.

3. Log in as Administrator, or as a member of the Administrators user group.

IMPORTANT! If you install the system using a computer that you provide, you must
log onto the Windows® operating system using a user account that belongs to the
Administrators user group.

4. In the desktop, double-click (StepOne software) or select

Start All Programs Applied Biosystems StepOne Software
<software name>
where <software name> is the current version of the StepOne software.
While the StepOne™ software loads:
• The software establishes communication with the instrument.
• The Windows XP operating system automatically detects the connection and
displays a Local Area Connection alert in the taskbar.

Note: You can disable the network connection messages displayed when the
StepOne™ software and instrument communicate. See “(Optional) Disable the
Local Area Connection Messages” on page 63 for more information.

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5. In the Login dialog box, create a user name:

a. In the User Name field, enter a user name.

Note: You cannot use the following characters in the User Name field: space,
forward slash (/), backslash (\), greater than sign (>), less than sign (<), asterisk
(*), question mark (?), quotation mark ("), vertical line (|), colon (:), or
semicolon (;).

b. Click OK.
We recommend that you log in with a user name. If you log in with a user name, you
can set preferences in the software. The next time you log in to the software with the
same user name, the software uses the preferences you set as the defaults.

IMPORTANT! If you log in to the software as a Guest, you cannot set preferences.

6. If you are prompted to update the instrument firmware, click Upgrade Firmware Now.

7. When the StepOne™ software prompts you to perform the RNase P experiment,
click Open Instrument Maintenance Manager.

Wait while the StepOne™ software downloads the maintenance data from the
instrument. If successful, the StepOne™ software automatically opens the RNase P
Run wizard in the Instrument Maintenance Manager.
If the StepOne™ software displays the “Instrument Connection Failed” message:
a. Confirm that the computer and instrument are connected by the yellow
StepOne system cable as explained in step 5 on page 54.
b. Power off the instrument, then after 30 sec power on the instrument.

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c. When the instrument displays the main screen, click Retry.

If the problem persists, contact Support as explained in “How to Obtain
Support” on page 14.

(Optional) Disable During normal operation, the Windows® operating system displays Local Area
the Local Area Connection messages when the StepOne™ software and instrument communicate. You
Connection can disable these messages by changing the network connection settings for the Ethernet
Messages network interface adapter as explained below.

Alert displayed when instrument communication…

begins ends

To disable the network connection alerts for the instrument connection:

1. In the desktop, select Start Control Panel, then double-click

Network Connections.

2. Right-click Local Area Connection, then select Properties.

3. Deselect Show icon in notification area when connected.

4. Deselect Notify me when this connection has limited or no connectivity.

5. Click OK.

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6. Close the Network Connections window.

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Perform the RNase P Experiment

Perform the RNase P Experiment

After you establish system communication, confirm the function of the system by
running a TaqMan® RNase P Fast Instrument Verification Plate.

Materials • TaqMan® RNase P Fast Instrument Verification Plate

Required • Powder-free gloves
• Safety glasses
• Centrifuge with reaction plate adapter

When to Perform We recommend performing an RNase P experiment:

the RNase P • After installing the system.
Experiment
• After moving the instrument to another location.
• As needed to confirm the function of the instrument, depending on your laboratory
and regulatory requirements.

Purpose of the The TaqMan® RNase P Fast Instrument Verification Plate experiment verifies the
Experiment performance of the instrument. The RNase P plate is preloaded with the reagents necessary
for the detection and quantitation of genomic copies of the human RNase P gene (a single-
copy gene encoding the RNase moiety of the RNase P enzyme).
Each well contains:
• 1✕ TaqMan® Fast Universal PCR Master Mix, No AmpErase® UNG
• RNase P primers
• FAM™ dye-labeled probe
• Known concentration of human genomic DNA template
The figure below illustrates the arrangement of the standard and unknown populations on
the RNase P plate. The RNase P plate contains five replicate groups of standards (1250,
2500, 5000, 10,000, and 20,000 copies), two unknown populations (5000 and 10,000
copies), and negative control wells (NC).

5K-copy Unknown
Population 1
STD 2.5K

STD 10K

5K-copy 5K-copy
NC

Unknown Unknown NC STD 1250 STD 2500

Population 1 Population 1
STD 5000 STD 10000 STD 20000
STD 1.25K

STD 20K

10K-copy Unknown
STD 5K

10K-copy 10K-copy
Unknown Unknown
Population 2 Population 2 Population 2

TaqMan® 48-well RNase P Plate for TaqMan® 96-well RNase P Plate for
the StepOne™ System the StepOnePlus™ System

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After the run, the StepOne™ software:

1. Generates a standard curve from the averaged threshold cycle (CT) values of the
replicate groups of standards.

2. Calculates the concentration of the two unknown populations using the standard
curve.

3. Calculates the following to assess the instrument performance:

[ ( CopyUnk2 ) – 3 ( σ CopyUnk2 ) ] > [ ( CopyUnk 1 ) + 3 ( σ CopyUnk1 ) ]

where:
• CopyUnk1 = Average copy number of unknown #1 (5,000-copy population)
• σCopyUnk1 = Standard deviation of unknown #1 (5,000-copy population)
• CopyUnk2 = Average copy number of unknown #2 (10,000-copy population)
• σCopyUnk2 = Standard deviation of unknown #2 (10,000-copy population)

Installation Specification
The instrument passes the installation specification if the inequality holds and the
instrument successfully distinguishes between 5,000 and 10,000 copies with greater than
99.7% confidence.
To meet the installation specification, you can omit a limited number of outlier wells
from the 5,000- and 10,000-copy unknown populations. The number of wells that you
can remove depends on the instrument that you are installing.

Maximum number of outlier wells that can be

removed from each…
Instrument Total
Negative
Standard
Unknown Population Controls
(STD)
(NC)

StepOnePlus™ System 6 0 0 12

StepOne™ System 2 0 0 4

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Set Up the Experiment

Prepare the TaqMan® RNase P Fast Instrument Verification Plate for the run.

Prepare the IMPORTANT! Do not use an RNase P plate for another Thermo Fisher Scientific
RNase P Plate instrument to verify the performance of the StepOne™ system. RNase P plates for other
instruments contain the TAMRA™ dye, which is not supported by the StepOne™ system.

IMPORTANT! Wear powder-free gloves when you handle the RNase P plate.

1. Get the TaqMan® RNase P Fast Instrument Verification Plate from the freezer, then
allow the reaction plate to warm to room temperature (for approximately 5 min).

2. Remove the RNase P plate from its packaging.

3. Vortex the reaction plate for 5 sec.

4. Centrifuge the reaction plate for 2 min at less than1500 rpm.

IMPORTANT! The reaction plate must be well mixed and centrifuged.

5. Confirm that the liquid is at the bottom of each well of the reaction plate. If not,
centrifuge the reaction plate again at a greater rpm and for a longer time.

IMPORTANT! Do not allow the bottom of the RNase P plate to become dirty. Fluids
and other contaminants that adhere to the bottom of the reaction plate can
contaminate the sample block(s) and cause an abnormally high background signal.

Correct Incorrect

Liquid is at the • Not centrifuged with enough force, or

bottom of the well. • Not centrifuged for enough time

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Run the Experiment

After preparing the TaqMan® RNase P Fast Instrument Verification Plate, load the plate
into the instrument and start the run.

Load the PHYSICAL INJURY HAZARD. During instrument operation, the

Instrument temperature of the sample block(s) can exceed 100°C. If the instrument has been used
recently, keep your hands away until the sample block(s) reach room temperature.

IMPORTANT! Wear powder-free gloves when you handle the RNase P plate.

1. Open the instrument drawer.

2. Place the RNase P plate in the sample block(s) so that the A1 position is at the back-
left corner of the sample block(s).

StepOnePlus™ System StepOne™ System

A1 A1

3. Close the instrument drawer carefully.

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Start the Run 1. If the StepOne™ software automatically opened the RNase P Run dialog box, go to
step 2. Otherwise, display the wizard as follows:
a. Select Instrument Instrument Maintenance Manager.
b. In the Instrument Maintenance Manager, select RNase P in the navigation
column.
c. Click Start RNase P Run.

2. In the Setup screen of the RNase P Run wizard, select The RNase P plate is loaded
into the instrument.

3. Click Next.

4. In the Run screen of the RNase P Run wizard, click Start Run .

Note: Before starting the run, the instrument may take up to 15 min to heat the
heated cover to the correct temperature.

1a

1c

1b

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Analyze the Experiment

Review the data to confirm the results of the experiment.

Confirm the Note: After the StepOne™ software completes the RNase P run, it automatically
Results of the analyzes the run and displays the results in the Analysis screen.
Analysis
1. In the Analysis screen of the RNase P Run wizard, confirm the status of the run:
• Passed – The instrument passed the RNase P run. Go to step 5 on page 72.
• Failed – The instrument failed the RNase P run. Go to step 2 to screen the
experiment for outliers.
If the run fails, the automated analysis may have included outliers that caused the
initial analysis to fail. Experimental error may cause some wells to be amplified
insufficiently or not at all. These wells typically produce CT values that differ
significantly from the average for the associated replicate wells. If included in the
calculations, these outlying data (outliers) can result in erroneous measurements.

2. In the Amplification Plot, select Ct vs. Well from the Plot Type dropdown menu.

3. Confirm the uniformity of each replicate population on the reaction plate (controls,
standards, and unknowns) by comparing the groupings of CT values:
a. In the plate layout, select the wells containing the 10,000-copy unknown
population:
• StepOne™ System – Wells from columns 1, 2, 6, 7, and 8 in rows D, E, and F
• StepOnePlus™ System – Wells rows F, G, and H

5K-copy Unknown
Population 1
STD 2.5K

STD 10K

5K-copy 5K-copy NC STD 1250 STD 2500

NC

Unknown Unknown
Population 1 Population 1
STD 5000 STD 10000 STD 20000
STD 1.25K

10K-copy Unknown
STD 20K
STD 5K

10K-copy 10K-copy
Unknown
Population 2
Unknown
Population 2
Population 2

TaqMan® 48-well RNase P Plate for TaqMan® 96-well RNase P Plate for
the StepOne™ System the StepOnePlus™ System

b. In the plot, confirm that the CTs of the replicate population are equivalent.

Note: The numbers on the X-axis of the plot correspond to the wells of the
reaction plate. Beginning with well A1, the wells are numbered from left-to-
right, and top-to-bottom.

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c. If an outlier is present in the population, select the corresponding well in the

plate layout, then click Omit to remove the well from the analysis.

Maximum number of outlier wells that can be

removed from each…
Instrument Total
Unknown Standard Negative
Population (STD) Control (NC)

StepOnePlus™ System 6 0 0 12

StepOne™ System 2 0 0 4

IMPORTANT! If too many outliers are present, order another RNase P plate and
repeat the experiment.

d. Repeat steps 3a through 3c for each replicate population (unknowns, standards,

and negative controls) on the reaction plate.

4. Click Reanalyze to analyze the run without the outliers.

If the status of the RNase P Run is “Failed” after performing steps 2 through 4, repeat
the RNase P experiment using a different RNase P plate. If the problem persists,
contact Support as explained in “How to Obtain Support” on page 14.

3c 4

2
3a

3b

Outlier

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5. Review the standard curve:

a. Select the Standard Curve tab.

b. Click the upper-right corner of the Plate Layout to select all wells.

c. Confirm that the R2 value is greater than or equal to 0.990.

If the R2 value is less than 0.990, repeat the RNase P experiment using a different
RNase P plate. If the problem persists, contact Support as explained in “How to
Obtain Support” on page 14.

6. Click Finish, click Yes when prompted to save the experiment.

5a

5b

5c

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Unload the PHYSICAL INJURY HAZARD. During instrument operation, the

Instrument temperature of the sample block(s) can exceed 100°C. If the instrument has been used
recently, keep your hands away until the sample block(s) reach room temperature.

IMPORTANT! Wear powder-free gloves when you handle the RNase P plate.

1. Open the instrument drawer.

2. Remove the RNase P plate from the sample block(s).

3. Close the instrument drawer carefully.

4. Dispose of the RNase P plate.

IMPORTANT! Do not power off the instrument following a run. The instrument
automatically enters a hibernation mode when not in use. Power off the instrument
only when it will not be used for an extended period.

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After the Installation

After the Installation

After verifying the performance of the system by completing the RNase P experiment,
you can install additional software to the computer and/or connect the instrument to a
network.

Install Additional We recommend installing the following types of software to improve the function and
Software security of the system computer:
• Antivirus software
• Archival or file compression software
• Security software (firewall and encryption utilities)
• Performance optimizing software

Note: For more information, see “Select Protective Hardware and Software” on page 31.

To confirm that third-party software does not interfere with the StepOne™ software:

1. Install the software to the computer that contains the StepOne™ software.

2. Perform several test experiments using “dummy” plates (plates that do not contain
reagents).

Note: The goal of the test experiments is to run plates under conditions that match
normal instrument operation. Therefore, the characteristics of the test experiments
(plate layout and run method) must closely resemble your actual experiments.

3. Confirm that the system performs each test experiment without producing errors.
If the system performs the tests successfully, perform experiments normally. If the
system encounters errors during the test runs, the software may not be compatible
with the StepOne™ software.

Network the You can expand the functionality of the colocated instrument by connecting it to an
System Ethernet network. When the colocated instrument is part of a network, computers on the
network that are running the StepOne™ software can:
• Monitor the status of a run in progress
• Send and download experiments to and from the instrument

IMPORTANT! Computers linked to a colocated instrument over a network cannot control

the instrument.

To connect the instrument to a network, go to Chapter 5, “Connect the System

to a Network,” on page 101.

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Install the Standalone Layout

This chapter covers:

■ About the Standalone Installation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
■ Set Up the Computer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Install the Computer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Install the StepOne™ Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
■ Check for StepOne™ Software Updates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
■ Prepare the StepOne™ Software for Standalone Use. . . . . . . . . . . . . . . . . . . . . . . 86
■ Perform the RNase P Experiment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
Set Up the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Run the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
■ Analyze the RNase P Experiment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Unload the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Transfer the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Analyze the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
■ After the Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

Note: For more information about any of the topics discussed in this guide, access the
Help from within the Applied Biosystems StepOne™ Real-Time PCR System Software
by pressing F1, or clicking in the toolbar, or selecting Help StepOne Software
Help.

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About the Standalone Installation

About the Standalone Installation

This chapter explains how to install the system for standalone operation.

When to Perform In this layout, the instrument is not connected to the computer running the StepOne™
a Standalone software. Instead, a USB drive ( ) is used to transfer data between the system
Installation components. Install the system in the standalone layout when the computer and the
instrument must be placed in separate locations. See “Standalone Layout” on page 34 for
complete descriptions of the layouts.

Note: If you want to connect the computer to the instrument, perform the colocated
installation as described in Chapter 3, “Install the Colocated Layout.”

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About the Standalone Installation

Standalone Chapter 2, Set Up the Instrument

Installation
Workflow Set Up the Computer
1. Install the computer.
2. Install the StepOne™ software.
Note: Perform step 2 only if you did not order a
computer from us.

Check for Software Updates

Prepare the Software for Standalone Use

Perform the RNase P Experiment

1. Set up the experiment.
2. Run the experiment.

Analyze the RNase P Experiment

1. Transfer the experiment data.
2. Analyze the experiment.

Chapter 5, Connect the System

to a Network

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Set Up the Computer

After you install the StepOne™ or StepOnePlus™ instrument, set up the standalone
computer.

IMPORTANT! If you ordered a computer from us, you need only to unpack the computer
as explained on page 79. Computers shipped by us are ready for use with the instrument
and already contain the StepOne™ software.

Materials • CD, Applied Biosystems StepOne™ Real-Time PCR System Software

Required • Computer

IMPORTANT! If you did not order a computer from us, provide a computer that
satisfies the requirements listed in “Minimum Computer Requirements” on page 30.
• Screwdrivers, flathead and Phillips
• (Optional) Protective hardware to install to the computer
We recommend that you install one or more of the following electrical devices to the
computer to prevent loss of data and to protect the computer from damage resulting
from electrical hazards:
• Power line regulator
• Uninterruptible power supply (UPS)
• Surge protector
• Backup storage device
For more information, see “Select Protective Hardware and Software” on page 31.

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Install the Computer

After obtaining the required materials, install the computer.

Confirm the Site Confirm that the installation site meets the physical and environmental requirements for
Requirements the computer. See the documentation for your computer for the site requirements.

Place the PHYSICAL INJURY HAZARD. Improper lifting can cause painful
Computer and sometimes permanent back injury. Use proper lifting technique when lifting or
moving the computer. We recommend safety training for proper lifting techniques. Do
not attempt to lift or move the computer or the monitor without the assistance of others.
Depending on the weight of the computer, this may require two or more people.

1. If you have not done so already, unpack the monitor, computer, keyboard, and
mouse, and assemble the computer components as described in the computer
installation guide.
Guidelines for lifting and moving the computer:
• Make sure that you have a secure grip on the computer when lifting.
• Make sure that the pathway from the beginning of the lift to the final location is
clear of obstructions.
• Do not lift an object and twist your torso at the same time.
• Keep your spine in a neutral position while lifting with your legs.
• Coordinate lift and movement with all participants before lifting and carrying
the computer.
• Instead of lifting the computer/monitor from the packing box, tilt the box on its
side, then slide the contents out of the box.

2. Make the following connections:

Connect Do Not Connect

Power Mouse Keyboard Monitor Ethernet

or or or

3. If you ordered a laptop computer from us, install the PCMCIA Network Card as
described in the documentation accompanying the card.
After installing the PCMCIA Network Card, wait several minutes to allow the
computer to enable the device. The computer may require time to initialize the drivers
and register the network card with the operating system before it is available for use.

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4. If you ordered a tower computer from us, set the voltage appropriately for your
region as described in the computer installation guide.

5. (Optional) Install electrical protective devices to the computer.

Note: For more information, see “Select Protective Hardware and Software” on
page 31.

Power On the 1. Power on the computer and monitor.

Computer
2. Log onto the operating system:
• If you are using a computer that you provided, log on to the operating system as
a member of the Administrators user group.
• If you are using a computer from us, enter Administrator in the User field, then
click Login. Leave the Password field blank.

IMPORTANT! If you are installing a computer that you provided, log onto the
Windows® operating system using a user account that belongs to the Administrators
user group.

3. In the Getting Started with Windows® XP window, deselect Show this screen at
startup, then click Exit.

4. Determine the next step:

• If you are installing a computer that you provided, go to “Install the StepOne™
Software” on page 81.
• If you are installing a computer from us, go to “Check for StepOne™ Software
Updates” on page 85.

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Set Up the Computer

Install the StepOne™ Software

After setting up the computer, define the display properties, computer name, and the date
and time settings, then install the StepOne™ software.

IMPORTANT! If you ordered a computer from us, skip “Install the StepOne™ Software”
and go to “Check for StepOne™ Software Updates” on page 85. Computers that are
supplied by us already contain the StepOne™ software, and are ready for use with the
instrument.

Prepare the 1. Change the screen saver and power options settings:
Operating
a. Right-click the desktop, then select Properties.
System
b. In the Display Properties dialog box, select the Screen Saver tab.

c. In the Screen saver group box, select (None) in the dropdown menu.

d. Click Power.

e. In the Power Options Properties dialog box, select Home\Office Desk from the
Power schemes dropdown list.
f. Define the Plugged in power scheme settings:
• Monitor – Select After 20 mins from the Plugged in dropdown menu.
• Hard Disks – Select Never from the Plugged in dropdown menu.
• System standby – Select Never from the Plugged in dropdown menu.
g. Click OK to save the settings.

1b

1e

1c
1f

1d

1g

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2. Change the screen resolution:

a. In the Display Properties dialog box, select the Settings tab.

b. In the Screen Area group box, use the slider to select 1280 by 1024 pixels.

c. In the Colors group box, select Highest (32 bit) in the dropdown menu.

d. Click OK twice, then click Yes to accept the new resolution.

2a

2b
2c

2d

3. Define the date and time settings:

a. Right-click the time readout in the toolbar, then select Adjust Date/Time.

b. In the Date & Time tab, define the date and time settings.

c. Select the Time Zone tab.

d. Select a time zone from the dropdown menu, then select Automatically adjust
clock for daylight saving changes if necessary.
e. Click OK.

Note: Computers on a network synchronize their date/time settings with the server.

3c
3d

3b

3d

3e

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Set Up the Computer

4. Change the computer name:

a. In the desktop, right-click My Computer, then select Properties.
b. In the System Properties dialog box, select the Computer Name tab.

c. Click Change.

d. In the Computer Name field, enter the computer name.

Do not give the computer the same name as the instrument.

A shared name could lead to network conflicts if you install both devices to an
Ethernet network.

e. If necessary, define the Domain or Workgroup settings if you are going to

connect the computer to a network.
f. Click OK twice.

g. In the Network Identification dialog box, click OK.

Note: The computer name identifies the computer on the network and must be
different than that of the instrument. After you install the system, you can change
the computer name without disrupting the function of the StepOne™ software.

4b

4d

4e

4c

4f

4g

5. In the Systems Settings Change dialog box, click Yes to restart the computer.

6. Log onto the operating system as a member of the Administrators user group.

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Set Up the Computer

Install the IMPORTANT! You must be logged into the Windows® operating system as an
Software administrator to install the StepOne™ software.

Note: If you encounter errors during the installation of the StepOne™ software, uninstall
the software as explained in “Uninstall the StepOne™ Software” on page 160.

1. Insert the Applied Biosystems StepOne™ Real-Time PCR System Software CD into
the CD drive of the computer and wait for the installer to start.
If the installer does not automatically start, double-click (My Computer),
navigate to the CD drive, then double-click Setup.exe.

2. In the Welcome page of the InstallShield Wizard, click Next.

3. In the License Agreement page, click Yes to accept the agreement.

4. In the Choose Destination Location page, click Next to accept the default location.

Note: We recommend installing the StepOne™ software to the D drive; however,

you can install the software to another location.

5. In the Default Instrument Type page, select the type of instrument that you are
installing (StepOnePlus™ Instrument or StepOne™ Instrument), then click Next.

6. In the Start Copying Files page, confirm that the installation location displayed in
the Current Settings field is correct, then click Next to begin installing the
StepOne™ software.

7. When the StepOne™ software completes the installation, click Finish.

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Check for StepOne™ Software Updates

Check for StepOne™ Software Updates

Updates and/or patches may be available for the StepOne software. We recommend that
you check for software updates before you proceed with the installation.

Note: To periodically check for software updates after the StepOne or StepOnePlus
system is installed (as part of routine maintenance), see “Update the StepOne™ Software
or the Operating System” on page 154.

To check for software updates:

1. Go to: http://www.lifetechnologies.com/support and click the Instrument

Software, Patches & Updates link.

2. In the Instrument Software, Patches & Updates page, click the link for the
appropriate software.

3. Under Software Download, view the available downloads.

4. If there are updates and/or patches for StepOne software v2.1 or later, select the
appropriate option, then follow the prompts to begin the download.
You may be asked for your instrument serial number. The serial number is on the
back panel of your instrument, or can be obtained from the instrument touchscreen.

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Prepare the StepOne™ Software for Standalone Use

Prepare the StepOne™ Software for Standalone Use

After installing the StepOne™ software, set the software preferences so that the software
does not attempt to connect to the instrument at startup or confirm the status of the RNase
P experiment.

Start the 1. In the desktop, double-click (StepOne software) or select

Software Start All Programs Applied Biosystems StepOne Software
<software name>
where <software name> is the current version of the StepOne software.

2. In the Login dialog box, create a user name.

a. In the User Name field, enter a user name.

Note: You cannot use the following characters in the User Name field: space,
forward slash (/), backslash (\), greater than sign (>), less than sign (<), asterisk
(*), question mark (?), quotation mark ("), vertical line (|), colon (:), or
semicolon (;).

b. Click OK.
We recommend that you log in with a user name. If you log in with a user name, you
can set preferences in the software. The next time you log in to the software with the
same user name, the software uses the preferences you set as the defaults.

IMPORTANT! If you log in to the software as a Guest, you cannot set preferences.
You will not be able to perform the steps in “Set the Preferences” on page 87.

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3. When the Instrument Connection Failed dialog box appears, click Continue
without connection.

Set the 1. In the Main Screen, select Tools Preferences.

Preferences
2. In the Preferences dialog box, select the Startup tab.

3. Deselect Connect to default instrument.

4. Click OK.

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Perform the RNase P Experiment

Perform the RNase P Experiment

After you install the instrument, confirm the function of the system by running a
TaqMan® RNase P Fast Instrument Verification Plate.

Materials • TaqMan® RNase P Fast Instrument Verification Plate

Required • Powder-free gloves
• Safety glasses
• Centrifuge with plate adapter

When to Perform We recommend performing an RNase P experiment:

the RNase P • After installing the system.
Experiment
• After moving the instrument to another location.
• As needed to confirm the function of the instrument, depending on your laboratory
and regulatory requirements.

Purpose of the The TaqMan® RNase P Fast Instrument Verification Plate experiment verifies the
Experiment performance of the instrument. The RNase P plate is preloaded with the reagents necessary
for the detection and quantitation of genomic copies of the human RNase P gene (a single-
copy gene encoding the RNase moiety of the RNase P enzyme).
Each well contains:
• 1✕ TaqMan® Fast Universal PCR Master Mix, No AmpErase® UNG
• RNase P primers
• FAM™ dye-labeled probe
• Known concentration of human genomic DNA template
The figure below illustrates the arrangement of the standard and unknown populations on
the RNase P plate. The RNase P plate contains five replicate groups of standards (1250,
2500, 5000, 10,000, and 20,000 copies), two unknown populations (5000 and 10,000
copies), and negative control wells.

5K-copy Unknown
Population 1
STD 2.5K

STD 10K

5K-copy 5K-copy
NC

Unknown Unknown NC STD 1250 STD 2500

Population 1 Population 1
STD 5000 STD 10000 STD 20000
STD 1.25K

STD 20K

10K-copy Unknown
STD 5K

10K-copy 10K-copy
Unknown Unknown
Population 2 Population 2 Population 2

TaqMan® 48-well RNase P Plate for TaqMan® 96-well RNase P Plate for
the StepOne™ System the StepOnePlus™ System

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After the run, the StepOne™ software:

1. Generates a standard curve from the averaged threshold cycle (CT) values of the
replicate groups of standards.

2. Calculates the concentration of the two unknown populations using the standard
curve.

3. Calculates the following to assess the instrument performance:

[ ( CopyUnk2 ) – 3 ( σ CopyUnk2 ) ] > [ ( CopyUnk 1 ) + 3 ( σ CopyUnk1 ) ]

where:
• CopyUnk1 = Average copy number of unknown #1 (5,000-copy population)
• σCopyUnk1 = Standard deviation of unknown #1 (5,000-copy population)
• CopyUnk2 = Average copy number of unknown #2 (10,000-copy population)
• σCopyUnk2 = Standard deviation of unknown #2 (10,000-copy population)

Installation Specification
The instrument passes the RNase P experiment if the inequality holds and the instrument
successfully distinguishes between 5,000 and 10,000 copies with greater than 99.7%
confidence.
To meet the installation specification, you can omit a limited number of outlier wells
from the 5,000- and 10,000-copy unknown populations. The number of wells that you
can remove depends on the instrument that you are installing.

Maximum number of outlier wells that can be

removed from each…
Instrument Total
Negative
Unknown Population Standard Controls
(NC)

StepOnePlus™ System 6 0 0 12

StepOne™ System 2 0 0 4

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Perform the RNase P Experiment

Set Up the Experiment

Prepare the TaqMan® RNase P Fast Instrument Verification Plate for the run.

Prepare the IMPORTANT! Do not use an RNase P plate for another Thermo Fisher Scientific
RNase P Plate instrument to verify the performance of the StepOne™ system. RNase P plates for other
instruments contain the TAMRA™ dye, which is not supported by the StepOne™ system.

IMPORTANT! Wear powder-free gloves when you handle the RNase P plate.

1. Get the TaqMan® RNase P Fast Instrument Verification Plate from the freezer, then
allow the reaction plate to warm to room temperature (for approximately 5 min).

2. Remove the RNase P plate from its packaging.

3. Vortex the RNase P plate for 5 sec.

4. Centrifuge the RNase P plate for 2 min at less than1500 rpm.

IMPORTANT! The RNase P plate must be well mixed and centrifuged.

5. Confirm that the liquid is at the bottom of each well of the RNase P plate. If not,
centrifuge the plate again at a greater rpm and for a longer time.

IMPORTANT! Do not allow the bottom of the RNase P plate to become dirty. Fluids
and other contaminants that adhere to the bottom of the RNase P plate can
contaminate the sample block(s) and cause an abnormally high background signal.

Correct Incorrect

Liquid is at the • Not centrifuged with enough force, or

bottom of the well. • Not centrifuged for enough time

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Perform the RNase P Experiment

Run the Experiment

After preparing the TaqMan® RNase P Fast Instrument Verification Plate, load the plate
into the instrument and start the run.

Load the PHYSICAL INJURY HAZARD. During instrument operation, the

Instrument temperature of the sample block(s) can exceed 100°C. If the instrument has been used
recently, keep your hands away until the sample block(s) reaches room temperature.

IMPORTANT! Wear powder-free gloves when you handle the RNase P plate.

1. Open the instrument drawer.

2. Place the RNase P plate in the sample block(s) so that the A1 position is at the back-
left corner of the sample block(s).

StepOnePlus™ System StepOne™ System

A1 A1

3. Close the instrument drawer carefully.

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Perform the RNase P Experiment

Start the Run 1. Touch the instrument touchscreen to awaken it, then touch .

2. In the Main Menu, touch Tools Menu, then touch RNase P Wizard.

3. In the Welcome screen, touch Continue, then touch four times to bypass the
instructions screens.

4. In the Start Run screen, touch Start to begin the run.

Note: Before starting the run, the instrument may take up to 15 min to heat the
heated cover to the correct temperature.

Notes

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Analyze the RNase P Experiment

Analyze the RNase P Experiment

After setting up the computer and the instrument completes the run, analyze the RNase P
experiment.

Materials • Powder-free gloves

Required • Safety glasses
• USB Drive (from the system packing kit)

Unload the Instrument

Unload the RNase P plate from the instrument.

Remove the PHYSICAL INJURY HAZARD. During instrument operation, the

RNase P Plate temperature of the sample block(s) can exceed 100°C. If the instrument has been used
recently, keep your hands away until the sample block(s) reaches room temperature.

IMPORTANT! Wear powder-free gloves when you handle the RNase P plate.

1. Open the instrument drawer.

2. Remove the RNase P plate from the sample block(s).

3. Close the instrument drawer carefully.

4. Dispose of the RNase P plate.

IMPORTANT! Do not power off the instrument following a run. The instrument
automatically enters a hibernation mode when not in use. Power off the instrument
only when it will not be used for an extended period.

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Transfer the Experiment

After unloading the instrument, transfer the experiment data to the computer for analysis.

Transfer the Data 1. Connect a USB drive to the USB port.

to the USB Drive

2. In the RNase P Wizard, touch .

If the instrument cannot find the USB drive, touch OK, wait for 30 sec, then touch
Collect Results again.

3. When prompted that the data has been transferred successfully, touch OK, then
touch .

4. Remove the USB drive from the instrument.

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Transfer the Data 1. Connect the USB drive to one of the USB ports on the computer.
to the Computer
2. In the StepOne™ software, select Instrument Instrument Maintenance
Manager.

3. In the Instrument Maintenance Manager, click RNase P.

4. Select the RNase P Run:

a. In the RNase P Run screen, click Browse.

b. In the Open dialog box, navigate to the USB drive.

c. In the file name field, select the experiment.

d. Click Open.
The StepOne™ software automatically analyzes the run and displays the results in
the RNase P Run screen.

4a

4b

4c
3

4d

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Analyze the Experiment

Review the data to confirm the results of the experiment.

Confirm the 1. In the Analysis screen of the RNase P Run wizard, confirm the status of the run:
Results of the • Passed – The instrument passed the RNase P run. Go to step 5 on page 98.
Analysis • Failed – The instrument failed the RNase P run. Go to step 2 to screen the
experiment for outliers.
If the run fails, the automated analysis may have included outliers that caused the
initial analysis to fail. Experimental error may cause some wells to be amplified
insufficiently or not at all. These wells typically produce CT values that differ
significantly from the average for the associated replicate wells. If included in the
calculations, these outlying data (outliers) can result in erroneous measurements.

2. In the Amplification Plot, select Ct vs. Well from the Plot Type dropdown menu.

3. Confirm the uniformity of each replicate population on the RNase P plate (controls,
standards, and unknowns) by comparing the groupings of CT values:
a. In the plate layout, select the wells containing the 10,000-copy unknown
population:
• StepOne™ System – Wells from columns 1, 2, 6, 7, and 8 in rows D, E, and F
• StepOnePlus™ System – Wells rows F, G, and H

5K-copy Unknown
Population 1
STD 2.5K

STD 10K

5K-copy 5K-copy NC STD 1250 STD 2500

NC

Unknown Unknown
Population 1 Population 1
STD 5000 STD 10000 STD 20000
STD 1.25K

10K-copy Unknown
STD 20K
STD 5K

10K-copy 10K-copy
Unknown
Population 2
Unknown
Population 2
Population 2

TaqMan® 48-well RNase P Plate for TaqMan® 96-well RNase P Plate for
the StepOne™ System the StepOnePlus™ System

b. In the plot, confirm that the CTs of the replicate population are equivalent.

Note: The numbers on the X-axis of the plot correspond to the wells of the
reaction plate. Beginning with well A1, the wells are numbered from left-to-
right, and top-to-bottom.

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Analyze the RNase P Experiment

c. If an outlier is present in the population, select the corresponding well in the

plate layout, then click Omit to remove the well from the analysis.

Maximum number of outlier wells that can be

removed from each…
Instrument Total
Unknown Standard Negative
Population (STD) Control (NC)

StepOnePlus™ System 6 0 0 12

StepOne™ System 2 0 0 4

IMPORTANT! If too many outliers are present, order another RNase P plate and
repeat the experiment.

d. Repeat steps 3a through 3c for each replicate population (unknowns, standards,

and negative controls) on the reaction plate.

4. Click Reanalyze to analyze the run without the outliers.

If the status of the RNase P Run is “Failed” after performing steps 2 through 4, repeat
the RNase P experiment using a different RNase P plate. If the problem persists,
contact Support as explained in “How to Obtain Support” on page 14.

3c 4

2
3a

3b
Outlier

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5. Review the standard curve:

a. Select the Standard Curve tab.

b. Click the upper-right corner of the Plate Layout to select all wells.

c. Confirm that the R2 value is greater than or equal to 0.990.

If the R2 value is less than 0.990, repeat the RNase P experiment using a different
RNase P plate. If the problem persists, contact Support as explained in “How to
Obtain Support” on page 14.

5a
6

5b

5c

6. Click Save Status, then click Yes when prompted.

7. Touch the instrument touchscreen to awaken it, then touch Pass in the RNase P
wizard.

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After the Installation

After the Installation

After verifying the performance of the system, you can install additional software to the
computer and/or connect the instrument to an Ethernet network.

Install Additional We recommend installing the following types of software to improve the function and
Software security of the system computer:
• Antivirus software
• Archival or file compression software
• Security software (firewall and encryption utilities)
• Performance optimizing software

Note: For more information, see “Select Protective Hardware and Software” on page 31.

To confirm that third-party software does not interfere with the StepOne™ software:

1. Install the software to the computer that contains the StepOne™ software.

2. Perform several test experiments using “dummy” plates (plates that do not contain
reagents).

Note: The goal of the test experiments is to run plates under conditions that match
normal instrument operation. Therefore, the characteristics of the test experiments
(plate layout and run method) must closely resemble your actual experiments.

3. Confirm that the system performs each test experiment without producing errors.
If the system performs the tests successfully, perform experiments normally. If the
system encounters errors during the test runs, the software may not be compatible
with the StepOne™ software.

Network the If the standalone instrument is connected to a network, then computers on the network
System that are running the StepOne™ software can:
• Monitor the status of the run in progress
• Send and download experiments to and from the instrument instead of using the
USB drive
To connect the instrument to a network, go to Chapter 5, “Connect the System
to a Network,” on page 101.

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Connect the System to a Network

This chapter covers:

■ Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
■ Connect the Instrument to a Network . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
■ Set Up a Computer for Remote Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
■ Monitor the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

Note: For more information about any of the topics discussed in this guide, access the
Help from within the Applied Biosystems StepOne™ Real-Time PCR System Software
by pressing F1, or clicking in the toolbar, or selecting Help StepOne Software
Help.

IMPORTANT! This chapter does not provide adequate detail to integrate the
Applied Biosystems StepOne™ or StepOnePlus™ Real-Time PCR Instrument into all
possible network architectures. Because your network may contain advanced features
(such as a firewall or network domains), we recommend that you consult a network
administrator before connecting the Applied Biosystems StepOne™ or StepOnePlus™
Real-Time PCR System to your laboratory network.

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Overview

Overview
After installing the Applied Biosystems StepOne™ or StepOnePlus™ Real-Time PCR
System, you can connect the instrument to a local area network to enhance its
functionality.
This chapter describes how to:
• Set up the instrument for use on a network.
• Set up a computer for remote monitoring.
• Test the network connection by engaging the remote monitoring feature.

About Remote Once the instrument is connected to a network, any computer on the network can use the
Monitoring remote monitoring feature of the StepOne™ software to monitor the instrument. During a
run, the remote monitoring feature can provide run status, temperature, and amplification
data as the instrument collects them. See “Monitor the Instrument” on page 110 for more
information on remote monitoring.

IMPORTANT! The remote monitoring feature does not allow you to control of the
instrument. Control of the instrument is limited to the colocated computer when the
system is in a colocated layout, or the instrument touchscreen when the system is in a
standalone layout.

Network Setup Chapter 3, Chapter 4,

Workflow Install the Colocated Layout Install the Standalone Layout

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Connect the instrument to a network

1. Define the LAN Port IP settings.

Set up the computer for remote monitoring

1. Connect the computer to the network.
2. Install the StepOne™ software.

Test the network connection

1. Enable remote monitoring.

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Overview

Example Network The LAN and Computer Ports of the instrument allow you to add the system to a network
Layouts in several ways. The following examples describe two basic network layouts of the
system.

Networked Standalone Layout

In this example, a standalone instrument is connected to a network by the instrument
LAN Port ( ), which has been configured for dynamic host configuration protocol
(DHCP) or static IP operation. In this layout, any computer on the network can monitor
and exchange experiment data with the instrument; however, computers on the network
cannot control the instrument. Runs must be started from the instrument touchscreen.

Network
server

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Networked standalone
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Networked Colocated Layout

In this example, a colocated instrument is connected to a networked colocated computer
by the Computer Port ( ), and to a network by the LAN Port ( ) that has been
configured for DHCP or static IP operation. In this layout, computers on the network can
monitor and exchange experiment data with the instrument; however, the instrument is
controlled locally by the colocated computer.

IMPORTANT! Consult your IT department to network a colocated computer. To enable

the full functionality of the StepOne™ software, the colocated computer requires a
network connection.

Network
server

Networked Networked
Networked colocated colocated
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Overview

Instrument Ports The instrument supports two 10Base-T Ethernet connections: a Computer Port for
communication with the colocated computer, and a Local Area Network (LAN) Port for
network communication.

Port Purpose Supports

The connection for the colocated computer, Static IP network service

which controls the instrument. only with subnet mask and
default gateway settings
Note: We do not support colocated layouts in
which the Computer Port does not directly
connect the instrument to the colocated
computer.

The connection for a local area network. • Static IP network service

with subnet mask and
When the instrument is connected to a
default gateway settings
network, computers on the network that run the
StepOne™ software can: or
• Remote monitor the instrument as it • Dynamic host
performs runs. configuration protocol
(DHCP) network service
• Send experiments to, and download
experiments from the instrument. • Managed data network
service (mDNS/DNS) for
IMPORTANT! Computers connected to the local domains ‡
instrument through the LAN port cannot control • IPv4 link-local (IPV4LL) in
the instrument. the RFC §

‡ Because managed data network service (mDNS) is limited to direct network connections, a
instrument set for mDNS may not be visible to other nodes that are separated by a router, hub,
or another network device.
§ Also known as Automatic Private IP Addressing (APIPA) or Internet Protocol Automatic
Configuration (IPAC). When the instrument is set for DHCP, Automatic Private IP Addressing
(APIPA) is automatically enabled, and the instrument provides an IP address when no address is
supplied by the DHCP server.

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Overview

Networking • Consult a Network Administrator

Guidelines and – We recommend that you consult a network administrator before connecting the
Best Practices system to your laboratory network.
– To enable the full functionality of the StepOne™ software, the colocated
computer requires a network connection.
• Confirm the Instrument Connections
Confirm that the instrument is connected to the network by the blue LAN Port ( ).
If the system is installed in a colocated layout, confirm that the instrument is
connected to the computer by the yellow Computer Port ( ).
• Limit Remote Monitoring to One Computer
Avoid using more than one computer to simultaneously remote monitor the
instrument. Although the system supports remote monitoring from multiple
computers, each connection taxes the instrument microprocessor. Too many
connections can overburden the instrument and result in instrument errors.

Note: The effects of an overburdened instrument are evident in the Temperature Plot
during a run. Symptoms can include extended hold times or brief, unexpected
plateaus in the instrument Temperature Plot.

• Observe Restrictions to mDNS and Auto Discovery

The instrument supports multicast domain name service (mDNS) but only when the
instrument and computer share a direct network connection and are within the same
subnet. Consequently, computers on the network that are separated from the
instrument by a router, hub, or other network device may not be able to access the
instrument by its host name.
The instrument also does not support auto discovery for mDNS. However, the
instrument does support name resolution but the instrument name must be unique
within the subnet.

Note: Confirm the uniqueness of the instrument name. The instrument does not test
the uniqueness of the instrument name when it is set.
• Add Instruments Using Lower Case Letters
When you add the instrument for remote monitoring (see “Enable Remote
Monitoring” on page 110), enter the instrument name using lower case letters only.

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Connect the Instrument to a Network

Connect the Instrument to a Network

After deciding how to connect the instrument to a network, set up the instrument
according to your network policies.

Materials Ethernet cable with RJ45 connectors

Required

Collect Required • Network policy for obtaining IP addresses: DHCP or static IP.
Information
IMPORTANT! When the instrument is set for DHCP, Automatic Private IP
Addressing (APIPA) is automatically enabled and the instrument will provide an IP
address when no address is supplied by a DHCP server.

• If the network requires a static IP address, obtain the IP address, subnet mask, and
gateway address for the instrument.

How to Obtain the MAC Address of the Instrument

If your systems administrator requests the Media Access Control (MAC) address for the
instrument, obtain the MAC address as follows:

1. Touch the instrument touchscreen to awaken it, then touch .

2. In the Main Menu, touch Settings Menu, then touch About the Instrument.
The MAC address for the instrument appears in the rights side of the About
Instrument screen.

3. When finished, touch Done.

4. In the Settings Menu, touch to return to the Main Menu.

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Connect the Instrument to a Network

Define the LAN 1. Touch the instrument touchscreen to awaken it, then touch .
Port IP Settings
2. In the Main Menu, touch Settings Menu, touch Admin Menu, then touch
Set IP Address.

3. Enter the IP settings for the network connection.

Network
Action
Service

DHCP Touch Assign IP address dynamically (using DHCP), touch Done, then
touch OK when prompted.

Static IP 1. Touch Static.

2. Enter the IP address, subnet mask, and default gateway settings. For
each setting, touch the field, enter the address, then touch Done.
3. Touch Done to save the settings, then touch OK when prompted.
The following figure shows an example of static IP network settings for the
LAN port of the instrument.

4. Touch to return to the Main Menu.

5. Connect the Ethernet cable between the:

• Blue LAN Port ( ) of the instrument, and the
• Ethernet port for your network

IMPORTANT! Do not connect the Ethernet cable to the yellow Computer Port
( ); this port is reserved for the colocated computer connection.

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Set Up a Computer for Remote Monitoring

Set Up a Computer for Remote Monitoring

After connecting the instrument to the network, connect the computer to the network and
install the StepOne™ software for remote monitoring.

Materials Ethernet cable with RJ45 connectors

Required
Computer Requirement
If you are connecting a computer that you provided to a network, confirm that the
computer contains a network interface card (NIC) or a free network port.

Collect Required • Network policy for obtaining IP addresses: DHCP or static IP

Information • If the network requires a static IP address, obtain the IP address, subnet mask, and
gateway address for the computer

Connect the IMPORTANT! We recommend that you arrange for a network administrator to connect
Computer to the your computer to the network. The following procedure does not provide adequate detail
Network for all network architectures.

1. Use the Ethernet cable to connect the computer to the nearest network port.

2. Power on the computer, then log in using an account that belongs to the
Administrators user group.

3. In the computer desktop, right-click My Network Places, then select Properties.

4. Right-click Local Area Connection, then select Properties.

5. Select Internet Protocol (TCP/IP), then click Properties.

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6. Set the Internet Protocol (TCP/IP) Properties:

Network
Action
Configuration

DHCP 1. Select Obtain an IP address automatically.

2. If the computer obtains DNS addresses:
• Automatically – Select Obtain DNS server address
automatically.
• Statically – Select Use the following DNS address, enter the
address of the preferred and alternate DNS servers if available.

Static IP 1. Select Use the following IP address.

2. In the IP Address field, enter the static IP address.
3. If necessary, enter a subnet mask.
4. If necessary, enter a static gateway address in the Default
Gateway field.

7. If your network requires advanced TCP/IP settings (such as WINS settings):

a. Click Advanced in the Internet Protocol (TCP/IP) Properties dialog box.

b. Define the IP Settings, DNS, and WINS tabs as instructed by your systems
administrator, then click OK.

8. Close all dialog boxes by clicking OK.

9. Restart the computer.

Note: The computer is now visible to other computers on the network.

Install the 1. If you have not already done so, install the StepOne™ software to the networked
StepOne™ computer (see “Install the Software” pages 59 or 84).
Software
Note: The StepOne™ software is required to monitor the instrument over the
network.

2. (Optional) Install protective software to the computer (see “Select Protective

Hardware and Software” on page 31).

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Monitor the Instrument

After connecting the instrument and a computer to the network, test the network
connection by enabling remote monitoring.

About Remote When the instrument is connected to the network, any computer on the network that is
Monitoring running the StepOne™ software can:
• Monitor the status of the instrument during and between runs
• Send an experiment to the instrument (see page 112)
• Download an experiment from the instrument (see page 112)
• Enable or change email notification settings for the instrument (see page 113)

Guidelines for Remote Monitoring

To ensure optimal performance of the remote monitoring feature, observe the following:
• Although the StepOne™ software can add multiple instruments for remote
monitoring, it can monitor only one instrument at a time.
• We do not recommend that an instrument be monitored by more than one computer
simultaneously.
• Unless you are sure that your instrument and computer exist on the same subnet, we
recommend that you use the IP address of the instrument to add it for remote
monitoring.

Enable Remote 1. In the StepOne™ software, select Instrument Remote Monitor.

Monitoring
2. In the Manage Instrument screen, click Add Instrument.

3. Add the instrument in the Add Instrument dialog box:

a. In the Profile Name field, enter the name of your instrument (from step 1b on
page 45).
b. In the Instrument Name, Host Name, or IP Address field, enter the host name
or IP Address of the instrument that you want to monitor.
c. Click Save & Exit.
The StepOne™ software displays an entry for the instrument in the left-most column
of the Manage Instruments screen.

4. Enable remote monitoring for the instrument:

a. In the Manage Instrument pane of the StepOne™ software, select the instrument
that you want to monitor.
b. In the entry for the instrument, click .

Note: You can also monitor the instrument by selecting your instrument from the
dropdown menu, then clicking Monitor.

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The StepOne™ software displays the status, attributes, and plot data for the selected
instrument in real time. If a communications warning appears, troubleshoot the
problem as explained in “Network Connection Problems” on page 161.
You will lose the software connection to the colocated instrument if you:
• Change the instrument that is connected directly to your computer
• Use the colocated instrument touchscreen to change the instrument name or
IP address
To reestablish the colocated connection, restart the StepOne™ software.

4a

4b

3a

3b

3c

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Send an 1. In the entry for your instrument, click .

Experiment to a
Monitored 2. Click Browse, then navigate to and select the experiment that you want send to the
Instrument instrument, then click Open.

3. Select your instrument from the Select Instrument dropdown menu.

4. Click Send Experiment, then click OK when prompted.

Download an 1. In the entry for your instrument, click .

Experiment from
a Monitored 2. Select your instrument from the Instrument dropdown menu.
Instrument
3. Select your experiment from the Experiment dropdown menu.

4. Click Browse, then navigate to and select the folder to which you want to send the
experiment, then click Select.

5. Click Download Experiment, then click OK when prompted

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Enable or Change The notification settings allow you to configure the StepOne™ software to alert you by
Notification email when the instrument begins and completes a run, or if an error occurs during a run.
Settings You can also set up the software to attach a completed run file to the Run Completed
email notification. The notifications settings feature is optional and does not affect
system performance.

IMPORTANT! The StepOne™ software will transmit email only while the instrument is
monitored. If the network connection is interrupted, the StepOne™ software will stop
transmitting updates.

To modify the notification settings:

1. Contact your systems administrator or information technology department for the:

• Network address of a Simple Mail Transfer Protocol (SMTP) server.
• A user name and password for the server, if required for access.
• The Secure Sockets Layer (SSL) setting of the server (on or off).

2. While monitoring an instrument, select Enable Run Notifications in the top-right

corner of the screen.

3. Click Change Notifications, change the Notification Settings as desired, then click
the close box.

4. Select Enable Notifications.

5. Select the events that generate notifications:

• Instrument Error – Causes the system to email recipients all errors
encountered by the instrument during each run.
• Run Started – Causes the system to email the recipients every time the
instrument starts a run.
• Run Completed – Causes the system to email the recipients every time the
instrument completes a run.

6. Click the Enter e-mail addresses for notifications field, then enter the email
address(es) that you want to receive email notifications.

7. Click the Outgoing Mail Server (SMTP) field, then enter the address of the SMTP
server.

8. Select whether or not to attach completed run files to the Run Completed email
notifications.

Note: This option applies only to email notifications that are generated when the
instrument completes a run (that is, Run Completed is selected in step 5).

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9. Set the authentication settings as needed.

If the SMTP server requires authentication:
a. Click Yes.

b. Click the User Name field, then enter the user name to access the server.

c. Click the Password field, then enter the password for the user account.

10. Click Test Configuration. If the notification settings are set up correctly, sample
emails are sent to the addresses entered in step 6.

7
8
9a

9b
9c

10

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Maintain the System

This chapter covers:

■ Regular Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
Perform a Spatial Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
Perform a Background Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
Perform a Dye Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
Archive and Back Up Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
■ Infrequent Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
Decontaminate the Sample Block(s). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Move the Instrument. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
Replace the External Fuses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
Ship the Instrument for Service . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
Update the StepOne™ Software or the Operating System . . . . . . . . . . . . . . . . . . 154

Note: For more information about any of the topics discussed in this guide, access the
Help from within the Applied Biosystems StepOne™ Real-Time PCR System Software
by pressing F1, or clicking in the toolbar, or selecting Help StepOne Software
Help.

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Regular Maintenance
Maintenance Perform routine maintenance of your Applied Biosystems StepOne™ or StepOnePlus™
Schedule Real-Time PCR Instrument and computer to ensure proper operation. The following
table displays the recommended maintenance schedule.

Perform See
Task
every... Page

Week Check the computer disk space. If necessary, archive or back up your 142
experiment files.

Power off the computer controlling the instrument, then after 30 sec —
power on the computer.

Clean the surface of the instrument with a lint-free cloth. —

Defragment the computer hard drive. —

Month Perform a background calibration. ‡ 122

1.5 Years Perform a spatial calibration. 118

Perform a dye calibration. 128

‡ You can run a background calibration to check for contamination. Also, you must run a
background calibration and a dye calibration if any part of the instrument optics are replaced or
moved.

Connection The spatial, background, and dye calibrations must be performed while the system is in a
Required for colocated layout. To set up the system for maintenance, connect the yellow StepOne
Maintenance system cable between the:
• Yellow Ethernet port ( ) of the instrument, and
• Ethernet port ( ) of the computer running the StepOne™ software

IMPORTANT! Do not connect the StepOne system cable to the blue LAN Port ( ),
which is reserved for a network connection.

Ethernet

Maintenance When a required maintenance task such as a calibration is overdue, the StepOne™
Notifications software and instrument display messages that prompt you to perform the necessary
procedure.

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How to View You can use the StepOne™ software to view a summary of maintenance data for your
Maintenance instrument. To view the maintenance information, select Instrument Instrument
Information Maintenance Manager in the StepOne™ software. For each calibration (spatial,
background, and dye), the software lists the following information:
• Status – Condition of the current calibration where:
– Current indicates that the calibration is up to date.
– Expired indicates that the calibration must be performed at the earliest possible
convenience.
– Not Run indicates that the calibration has not been run (RNase P only).
• Last Run – Date when current calibration was run.
• Expiry Date – Date when current calibration will expire.

Instrument Tools The Tools Menu of the instrument provides several features that test the temperature of
Menu Tests several instrument components against the product specification.

Test Description

Run Cycle Performance Test Verifies that the sample block(s) achieves the temperature
accuracy specification for the instrument.

Optics Verification Verifies that the instrument optics is operating to the

specifications of the instrument.

Statistics Displays the usage data for the instrument, including:

• Thermal cycles performed by the sample block(s).
• Degrees Celsius heated or cooled by the sample
block(s).
• Hours of operation for the instrument LED.
• RNase P verification run data.

Temperature Verification Service

We recommend that you perform an annual temperature verification of the instrument
depending on your usage and laboratory requirements. The temperature verification service
can be performed by us when the instrument is shipped for an annual service, or by the
service engineer at an additional cost. See “How to Obtain Support” on page 14 to contact
Support for pricing and further information.

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Perform a Spatial Calibration

Perform a spatial calibration every 18 months, or as often as necessary, depending on
instrument use.

Materials • Spectral calibration plate 1 from the spectral calibration kit for your instrument:
Required – StepOne™ Real-Time PCR System Spectral Calibration Kit (PN 4371433)
– StepOnePlus™ Real-Time PCR System Spectral Calibration Kit (PN 4371435)
• Safety glasses
• Powder-free gloves
• Centrifuge with reaction plate adapter
• StepOne system cable, yellow (from the system packing kit)

Purpose of the A spatial calibration maps the positions of the wells on the sample block(s) so that the
Calibration StepOne™ software can associate increases in fluorescence during a run with specific
wells of the reaction plate.

Prepare for the PHYSICAL INJURY HAZARD. During instrument operation, the
Calibration temperature of the sample block(s) can exceed 100°C. If the instrument has been used
recently, keep your hands away until the sample block(s) reaches room temperature.

IMPORTANT! Wear powder-free gloves when you handle calibration plates.

1. If you have not already done so, use the StepOne system cable to connect the:
• Yellow Ethernet port ( ) of the instrument to the
• Ethernet port ( ) of the computer running the StepOne™ software

IMPORTANT! Do not connect the StepOne system cable to the blue LAN Port ( ).

Ethernet

2. Get calibration plate 1 from the spectral calibration kit in the freezer.

3. Allow calibration plate 1 to warm to room temperature (approximately 5 min).

IMPORTANT! Do not remove calibration plate 1 from its packaging until you are
ready to run it. The fluorescent dyes in the wells of both spectral calibration plates
are photosensitive. Prolonged exposure to light can diminish the fluorescence signal
strength of the dyes.

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4. Remove calibration plate 1 from its packaging.

5. Vortex calibration plate 1 for 5 sec.

6. Centrifuge calibration plate 1 for 2 min at less than1500 rpm.

IMPORTANT! The calibration plate must be well mixed and centrifuged.

7. Confirm that the liquid is at the bottom of each well of calibration plate 1. If not,
centrifuge the calibration plate again at a greater rpm and for a longer time.

IMPORTANT! Do not allow the bottom of the calibration plate to become dirty.
Fluids and other contaminants that adhere to the bottom of the plates can
contaminate the sample block(s) and cause an abnormally high background signal.

Correct Incorrect

Liquid is at the • Not centrifuged with enough force, or

bottom of the well. • Not centrifuged for enough time

8. Load calibration plate 1 into the instrument:

a. Open the instrument drawer.

b. Place the calibration plate in the sample block(s) so that the A1 position is at
the back-left corner.
c. Close the instrument drawer carefully.

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Perform the 1. In the main screen of the StepOne™ software, select Instrument Instrument
Calibration Maintenance Manager.

2. In the navigation pane of the Instrument Maintenance Manager, click Spatial.

3. In the Instrument Maintenance Manager, click Start Calibration.

4. In the Setup screen of the Spatial Calibration dialog box, select The calibration
plate is loaded into the instrument, then click Next.

5. In the Run screen, click START RUN , then wait for the instrument to complete
the spatial calibration.

Remove the PHYSICAL INJURY HAZARD. During instrument operation, the

Calibration Plate temperature of the sample block(s) can exceed 100°C. If the instrument has been used
recently, keep your hands away until the sample block(s) reaches room temperature.

IMPORTANT! Wear powder-free gloves when you handle calibration plate.

1. Open the instrument drawer.

2. Remove calibration plate 1 from the sample block(s), place it inside its packaging
sleeve, and return it to the spectral calibration kit in the freezer.

3. Close the instrument drawer carefully.

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Analyze the 1. In the Analysis screen of the Spatial Calibration dialog box, confirm the status of the
Calibration Data calibration:
• Passed – The instrument passed the calibration. Go to step 2.
• Failed – The instrument failed the calibration. Contact Support as described in
“How to Obtain Support” on page 14.

2. Click Finish.

3. When prompted to save the calibration, click Yes.

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Perform a Background Calibration

Perform a background calibration monthly, or as often as necessary, depending on
instrument use.

Materials • Background plate from the spectral calibration kit for your instrument:
Required – StepOne™ Real-Time PCR System Spectral Calibration Kit (PN 4371433)
– StepOnePlus™ Real-Time PCR System Spectral Calibration Kit (PN 4371435)

Note: Alternatively, create a background plate as described in “How to Create a

Background Plate” on page 127.

• Powder-free gloves
• Safety glasses
• Centrifuge with reaction plate adapter
• StepOne system cable, yellow (from the system packing kit)

Calibration • Make sure the centrifuge you use is clean. Before centrifuging, wipe down the
Guidelines bucket using a tissue.
• Handle the calibration plates with care to prevent contamination. To avoid
contaminating the reaction plate, do not place plates on a lab bench. Always return
the calibration plate to its original bag.

Purpose of the A background calibration measures the level of background fluorescence in the
Calibration instrument. During a background calibration run, the system:
• Performs continuous reads of a background plate containing PCR buffer for 10 min
at 60°C.
• Averages the spectra recorded during the run and extracts the resulting spectral
component to a calibration file.
The StepOne™ software uses the calibration file during subsequent runs to remove the
background fluorescence from the run data.

Background Fluorescence
Fluorescence data collected by the system includes a fluorescent spectral component
inherent to the system, commonly referred to as background fluorescence. This
background fluorescence is a composite signal found in all signal data that consists of
fluorescence from several sources, including:
• Background electronic signal
• Contaminants in the sample block(s)
• The plastic consumable (plates and caps)

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Prepare for the PHYSICAL INJURY HAZARD. During instrument operation, the
Calibration temperature of the sample block(s) can exceed 100°C. If the instrument has been used
recently, keep your hands away until the sample block(s) reaches room temperature.

IMPORTANT! Wear powder-free gloves when you handle the background plate.

1. If you have not already done so, use the StepOne system cable to connect the:
• Yellow Ethernet port ( ) of the instrument to the
• Ethernet port ( ) of the computer running the StepOne™ software

IMPORTANT! Do not connect the StepOne system cable to the blue LAN Port ( ).

Ethernet

2. Get the background plate from the spectral calibration kit in the freezer.

3. Allow the background plate to warm to room temperature (at least 5 min).

4. Remove the background plate from its packaging.

IMPORTANT! Do not discard the packaging sleeve. The background plate can be
used up to three times if it is stored in its original packaging sleeve.

5. Vortex the background plate for 5 sec.

6. Centrifuge the background plate for 2 min at less than1500 rpm.

IMPORTANT! The background plate must be well mixed and centrifuged.

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7. Confirm that the liquid is at the bottom of each well of the background plate. If not,
centrifuge the background plate again at a greater rpm and for a longer time.

IMPORTANT! Do not allow the bottom of the background plate to become dirty.
Fluids and other contaminants that adhere to the bottom of the background plate can
contaminate the sample block(s) and cause an abnormally high background signal.

Correct Incorrect

Liquid is at the • Not centrifuged with enough force, or

bottom of the well. • Not centrifuged for enough time

8. Load the background plate into the instrument:

a. Open the instrument drawer.

b. Place the background plate in the sample block(s) so that the A1 position is at
the back-left corner.
c. Close the instrument drawer carefully.

Perform the 1. In the main screen of the StepOne™ software, select Instrument
Calibration Instrument Maintenance Manager.

2. In the navigation pane of the Instrument Maintenance Manager, click Background.

3. Click Start Calibration.

4. In the Setup screen of the Background Calibration dialog box, select The
background plate is loaded into the instrument, then click Next.

5. In the Run screen, click START RUN , then wait for the instrument to complete
the background calibration.

Note: If the StepOne™ software displays messages during the run, troubleshoot the
errors as described in “Background Calibration Failure” on page 164.

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Remove the PHYSICAL INJURY HAZARD. During instrument operation, the

Background Plate temperature of the sample block(s) can exceed 100°C. If the instrument has been used
recently, keep your hands away until the sample block(s) reaches room temperature.

IMPORTANT! Wear powder-free gloves when you handle the background plate.

1. Open the instrument drawer.

2. Remove the background plate from the sample block(s), place it inside its packaging
sleeve, and return it to the spectral calibration kit in the freezer.

Note: Do not discard the background plate. If you store the background plate in its
packaging sleeve at –20 to –25°C, you can reuse it three times after you open it.

3. Close the instrument drawer carefully.

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Analyze the 1. In the Analysis screen of the Background Calibration dialog box, confirm the status
Calibration Data of the calibration:
• Passed – The instrument passed the calibration. Go to step 2.
• Failed – The instrument failed the calibration. Troubleshoot the error as
described in “Background Calibration Failure” on page 164.

2. Select all wells of the plate layout.

3. Inspect the raw data for irregular spectral peaks.

If one or more wells produce raw spectra that diverge significantly from that of the
neighboring wells, the background plate or the sample block(s) could contain a
fluorescent contaminant. If you suspect contamination, troubleshoot the irregular
signal as explained in “Background Calibration Failure” on page 164.

4. If all spectra are acceptable, click Finish.

5. When prompted to save the background calibration, click Yes.

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How to Create a Whenever possible, use a background plate included with the spectral calibration kit. The
Background Plate background plate contains a buffer that accurately models the reagents used for PCR, and
produces high-quality calibration data. However, if a background plate from a spectral
calibration kit is not available, you can create one as explained below.

Materials Required
• Deionized water
• MicroAmp® Optical Adhesive Film or MicroAmp® 8-Cap Strip (Flat Caps only)
• MicroAmp® Optical Reaction Plate
• Pipettor, 200-µL and pipette tips
• Powder-free gloves
• Safety glasses

Create the Background Plate

IMPORTANT! Wear powder-free gloves while creating the background plate.

1. Remove a reaction plate from its box and place it on a clean, dry surface.

2. Pipette 30 µL of deionized water to each well of the reaction plate.

3. Seal the reaction plate using an optical adhesive film or optical flat caps.

4. Use the reaction plate to perform the background calibration.

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Perform a Dye Calibration

Perform a dye calibration at least every 18 months, or as often as necessary depending on
instrument use. Because the age and use of the instrument can affect spectral readings,
you may need to perform the calibration more frequently.

IMPORTANT! Perform a background calibration before performing a dye calibration (see

“Perform a Background Calibration” on page 122).

Materials • Spectral Calibration Plates from the spectral calibration kit for your instrument:
Required – StepOne™ Real-Time PCR System Spectral Calibration Kit (PN 4371433)
– StepOnePlus™ Real-Time PCR System Spectral Calibration Kit (PN 4371435)
• Safety glasses
• Powder-free gloves
• Centrifuge with reaction plate adapter
• StepOne system cable, yellow (from the system packing kit)

Custom Dye Plates

The StepOne™ and StepOnePlus™ instrument supports the use of custom dyes (dyes not
supplied by Thermo Fisher Scientific) that are excited with a light source at 470 nm and
fluoresce within the spectral range it supports (500 to 650 nm). See “How to Calibrate a
Custom Dye” on page 139 for more information.

Purpose of the During a dye calibration run, the system:

Calibration • Collects spectral data from a series of dye standards.
• Stores the spectral information for the dye standards in the spectra run file, a
calibration file in the StepOne™ software directory.
The StepOne™ software uses spectral data during subsequent runs to characterize dyes
and distinguish the individual contribution of each dye in the collective fluorescence
collected by the instrument during a run. After each run, the StepOne™ software stores
run data in the form of a raw spectral signal for each reading. It determines the
contribution of each fluorescent dye used in the sample by comparing the raw spectra to
the spectral file. After analysis, the StepOne™ software stores the spectral data with the
collected fluorescence data for each experiment.

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Supported Dyes StepOnePlus™ System Dyes

The StepOnePlus™ instrument uses the following dye set for calibration: FAM™ dye,
JOE™ dye, NED® dye, ROX™ dye, TAMRA® dye, VIC® dye, and SYBR® Green dye.
The following figure shows the emission spectrum for each dye, and the filter at which
each dye is read.

Filters 1 2 3 4
Wavelength 500 600 700
(nm)
Emission
Spectra

Dyes ~520 nm ~550 nm ~580 nm ~610 nm

• FAM • JOE • NED • ROX
• SYBR Green • VIC • TAMRA

StepOne™ System Dyes

The StepOne™ instrument uses the following dye set for calibration: FAM™ dye,
JOE™ dye, ROX™ dye, VIC® dye, and SYBR® Green dye. The following figure shows
the emission spectrum for each dye, and the filter at which each dye is read.

Filters 1 2 3
Wavelength 500 600 700
(nm)
Emission
Spectra

Dyes ~520 nm ~550 nm ~610 nm

• FAM • JOE • ROX
• SYBR Green • VIC

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Custom Dyes
The system can be used to run assays designed with custom dyes (dyes not supplied by
Thermo Fisher Scientific). However, before using custom dyes with the instrument, you
must create and run a custom calibration plate. The purpose of the custom plate is similar
to that of a spectral calibration plate. The StepOne™ software uses the custom
calibration plate to create a spectral standard to distinguish the custom dye in the
fluorescence data collected during the run.

IMPORTANT! To use a custom dye on your system, it must be excited with light at
470 nm and fluoresce within the 500 to 650 nm spectral range measured by the
StepOne™ and StepOnePlus™ instrument.

IMPORTANT! We do not recommend the use of TAMRA™ dye as reporter or quencher

with the StepOne™ system. TAMRA™ dye may be used as a reporter or quencher with
the StepOnePlus™ system.

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About the The product of a dye calibration is a collection of spectral profiles that represent the
Analysis fluorescence signature of each dye standard. Each profile consists of a set of spectra that
correspond to the fluorescence collected from the wells of the spectral calibration plate.
The StepOne™ software plots the resulting data for each spectral profile in a graph of
fluorescence versus filter.
When the StepOne™ software extracts the calibration data from a dye run, it evaluates the
fluorescence signal generated by each well in terms of the collective spectra for the entire
calibration plate. Dye spectra are generally acceptable if they peak within the same filter
as their group but diverge slightly at other wavelengths (see below).
The StepOne™ software can compensate for some differences in a spectral profile by
replacing (auto-repairing) the spectra of unacceptable wells with the spectra of other
wells on the reaction plate. However, the software allows only a few replacements and
may reject the calibration if the spectra between neighboring wells vary significantly.

Note: Because the wells in a calibration plate contain dyes at identical concentrations,
the resulting signals for the wells containing each dye should be similar. Among wells
containing the same dye, variations in spectral position and peak position are caused by
minor differences in the optical and excitation energy between the individual wells.

Acceptable Spectra Unacceptable Spectra

Spectra peak at the same wavelength Spectra peak at the different
and do not diverge significantly wavelengths

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Prepare for the IMPORTANT! Wear powder-free gloves when you handle the calibration plates.
Calibration
1. If you have not already done so, use the StepOne system cable to connect the:
• Yellow Ethernet port ( ) of the instrument to the
• Ethernet port ( ) of the computer running the StepOne™ software

IMPORTANT! Do not connect the StepOne system cable to the blue LAN Port ( ).

Ethernet

2. Get calibration plate 1 from the spectral calibration kit in the freezer.

3. Allow calibration plate 1 to warm to room temperature (~5 min).

IMPORTANT! Do not remove calibration plate 1 from its packaging until you are
ready to run it. The fluorescent dye in the wells of each calibration plate is
photosensitive. Prolonged exposure to light can diminish the fluorescence signal
strength of the dyes.

4. Remove calibration plate 1 from its packaging.

5. Vortex calibration plate 1 for 5 sec.

6. Centrifuge calibration plate 1 for 2 min at less than1500 rpm.

IMPORTANT! The calibration plate must be well mixed and centrifuged.

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7. Confirm that the liquid is at the bottom of each well of the calibration plate. If not,
centrifuge the plate again at a greater rpm and for a longer time.

IMPORTANT! Do not allow the bottom of the calibration plates to become dirty.
Fluids and other contaminants that adhere to the bottom of the plates can
contaminate the sample block(s) and cause an abnormally high background signal.

Correct Incorrect

Liquid is at the • Not centrifuged with enough force, or

bottom of the well. • Not centrifuged for enough time

8. Load calibration plate 1 into the instrument:

a. Open the instrument drawer.

b. Place calibration plate 1 in the sample block(s) so that the A1 position is at the
back-left corner.
c. Close the instrument drawer carefully.

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Perform the PHYSICAL INJURY HAZARD. During instrument operation, the

Calibration temperature of the sample block(s) can exceed 100°C. If the instrument has been used
recently, keep your hands away until the sample block(s) reaches room temperature.

1. In the main menu of the StepOne™ software, select Instrument

Instrument Maintenance Manager.

2. In the Instrument Maintenance Manager, click Dye in the navigation pane.

3. In the Dye screen, select System Dye Calibration.

4. Click Start Calibration.

5. In the Setup screen, select Plate 1 is loaded into the instrument.

6. Click Next.

7. In the Run screen, click Start Run .

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8. While the instrument is running calibration plate 1, prepare calibration plate 2 by

performing steps 4 through 7 on page 133.

9. When the wizard prompts you to do so, load and run calibration plate 2:
a. Open the instrument drawer.

b. Remove the calibration plate 1 from the sample block(s) and discard it.

c. Place calibration plate 2 in the sample block(s) so that the A1 position is at the
back-left corner.
d. Close the instrument drawer carefully.

e. In the Dye Calibration Setup wizard, select Plate 2 is loaded in the

instrument, click Continue Calibration.

10. When the instrument completes the calibration, unload calibration plate 2:
a. Open the instrument drawer.

b. Remove the calibration plate 2 from the sample block(s) and discard it.

c. Close the instrument drawer carefully.

Analyze the 1. After the calibration is complete, confirm the status of the calibration:
Calibration Data • Passed – The instrument passed the calibration. Go to step 2a.
• Failed – The instrument failed the calibration. Troubleshoot the error as
described in “Dye Calibration Failure” on page 166.

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2. For each dye present on the plate, confirm the grouping of the dye spectra:
a. In the plate layout, press Ctrl, then select the wells that contain the same dye.

b. Inspect the raw data. For each spectrum, confirm that the peak is:
• Within the detectable range for the instrument.
• Free of irregular spectral peaks.
• Present in the correct channel for the dye (see “StepOne™ System Dye
Spectra” on page 137 or “StepOnePlus™ System Dye Spectra” on page 138).
If a spectrum does not comply with the criteria above, troubleshoot the problem as
described in “Dye Calibration Failure” on page 166.

Note: Among wells containing the same dye, variations in spectral position and
peak position are caused by minor differences in the optical and excitation energy
between the individual wells.

3. If all spectra are acceptable, click Plate 2 from the View Details menu.

4. Inspect the raw data for the as described in step 2b.

5. If all spectra are acceptable, click Finish.

6. When prompted to save the dye calibration, click Yes.

3
2a

2b/4

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(~610 nm)
Filter 3

ROX
(~550 nm)
Filter 2

JOE

VIC
StepOne™ System Dye Spectra

SYBR Green
(~520 nm)
Filter 1

FAM
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(~610 nm)
Filter 4

ROX
(~580 nm)
Filter 3

TAMRA
NED
(~550 nm)
Filter 2

JOE

VIC
StepOnePlus™ System Dye Spectra

SYBR Green
(~520 nm)
Filter 1

FAM
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How to Calibrate Materials Required

a Custom Dye • Centrifuge with reaction plate adaptor
• Custom dye(s)
• Deionized water
• MicroAmp® Optical Adhesive Film or MicroAmp® 8-Cap Strip (Flat Caps only)
• MicroAmp® Optical Reaction Plate of the size appropriate for your instrument
• Pipettors and pipette tips (200-µL and 1000-µL)
• Powder-free gloves
• Safety glasses
• Tubes (2-ml and 10-ml)

Prepare the Custom Spectral Calibration Plate

1. Create a calibration plate for the custom dye:

a. Prepare 1.5 mL of the custom dye at the desired concentration.

b. Pipette 30 µL of the diluted custom dye to all wells of a reaction plate.

c. Seal the wells of the reaction plate using an adhesive film or optical flat caps.

d. Vortex the calibration plate for 5 sec.

e. Centrifuge calibration plate for 2 min at less than1500 rpm.

IMPORTANT! The calibration plate must be well mixed and centrifuged.

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f. Confirm that the liquid in each well of the calibration plate is at the bottom of
the well. If not, centrifuge the calibration plate again at a greater rpm and for a
longer time.

IMPORTANT! Do not allow the bottom of the calibration plate to become dirty.
Fluids and other contaminants that adhere to the bottom of the plate can contaminate
the sample block(s) and cause an abnormally high background signal.

Correct Incorrect

Liquid is at the • Not centrifuged with enough force, or

bottom of the well. • Not centrifuged for enough time

2. Load the custom calibration plate into the instrument:

a. Open the instrument drawer.

b. Place the custom calibration plate in the sample block(s) so that the A1 position
is at the back-left corner.
c. Close the instrument drawer carefully.

Run the Custom Spectral Calibration Plate

1. In the main screen of the StepOne™ software, select Instrument

Instrument Maintenance Manager.

2. In the Instrument Maintenance Manager:

a. In the navigation pane, click Dye.

b. In the Dye screen, select Custom Dye Calibration.

c. Click Start Calibration.

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3. In the Setup screen of the Dye Calibration dialog box, select a custom dye from the
list or create the custom dye as follows:
a. Click New Dye.

b. In the Dye Manager dialog box, click New.

c. Complete the New Dye dialog box, then click OK.

Field/Option Action

Name Enter a name for the custom dye.

Wavelength Enter the wavelength at which the dye fluoresces.

Type Select:
• Reporter if the dye works in conjunction with a quencher dye
to report an increase of PCR product.
• Quencher if the dye suppresses the fluorescence of a reporter
dye until amplification of PCR product.
• Both if the dye reports an increase of PCR product without the
aid of a quencher dye.

d. Click Close.

4. In the Setup screen of the Dye Calibration dialog box, enter a temperature setting for
the calibration.

Note: Set the temperature to match the temperature at which you intend to collect
data. For example, the temperature for all Thermo Fisher Scientific system dyes is
60°C because data collection for TaqMan® reagents occurs during the 60°C
extension step of the PCR.

5. Select The custom dye plate is loaded in the instrument, then click Next.

6. In the Run screen, click Start Run, then wait for the instrument to complete the dye
calibration.

Note: If the StepOne™ software displays messages during the run, troubleshoot the
errors as described in “Dye Calibration Failure” on page 166.

7. When the instrument displays the Main Menu, unload the custom calibration plate.

8. Analyze the custom spectral calibration plate as explained in “Analyze the

Calibration Data” on page 135.

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Archive and Back Up Data

Develop a strategy that addresses the following data management issues for the
instrument.

Check Disk If you perform real-time experiments on your system, or collect real-time data for
Space Regularly genotyping experiments, check the available space on your hard drive weekly. When the
hard drive is within 20% of maximum capacity, transfer the older data to a backup
storage device.

Archive Older Conserve space on the computer hard drive by using a data compression utility to archive
Experiments unused or older experiments. Several commercially available compression utilities are
available, such as the PKZIP and ARC archive formats common to the Microsoft®
Windows® operating system.

Back Up Back up the experiments generated by your system to:

Experiments • Protect against potential data loss of data caused by an unforeseen failure of the
Regularly computer or its hard drive(s).
• Conserve space on the hard drive and to optimize performance, if you remove old
data after backing up.
See “Select Protective Hardware and Software” on page 31 for more information on
backup storage devices.

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Infrequent Maintenance
Maintenance Perform the following procedures as needed to resolve problems as they arise:
■ Decontaminate the Sample Block(s) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
■ Move the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
■ Replace the External Fuses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
■ Ship the Instrument for Service . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
■ Update the StepOne™ Software or the Operating System . . . . . . . . . . . . . . . . 154
■ Update the StepOne™ Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

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Decontaminate the Sample Block(s)

If you perform a background calibration and observe high fluorescent background that
you suspect to be contamination, decontaminate the sample block(s).

Materials • Bleach solution (10%)

Required • Cotton or nylon swabs and lint-free cloths
• Deionized water
• EtOH solution (95%)
• Pipettor and pipette tips (100-µL)
• Powder-free gloves
• StepOne system cable, yellow (from the system packing kit)

Guidelines for We recommend the use of 10% bleach solution for resolving
Bleach Solution fluorescent contamination on the instrument sample block(s); however, excessive use of
Use the solution can corrode the block material.

To prevent block degradation:

• Use bleach solution to decontaminate the sample block(s) as a last resort. Rinse the
sample block(s) with bleach only after treatments of deionized water and 95%
ethanol fail to remove the contamination.
• Use deionized water to rinse the sample block(s) thoroughly after treating the block
with bleach solution. Thoroughly removing residual bleach from the metal surfaces
with water minimizes the long term effects of bleach.

Clean the PHYSICAL INJURY HAZARD. During instrument operation, the

Contaminated temperature of the sample block(s) can exceed 100°C. If the instrument has been used
Wells recently, keep your hands away until the sample block(s) reaches room temperature.

Prepare the 1. If you have not already done so, use the StepOne system cable to connect the:
Instrument • Yellow Ethernet port ( ) of the instrument to the
• Ethernet port ( ) of the computer running the StepOne™ software

IMPORTANT! Do not connect the StepOne system cable to the blue LAN Port ( ).

Ethernet

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2. Using the data from the background calibration, identify the location of the
contaminated wells on the sample block(s).

3. Turn off and unplug the system. Allow it to cool for 15 min.

4. Open the instrument drawer.

5. Clean each contaminated well of the sample block(s):

a. Pipette a small volume of water into the contaminated well, slowly pipette the
water up and down several times, then expel the water into a waste beaker.
b. Use a cotton swab to scrub inside of the contaminated well.

c. Use a lint-free cloth to absorb the excess deionized water in the well.

d. Repeat steps 5a through 5d.

6. Close the instrument drawer, then plug in and power on the instrument.

7. Perform a background calibration to confirm that you have eliminated the

contamination.

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8. If the contamination persists, clean the wells with a 95% EtOH solution:

CHEMICAL HAZARD. Ethanol is a flammable liquid and

vapor. Exposure causes eye, skin, and respiratory tract irritation and may cause
central nervous system depression and liver damage. Read the SDS, and follow the
handling instructions. Wear appropriate protective eyewear, clothing, and gloves.

a. Perform steps 2 through 5, substituting a 95% EtOH solution in place of the

deionized water.
b. Repeat step 5 using deionized water to rinse the wells of the sample block(s).

c. Perform steps 6 and 7 to confirm that you have eliminated the contamination.

IMPORTANT! Use deionized water to rinse wells after they have been treated with
bleach or EtOH solution.

9. If the contamination persists, clean the wells with a 10% bleach solution:

CHEMICAL HAZARD. Sodium hypochlorite (bleach) is a

liquid disinfectant that can be corrosive to the skin and can cause skin
depigmentation. Read the SDS, and follow the handling instructions. Wear
appropriate protective eyewear, clothing, and powder-free gloves.

a. Perform steps 2 through 5, substituting a 10% bleach solution in place of the

deionized water.
b. Repeat step 5 using deionized water to rinse the wells of the sample block(s).

c. Perform steps 6 and 7 to confirm that you have eliminated the contamination.

IMPORTANT! Use deionized water to rinse wells after they have been treated with
bleach or EtOH solution.

10. If the contamination is still present, contact technical support.

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Move the Instrument

Perform this procedure to safely move the instrument short distances (for example,
between laboratories in the same building).

Note: If you want to transport the instrument via ground or air shipping, package the
instrument for shipping as explained in “Ship the Instrument for Service” on page 152.

Materials Original packing plate or a MicroAmp® Optical Reaction Plate

Required

Prepare the 1. Load the packing plate or empty reaction plate into the instrument:
Instrument
a. Open the instrument drawer.

b. Place the packing plate or empty reaction plate onto the sample block(s).

c. Close the instrument drawer.

2. Raise the sample block(s) to secure it for transport:

a. Touch the instrument touchscreen to awaken it, then touch .
b. In the Main Menu, touch Tools Menu, touch Ship Prep, then touch Ship Prep.

c. Wait for the instrument to raise the sample block(s), then power off the
instrument when prompted.

3. Disconnect the power cord from the back of the instrument.

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Move and PHYSICAL INJURY HAZARD. Do not attempt to lift the

Reconnect the instrument or any other heavy objects unless you have received related training. Incorrect
Instrument lifting can cause painful and sometimes permanent back injury. Use proper lifting
techniques when lifting or moving the instrument. At least two people are required to lift
the instrument.

IMPORTANT! Moving your instrument can create subtle changes in the alignment of the
instrument optics. We recommend that you run a TaqMan® RNase P Fast Instrument
Verification Plate after you move the instrument to confirm the performance of the
system.

1. Move your instrument according to the guidelines in step 6 on page 40.

Do not carry the instrument by the instrument drawer. Carrying

the instrument by the drawer can damage the instrument optics.

Yes No

2. Install the components of the system according the chosen layout.

Perform the installation

Layout Description
described in…

Colocated In this layout, the instrument is connected to Chapter 3, “Install the

the colocated computer by the yellow Colocated Layout,” on
StepOne system cable. page 49

Standalone In this layout, the instrument is not directly Chapter 4, “Install the
connected to the computer running the Standalone Layout,” on
StepOne™ software. page 75

If the RNase P experiment performed at the end of the installation:

• Passes, then you have successfully moved the instrument.
• Fails, recalibrate the instrument:
a. Perform a spatial calibration (see “Perform a Spatial Calibration” on page 118).

b. Perform a background calibration (see “Perform a Background Calibration” on

page 122).
c. Perform a dye calibration (see “Perform a Dye Calibration” on page 128).

d. Perform another RNase P experiment (see “Perform the RNase P Experiment”

on page 65 or page 88).

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Replace the External Fuses

Replace the fuses of the instrument as needed.

Materials • Fuses, 10 A 250 VAC Slo-Blo®

Required • Small flatblade screwdriver

About the Fuses

The instrument uses two 10 A 250 VAC fuses to protect the system electronics from
power surges. When replacing one fuse, we recommend that you replace the companion
fuse, regardless of its condition.

Note: Acceptable AC line voltage tolerances are 100, 120, 220, 230 ±10%; 240 VAC
+6%/–10%, 50/60 Hz ± 1%.

FIRE HAZARD. For continued protection against the risk of fire,

replace fuses only with Listed and Certified fuses of the same type and rating as those
currently in the instrument.

SHOCK HAZARD. To protect yourself against shock hazards, use a

properly wired three-terminal outlet. Do not use an adapter to a two-terminal outlet since
this does not provide positive ground protection.

Replace the ELECTRICAL SHOCK HAZARD. Severe electrical shock, which

Fuses could cause physical injury or death, can result from working on an instrument when the
high voltage power supply is operating. To avoid electrical shock, disconnect the power
supply to the instrument, unplug the power cord, and wait at least 1 min before working
on the instrument.

1. Power off the instrument.

2. Disconnect the power cord from the back of the instrument.

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3. Remove the power entry module and fuses from the power entry module:
a. Insert a small flat-tip screwdriver into one of two slots in the right side of the
power entry module containing the fuses, and pry open the door.
b. Insert the screwdriver into either side slot on the fuse compartment and eject
the power entry module.
c. Replace the burnt fuse(s) in the power entry module with a new 10 A fast-
acting 250 VAC fuse.

4. Replace the power entry module:

a. Lightly squeeze both mounted fuses between your thumb and forefinger and
insert the power entry module into the fuse compartment until it is fully seated.
b. Press the power entry module door until it locks in place.

5. Connect the power cord to the back of the instrument.

6. Power on the instrument, then wait for it to perform a diagnostic of all major system
components. If the diagnostic is successful, the instrument displays the main screen.
If the instrument detects a problem, it displays an error code for the related failed
test. Repeat the procedure above without replacing the fuses to reseat the fuse
holder. If the problem persists, contact Support as explained in “How to Obtain
Support” on page 14.

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Ship the Instrument for Service

If the instrument requires the attention of a service representative, decontaminate the
instrument and package it for transportation to Thermo Fisher Scientific.

Materials MicroAmp® Optical Reaction Plate

Required

Prepare for 1. Contact the Customer Care Center (see “How to Obtain Support” on page 14) and
Shipment request a Certificate of Decontamination.

2. Decontaminate the instrument according to the guidelines in the Certificate of

Decontamination.

3. Sign the completed Certificate of Decontamination and fax or e-mail it to the

Customer Care Center.

IMPORTANT! Do not dispose the completed Certificate of Decontamination. You

must include a copy in the package containing the instrument.

4. Raise the sample block(s) to secure it for transport:

a. Load the empty reaction plate into the instrument.

b. Touch the instrument touchscreen to awaken it, then touch .

c. In the Main Menu, touch Tools Menu, touch Ship Prep, then touch Ship Prep.

d. Wait for the instrument to raise the sample block(s), then power off the
instrument when prompted.

5. Disconnect the power cord from the back of the instrument.

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6. Using the packaging material provided by Thermo Fisher Scientific, create a

package to ship the instrument that includes the:
• StepOne™ or StepOnePlus™ instrument
• Certificate of Decontamination (from step 3b on page 152)

IMPORTANT! Shipping an instrument swithout a completed Certificate of

Decontamination will delay service of the instrument.

• Payment for the service in one of the following forms:

• Purchase order, or
• Company letterhead with the words “verbal purchase order,” or
• Visa or MasterCard credit card information
• Your address and contact information, including:
• Billing address
• Return shipping address
• Name and phone number of a contact

IMPORTANT! Do not include accessories or cords in the package.

7. Affix the postage provided to the package.

8. Inform the courier to arrange pickup, then ship the package to the location specified
by the Customer Care Center.

Install the When you receive the serviced instrument, install it as you would a new instrument by
Serviced completing Chapters 1 through 5 of this guide.
Instrument

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Update the StepOne™ Software or the Operating System

Visit the For the latest services and support information for the StepOne™ and StepOnePlus™
StepOne™ and systems, go to:
StepOnePlus™ info.appliedbiosystems.com/stepone
System Website
The website contains the latest software and documentation updates for the instrument
and analysis software.
At the system support page, you can:
• Submit a question directly to Technical Support
• Order user documents, SDSs, certificates of analysis, and other related documents
• Download documents in Adobe portable document format (PDF)
• Obtain information about customer training
• Download software updates and patches for the instrument

Update the Prepare for the Update

StepOne™ If you want to update the StepOne™ Software, prepare your computer by exporting the
Software application libraries and backing up your experiment files.

1. Back up the application libraries:

a. In the main menu of the StepOne™ software, select Tools <desired library>.
b. When the library dialog box opens, select the element(s) that you want to
export, then click Export.
c. In the Export dialog box, click Save to archive the selected records.

d. Repeat steps 1 through 3 for the remaining libraries to archive them.

2. Back up all experiment files by creating a copy of the directory that you are using to
store files.
The default directory for StepOne or StepOnePlus™ experiments is:
D:\Applied Biosystems\<software name>\experiments
where <software name> is the current version of the StepOne software.

3. Back up the instrument data:

a. Connect a USB drive to the USB port.

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b. Touch the instrument touchscreen to awaken it, then touch .

c. In the Main Menu, touch Settings Menu, then touch Admin Menu,
touch Admin Menu, then touch Back Up Experiments and Settings.
d. Touch Back Up, then wait for the instrument to transfer the data.

e. When the transfer is complete, touch Done, then remove the USB drive.

Check for Updates

1. Go to:
http://info.appliedbiosystems.com/stepone
to open the StepOne and StepOnePlus system product page.

2. Under Software Download, view the available downloads.

3. If there are updates and/or patches for v2.1 or later, select the appropriate option,
then follow the prompts to begin the download.
You may be asked for your instrument serial number. The serial number is on the
back panel of your instrument, or can be obtained from the instrument touchscreen.

Determine the Do not upgrade or update the Microsoft Windows® operating system of the computer that
Compatibility of runs the StepOne™ software without first consulting the software release notes or the
an Operating Thermo Fisher Scientific website. Future versions of the Windows® operating system
System Upgrade and updates to the current operating system may conflict with the StepOne™ software
or Update and render the software inoperable.

1. From the desktop, browse to and open:

<drive>:\Applied Biosystems\<software name>
where:
– <drive> is the computer hard drive on which the StepOne software is installed.
The default installation drive for the software is the D drive.
– <software name> is the current version of the StepOne software.

2. Double-click release-notes.html, then read the StepOne™ Software Release Notes

for the compatibility of interest.

3. If the release notes do not mention the compatibility:

a. Go to:
info.appliedbiosystems.com/stepone
b. Search the system website for the compatibility of interest.

4. If the website does not contain the information of interest, contact Support for the
information (see “How to Obtain Support” on page 14).

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How to Troubleshoot the Installation

This appendix covers:

■ StepOne™ Software Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
■ Network Connection Problems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
■ Background Calibration Failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
■ Dye Calibration Failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166

Note: For more information about any of the topics discussed in this guide, access the
Help from within the Applied Biosystems StepOne™ Real-Time PCR System Software
by pressing F1, or clicking in the toolbar, or selecting Help StepOne Software
Help.

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StepOne™ Software Problems

StepOne™ Software Problems

If you encounter errors during the installation of the StepOne™ software, uninstall the
software before reinstalling it.

Uninstall the 1. Log onto the operating system as Administrator, or as a member of the
StepOne™ Administrators user group.
Software
IMPORTANT! If you are installing the StepOne™ software, you must log onto the
Windows® operating system using a user account that belongs to the Administrators
user group.

2. Remove the StepOne™ software:

a. In the desktop, select Start Control Panel Add/Remove Programs.
b. In the Add/Remove Programs dialog box, select StepOne™ software, then
click Change/Remove.
c. In the Welcome page of the InstallShield Wizard dialog box, select Remove.

d. In the Confirm Uninstall dialog box, click OK.

e. When the uninstallation is complete, then click Finish.

f. Close the Add/Remove Programs dialog box.

3. Restart the computer and log in as Administrator, or as a member of the

Administrators user group.

4. Reinstall the StepOne™ software.

If the StepOne™ software installation continues to fail, reimage the computer and
repeat the installation.

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Network Connection Problems

Network Connection Problems

Use this topic to resolve problems when setting up the instrument for remote monitoring.

Troubleshooting
Start StepOne™ software cannot add instrument for
Workflow remote monitoring

Connect the Ethernet

Is the instrument
cable for the network
connected to the
connection to the blue
network by the blue No LAN Port at rear of the
LAN Port?
instrument.
Yes
Determine the IP address and network
settings of the instrument LAN port as
explained in step 1 on page 162.

Confirm with your network administrator

Is the LAN Port of the that the static IP address, subnet mask,
instrument set up for and default gateway settings are correct.
static IP or DHCP? Static IP Correct the network settings as directed
by your network administrator.
DHCP
Determine the subnet address as
explained in “How to Determine the Subnet
Address” on page 163.

Are the instrument and

computer located on Add and monitor the instrument using the
the same subnet? No IP address of the LAN Port.

Yes
Confirm the network connection by
pinging the instrument as explained in
“How to Ping the Instrument” on page 162.

Can you “ping” Consult your network administrator to

the instrument from determine the source of the failed
a computer on the No network connection.
network?
Yes

Can you add or monitor When you add the instrument for remote
the instrument using the monitoring, enter the instrument name
StepOne™ software? No using lower case letters only.

If you cannot use the instrument name to

add the instrument for remote monitoring,
try using the IP address of the instrument.

Unplug the instrument from the network,

then ping the IP address. If you can ping
Contact Support as explained
the address after removing the instrument
in “How to Obtain Support” on
from the network, then another device is
page 14.
using the same IP address and you must
reassign it to resolve the conflict.

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Network Connection Problems

How to Ping the You can confirm the connection between a network computer and instrument by pinging the
Instrument instrument name or IP address from the computer. The ping command issued from the DOS
command shell causes the computer to send a series of 32-byte data packets to the instrument.
If the network connection between the computer and the instrument is unobstructed, the
instrument sends data packets in reply.
1. Determine the IP address of the instrument:
a. Touch the instrument touchscreen to awaken it, then touch .
b. In the Main Menu, touch Settings Menu Admin Menu Set IP Address.
c. Record the IP address in the IP Address field.

2. Ping the instrument:

a. Power on the instrument and wait for the instrument to boot.
b. In the computer desktop, select Start Programs Accessories
Command Prompt.
c. In the Command Prompt window, enter ping <IP address>, then
press Enter.

If the DOS prompt… Then…

replies to the pings the computer can communicate with the IP address.

displays “request Wait a few minutes for the network to update the IP address of
timed out” the instrument, then ping the instrument again.
If you still cannot ping the instrument after two attempts,
consult your network administrator to determine the source of
the failed network connection.

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Network Connection Problems

Resolving an IP Address Conflict

If you suspect that the IP address used by either port of the instrument may conflict with
another device on your network, you can use the following procedure to test for the
conflict.
1. Unplug the instrument from the network.
2. Ping the IP address of the instrument LAN port as explained above.
If you can ping the IP address, then another device on the network is already using
the address and you must reassign the IP address of the instrument to resolve the
conflict.

How to 1. Touch the instrument touchscreen to awaken it, then touch .

Determine the 2. In the Main Menu, touch Settings Menu Admin Menu Set IP Address.
Subnet Address
3. Record the IP address in the IP Address field. The first three numbers of the IP
address are the subnet address.
For example, the italicized numbers in the following IP address are the subnet
address:
192.168.100.140
If the subnet address for the instrument is different than that of the computer used for
remote monitoring, then you cannot use the instrument name to add the instrument for
remote monitoring. The mDNS limitation explained in “Networking Guidelines and Best
Practices” on page 105 restricts the use of the instrument name as an identifier to
network devices on the same subnet.
Note: To determine the subnet address of the computer that you want to use to monitor
the instrument, select Start Help and Support in the computer desktop, then use the
Microsoft Windows Help System search for the procedure appropriate for the operating
system.

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Background Calibration Failure

Background Calibration Failure

Use this topic to troubleshoot failed background calibrations.

Troubleshooting Start
Workflow
Does the instrument Repeat the calibration. If the problem persists,
complete the contact Support as explained in “How to Obtain
background run? No Support” on page 14.

Yes
One or more wells 1. Repeat the calibration using the
of the background same background plate.
Does the
plate have produced
calibration pass? No 2. If the calibration fails again,
spectra that exceed
repeat the calibration using a
the maximum limit
different background plate.
for the instrument.
3. If the calibration fails again,
determine the source of the
contamination as explained in
“Identify the Source and Location
of Contamination” below.

Identify the Signals that exceed the limit of normal background fluorescence may indicate the
Source and presence of fluorescent contamination on the calibration plate or the sample block(s).
Location of Common contaminants include: ink residue from permanent pens, powder from
Contamination disposable gloves, and dust.

To determine the source and location of the contamination:

1. While viewing the raw spectra, locate the contaminated well position(s) by selecting
successively smaller regions of the plate layout.

2. Rotate the background plate 180°, then perform the background calibration again.

3. Repeat step 1 to locate the contamination. If the position(s) of the contamination in

steps 1 and 3 are:
• Identical – The sample block(s) are contaminated. Decontaminate the sample
block(s) as explained on page 145.
• Reversed – The background plate is contaminated. Discard the background
plate and perform the background run using a new background plate.
If the calibration fails after you have replaced the background plate and decontaminated
the sample block(s), perform the following test:

1. Load a plate covered by a piece of black paper into the instrument.

2. Perform the background run as explained in “Perform a Background Calibration” on

page 122.

3. After the run is complete, select all wells of the plate layout.

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Background Calibration Failure

4. View the Spectral plot for the peak(s). If the peak is:
• Visible – The optics of your instrument may be contaminated. Contact Support
as explained in “How to Obtain Support” on page 14.
• Absent – The sample block(s) are contaminated. Decontaminate the sample
block(s) as explained in “Decontaminate the Sample Block(s)” on page 145.

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Dye Calibration Failure

Dye Calibration Failure

Use this topic to troubleshoot failed dye calibrations.

Troubleshooting Start
Workflow
Reattempt the calibration. If the
Does the instrument
problem persists, contact Support
complete the dye
No as explained in “How to Obtain
calibration?
Support” on page 14.
Yes
One or more wells
Review the spectra for
produced spectra:
Does the irregularities as explained in
• Beyond the detectable “Analyze the Calibration Data”
calibration pass? No range for the system, or on page 135.
• In more than one filters
Yes

If the peaks of one or more spectra appear in the wrong

Do the dye spectra filter, confirm that:
peak in the correct • The spectral calibration plate was run in the correct
filters? No orientation (the plate was not rotated 180° when run).
• The correct dyes were loaded onto the spectral
calibration plate, if you are running a custom plate.

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Dye Calibration Failure

Common The following table displays irregular spectra common to dye calibrations.
Irregularities

Spectra Possible Cause Action

One or more raw spectra are • The spectral calibration plate has 1. Unload the instrument and view the wells of the
at or below the detectable been centrifuged insufficiently. spectral calibration plate. If the liquid in the wells
threshold for the calibration. • The spectral calibration plate are not:
contains old or insufficient reagents. – At the bottom of the wells, centrifuge the plate
• If you are running a custom spectral for a longer time, then repeat the calibration.
calibration plate, the dye may not – Equivalent in volume, the plate is not sealed
be present at a sufficient and the reagents have evaporated. Discard it
concentration. and run another.
2. If the spectral calibration plate appears to be
normal, discard the plate and run another.
3. If the problem persists, contact Support as
explained in “How to Obtain Support” on page 14.
Note: If you are running a custom spectral
calibration plate, create another plate but increase
the concentration of the dye that produced
insufficient signal.

One or more raw spectra • Fluorescent contamination is Confirm that the presence of contamination by
exceed the maximum limit for present on the sample block(s) or performing a background calibration as explained
the instrument. spectral calibration plate. in “Perform a Background Calibration” on
• If you are running a custom spectral page 122. If the background calibration does not
calibration plate, the dye may be show sample block contamination, the spectral
too concentrated. calibration plate may be contaminated.
Note: If you are running a custom spectral
The spectra contain peaks in Fluorescent contamination is present calibration plate, create another plate but decrease
more than one filter. on the sample block(s) or spectral the concentration of the dye that exceeds the
calibration plate. detectable limit.

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 Glossary

Advanced Setup In the StepOne™ software, a feature that allows you to set up your experiment according
to your experiment design. Advanced Setup provides you with maximum flexibility in the
design and setup of your experiment.

AIF See assay information file (AIF).

allele For a given target, any of the different sequences that occurs in the population.

allelic discrimina- Display of data collected during the post-PCR read. The allelic discrimination plot is a
tion plot graph of the normalized reporter signal from the allele 1 probe plotted against the
normalized reporter signal from the allele 2 probe.

amplicon A segment of DNA amplified during PCR.

amplification Part of the instrument run in which PCR produces amplification of the target. For
quantitation experiments, fluorescence data collected during amplification are displayed
in an amplification plot, and the data are used to calculate results. For genotyping or
presence/absence experiments, fluorescence data collected during amplification are
displayed in an amplification plot, and the data can be used for troubleshooting.

amplification Calculation of efficiency of the PCR amplification. The amplification efficiency is

efficiency (EFF%) calculated using the slope of the regression line in the standard curve. A slope close to
−3.32 indicates optimal, 100% PCR amplification efficiency. Factors that affect
amplification efficiency:
• Range of standard quantities – To increase the accuracy and precision of the
efficiency measurement, use a broad range of standard quantities, 5 to 6 logs
(105 to 106 fold).
• Number of standard replicates – To increase the precision of the standard
quantities and decrease the effects of pipetting inaccuracies, include replicates.
• PCR inhibitors – PCR inhibitors in the reaction can reduce amplification and alter
measurements of the efficiency.

amplification plot Display of data collected during the cycling stage of PCR amplification. Can be viewed
as:
• Baseline-corrected normalized reporter (ΔRn) vs. cycle
• Normalized reporter (Rn) vs. cycle
• Threshold cycle (CT) vs. well

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amplification stage Part of the instrument run in which PCR produces amplification of the target. The
amplification stage is called a cycling stage in the thermal profile and consists of
denaturing, primer annealing, and polymerization steps that are repeated.
For quantitation experiments, fluorescence data collected during the amplification stage
are displayed in an amplification plot, and the data are used to calculate results. For
genotyping or presence/absence experiments, fluorescence data collected during the
amplification stage are displayed in an amplification plot, and the data can be used for
troubleshooting. See also cycling stage.

assay In the StepOne™ and StepOnePlus™ systems, a PCR reaction mix that contains primers to
amplify a target and a reagent to detect the amplified target.

Assay ID Identifier assigned by Thermo Fisher Scientific to TaqMan® Gene Expression Assays and
TaqMan® SNP Genotyping Assays.

assay information Data file on a CD shipped with each assay order. The file name includes the number from
file (AIF) the barcode on the plate. The information in the AIF is provided in a tab-delimited format.

assay mix PCR reaction component in Thermo Fisher Scientific TaqMan® Gene Expression Assays
and TaqMan® SNP Genotyping Assays. The assay mix contains primers designed to
amplify a target and a TaqMan® probe designed to detect amplification of the target.

AutoDelta In the run method, a setting to increase or decrease the temperature and/or time for a step
with each subsequent cycle in a cycling stage. When AutoDelta is enabled for a cycling
stage, the settings are indicated by an icon in the thermal profile:
• AutoDelta on:
• AutoDelta off:

automatic baseline An analysis setting in which the software calculates the baseline start and end values for
the amplification plot. You can apply the automatic baseline setting to specific wells in
the reaction plate. See also baseline.

automatic CT An analysis setting in which the software calculates the baseline start and end values and
the threshold in the amplification plot. The software uses the baseline and threshold to
calculate the threshold cycle (CT). See also threshold cycle (CT).

baseline In the amplification plot, a line fit to the fluorescence levels during the initial stages of
PCR, when there is little change in fluorescence signal.

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baseline-corrected The magnitude of normalized fluorescence signal generated by the reporter:

normalized reporter
(ΔRn) 1. In experiments that contain data from real-time PCR, the magnitude of normalized
fluorescence signal generated by the reporter at each cycle during the PCR
amplification. In the ΔRn vs. Cycle amplification plot, ΔRn is calculated at each
cycle as:
ΔRn (cycle) = Rn (cycle) − Rn (baseline), where Rn = normalized reporter

2. In genotyping experiments and presence/absence experiments, the difference in

normalized fluorescence signal generated by the reporter between the pre-PCR read
and the post-PCR read. In the allelic discrimination plot (genotyping experiments)
and the presence/absence plot (presence/absence experiments), ΔRn is calculated
as:
ΔRn = Rn (post-PCR read) − Rn (pre-PCR read), where Rn = normalized reporter
See also normalized reporter (Rn).

biological replicate Reactions that contain identical components and volumes, but evaluate separate samples
of the same biological source (for example, samples from three different mice of the same
strain, or separate extractions of the same cell line or tissue sample).
When using biological replicate groups in a comparative CT study, the values displayed
in the Biological Replicates tab are calculated by combining the results of the separate
biological samples and treating this collection as a single population (that is, as one
sample). For ΔCT computations (normalizing by the endogenous control) in a singleplex
experiment, the separate biological samples are treated as unpaired data when computing
variability estimates of the single biological replicate. You can observe individual
contributions of the separate biological samples to the single biological replicate results
in the Technical Replicates tab.

Note: To view the Biological Replicates and Technical Replicates tabs, from the Study
Menu pane, select Analysis Gene Expression.

blocked IPC In presence/absence experiments, a reaction that contains IPC blocking agent, which
blocks amplification of the internal positive control (IPC). In the StepOne™ software, the
task for the IPC target in wells that contain IPC blocking agent. See also negative control-
blocked IPC wells.

calibrator See reference sample.

chemistry See reagents.

colocated layout A system layout in which the StepOne™ or StepOnePlus™ instrument is directly
connected to a colocated computer by the yellow cable. In this layout, you can control the
instrument with the StepOne™ software on the colocated computer or with the instrument
touchscreen.

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comparative CT Method for determining relative target quantity in samples. With the comparative CT
(ΔΔCT) method (ΔΔCT) method, the StepOne™ software measures amplification of the target and of the
endogenous control in samples and in a reference sample. Measurements are normalized
using the endogenous control. The software determines the relative quantity of target in
each sample by comparing normalized target quantity in each sample to normalized target
quantity in the reference sample.

CT See threshold cycle (CT).

custom dye Dye that is not supplied by Thermo Fisher Scientific. Custom dyes may be adapted for
use in experiments on the StepOne™ and StepOnePlus™ systems. When using custom
dyes, the custom dye should be added to the Dye Library and a custom dye calibration
performed.

IMPORTANT! We do not recommend the use of TAMRA™ dye as reporter or quencher

with the StepOne™ system. TAMRA dye may be used as a reporter or quencher with the
StepOnePlus™ system.

cycle threshold See threshold cycle (CT).

cycling stage In the thermal profile, a stage that is repeated. A cycling stage is also called an
amplification stage. For cycling stages, you can enable AutoDelta settings. See also
amplification stage.

data collection A process during the instrument run in which an instrument component detects
fluorescence data from each well of the reaction plate. The instrument transforms the
signal to electronic data, and the data are saved in the experiment file. In the StepOne™
software, a data collection point is indicated by an icon in the thermal profile:
• Data collection on:
• Data collection off:

delta Rn (ΔRn) See baseline-corrected normalized reporter (DRn).

derivative reporter The negative first-derivative of the normalized fluorescence generated by the reporter
(−Rn′) during PCR amplification. In the derivative reporter (–Rn′) vs. temperature melt curve,
the derivative reporter signal is displayed in the y-axis.

Design Wizard A feature in the StepOne™ software that helps you set up your experiment by guiding you
through best practices as you enter your experiment design.

diluent A reagent used to dilute a sample or standard before adding it to the PCR reaction. The
diluent can be water or buffer.

Diluted Sample In the StepOne™ software, a field displayed on the Sample Dilution Calculations tab of
Concentration the Reaction Setup screen. For this field, enter the sample concentration you want to use
(10✕ for Reaction to add to the reaction mix for all samples in the experiment. “10✕ for Reaction Mix”
Mix) indicates that the software assumes the sample or standard component of the reaction mix
is at a 10✕ concentration. For example, if the diluted sample concentration is 50.0 ng/μL
(10✕), the final sample concentration in the reaction is 5 ng/μL (1✕).

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dilution factor See serial factor.

dissociation curve See melt curve.

EFF% See amplification efficiency (EFF%).

endogenous A target or gene that should be expressed at similar levels in all samples you are testing.
control Endogenous controls are used in relative standard curve and comparative CT (ΔΔCT)
experiments to normalize fluorescence signals for the target you are quantifying.
Housekeeping genes can be used as endogenous controls. See also housekeeping gene.
When using multiple endogenous controls, the software treats all endogenous controls as
a single population, and calculates the experiment-appropriate mean to establish a single
value against which the target of interest is normalized. In comparative CT experiments,
the mean calculated is the arithmetic mean of the CT values. In relative standard curve
experiments, the CT values are converted to relative quantities prior to normalization; the
mean calculated is subsequently the geometric mean of the relative quantities.

Note: Arithmetic and geometric means are related and equivalent due to logarithmic
transformation of the data.

Variability estimates for multiple endogenous controls are computed separately. The final
variability estimate is a pooled combination of the individual variability estimates
(similar to computing pooled standard deviations).

endpoint read See post-PCR read.

experiment Refers to the entire process of performing a run using the StepOne™ or StepOnePlus™
systems, including setup, run, and analysis. The types of experiments you can perform
using the StepOne™ and StepOnePlus™ systems:
• Quantitation - standard curve
• Quantitation - relative standard curve
• Quantitation - comparative CT (ΔΔCT)
• Melt curve
• Genotyping
• Presence/absence

experiment name Entered during experiment setup, the name that is used to identify the experiment.
Experiment names cannot exceed 100 characters and cannot include any of the following
characters: forward slash (/), backslash (\), greater than sign (>), less than sign (<),
asterisk (*), question mark (?), quotation mark ("), vertical line (|), colon (:),
semicolon (;), and sign (&), percent sign (%), dollar sign ($), at sign (@), circumflex (^),
left parenthesis ( ( ), right parenthesis ( ) ), or exclamation point (!).

IMPORTANT! If you run the instrument in standalone mode from the instrument
touchscreen, you cannot enter more than 32 characters in the Experiment Name field and
you cannot include spaces in the name.

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experiment type The type of experiment you are performing using the StepOne™ or StepOnePlus™ system:
• Standard curve
• Comparative CT (ΔΔCT)
• Relative standard curve
• Melt curve (not available in the Design Wizard)
• Genotyping
• Presence/absence
The experiment type you select affects the setup, run, and analysis.

forward primer Oligonucleotide that flanks the 5′ end of the amplicon. The reverse primer and the
forward primer are used together in PCR reactions to amplify the target.

holding stage In the thermal profile, a stage that includes one or more steps. You can add a holding stage
to the thermal profile to activate enzymes, to inactivate enzymes, or to incubate a reaction.

housekeeping gene A gene that is involved in basic cellular functions and is constitutively expressed.
Housekeeping genes can be used as endogenous controls. See also endogenous control.

internal positive In presence/absence experiments, a short synthetic DNA template that is added to PCR
control (IPC) reactions. You can use the IPC to distinguish between true negative results (that is, the
target is absent in the samples) and negative results caused by PCR inhibitors, incorrect
assay setup, or reagent or instrument failure.

inventoried assays TaqMan® Gene Expression Assays and TaqMan® SNP Genotyping Assays that have been
previously manufactured, passed quality control specifications, and stored in inventory.

IPC In presence/absence experiments, abbreviation for internal positive control (IPC). In the
StepOne™ software, the task for the IPC target in wells that contain the IPC and do not
contain IPC blocking agent. See also internal positive control (IPC).

IPC blocking agent Reagent added to PCR reactions to block amplification of the internal positive control
(IPC).

IPC+ See negative control-IPC wells.

made-to-order TaqMan® Gene Expression Assays or TaqMan® SNP Genotyping Assays that are
assays manufactured at the time of order. Only assays that pass manufacturing quality control
specifications are shipped.

manual baseline An analysis setting in which you enter the baseline start and end values for the
amplification plot. You can apply the manual baseline setting to specific wells in the
reaction plate.

manual CT An analysis setting in which you enter the threshold value and select whether to use
automatic baseline or manual baseline values. The software uses the baseline and the
threshold values to calculate the threshold cycle (CT).

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melt curve A plot of data collected during the melt curve stage. Peaks in the melt curve can indicate
the melting temperature (Tm) of the target or can identify nonspecific PCR amplification.
In the StepOne™ software, you can view the melt curve as normalized reporter (Rn) vs.
temperature or as derivative reporter (−Rn′) vs. temperature. Also called dissociation
curve.

melt curve stage In the thermal profile, a stage with a temperature increment to generate a melt curve.

melting In melt curve experiments, the temperature at which 50% of the DNA is double-stranded
temperature (Tm) and 50% of the DNA is dissociated into single-stranded DNA. The Tm is displayed in the
melt curve.

multicomponent A plot of the complete spectral contribution of each dye for the selected well(s) over the
plot duration of the PCR run.

negative control In the StepOne™ software, the task for targets or SNP assays in wells that contain water
(NC) or buffer instead of sample. No amplification of the target should occur in negative control
wells. Previously called no template control (NTC).

negative control- In presence/absence experiments, wells that contain IPC blocking agent instead of sample
blocked IPC wells in the PCR reaction. No amplification should occur in negative control-blocked IPC wells
because the reaction contains no sample and amplification of the IPC is blocked.
Previously called no amplification control (NAC).

negative control- In presence/absence experiments, wells that contain IPC template and buffer or water
IPC wells instead of sample. Only the IPC template should amplify in negative control-IPC wells
because the reaction contains no sample. Previously called IPC+.

no amplification See negative control-blocked IPC wells.

control (NAC)

no template control See negative control (NC).

(NTC)

nonfluorescent Molecules that are attached to the 3′ end of TaqMan® probes. When the probe is intact,
quencher-minor the nonfluorescent quencher (NFQ) prevents the reporter dye from emitting fluorescence
groove binder signal. Because the NFQ does not fluoresce, it produces lower background signals,
(NFQ-MGB) resulting in improved precision in quantitation. The minor groove binder (MGB)
increases the melting temperature (Tm) without increasing probe length. It also allows the
design of shorter probes.

normalized quantity Quantity of target divided by the quantity of endogenous control.

normalized reporter Fluorescence signal from the reporter dye normalized to the fluorescence signal of the
(Rn) passive reference.

omit well An action that you perform before reanalysis to omit one or more wells from analysis.
Because no algorithms are applied to omitted wells, omitted wells contain no results.

outlier For a set of data, a datapoint that is significantly smaller or larger than the others.

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passive reference A dye that produces fluorescence signal. Because the passive reference signal should be
consistent across all wells, it is used to normalize the reporter dye signal to account for
non-PCR related fluorescence fluctuations caused by minor well-to-well differences in
concentrations or volume. Normalization to the passive reference signal allows for high
data precision.

plate layout An illustration of the grid of wells and assigned content in the reaction plate. In StepOne™
systems, the grid contains 6 rows and 8 columns. In StepOnePlus™ systems, the grid
contains 8 rows and 12 columns.
In the StepOne™ software, you can use the plate layout as a selection tool to assign well
contents, to view well assignments, and to view results. The plate layout can be printed,
included in a report, exported, and saved as a slide for a presentation.

point One standard in a standard curve. The standard quantity for each point in the standard
curve is calculated based on the starting quantity and serial factor.

positive control In genotyping experiments, a DNA sample with a known genotype, homozygous or
heterozygous. In the StepOne™ software, the task for the SNP assay in wells that contain
a sample with a known genotype.

post-PCR read Used in genotyping and presence/absence experiments, the part of the instrument run that
occurs after amplification. In genotyping experiments, fluorescence data collected during
the post-PCR read are displayed in the allelic discrimination plot and used to make allele
calls. In presence/absence experiments, fluorescence data collected during the post-PCR
read are displayed in the presence/absence plot and used to make detection calls. Also
called endpoint read.

pre-PCR read Used in genotyping and presence/absence experiments, the part of the instrument run that
occurs before amplification. The pre-PCR read is optional but recommended.
Fluorescence data collected during the pre-PCR read can be used to normalize
fluorescence data collected during the post-PCR read.

primer mix PCR reaction component that contains the forward primer and reverse primer designed to
amplify the target.

primer/probe mix PCR reaction component that contains the primers designed to amplify the target and a
TaqMan® probe designed to detect amplification of the target.

pure dye See custom dye and system dye.

quantitation In quantitation experiments, the method used to determine the quantity of target in the
method samples. In StepOne™ and StepOnePlus™ systems, there are three types of quantitation
methods: standard curve, relative standard curve, and comparative CT (ΔΔCT).

quantity In quantitation experiments, the amount of target in the samples. Absolute quantity can
refer to copy number, mass, molarity, or viral load. Relative quantity refers to the fold-
difference between normalized quantity of target in the sample and normalized quantity
of target in the reference sample.

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quencher A molecule attached to the 3′ end of TaqMan® probes to prevent the reporter from
emitting fluorescence signal while the probe is intact. With TaqMan® reagents, a
nonfluorescent quencher-minor groove binder (NFQ-MGB) can be used as the quencher.
With SYBR® Green reagents, no quencher is used.

IMPORTANT! We do not recommend the use of TAMRA™ dye as reporter or quencher

with the StepOne™ system. TAMRA dye may be used as a reporter or quencher with the
StepOnePlus™ system.

QuickStart A feature in StepOne™ and StepOnePlus™ systems that allows you to run an experiment
without entering plate setup information. QuickStart requires a colocated layout with the
instrument powered on and an intact instrument-computer connection.

R2 value Regression coefficient calculated from the regression line in the standard curve. The R2
value indicates the closeness of fit between the standard curve regression line and the
individual CT data points from the standard reactions. A value of 1.00 indicates a perfect
fit between the regression line and the data points.

ramp The rate at which the temperature changes during the instrument run. Except for the melt
curve step, the ramp is defined as a percentage. For the melt curve step, the ramp is
defined as a temperature increment. In the graphical view of the thermal profile, the ramp
is indicated by a diagonal line.

ramp speed Speed at which the temperature ramp occurs during the instrument run. Available ramp
speeds include fast and standard.
• For optimal results using the fast ramp speed, we recommend using fast reagents in
your PCR reactions.
• For optimal results using the standard ramp speed, we recommend using standard
reagents in your PCR reactions.

IMPORTANT! Fast reagents are not supported for presence/absence experiments.

raw data plot A plot of raw fluorescence signal (not normalized) for each optical filter.

reaction mix A solution that contains all components to run the PCR reaction, except for the template
(sample, standard, or control).

reagents The PCR reaction components you are using to amplify the target and to detect
amplification. Types of reagents used on the StepOne™ and StepOnePlus™ systems:
• TaqMan® reagents
• SYBR® Green reagents
• Other reagents

real-time PCR Process of collecting fluorescence data during PCR. Data from the real-time PCR are used
to calculate results for quantitation experiments or to troubleshoot results for genotyping
or presence/absence experiments.

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reference sample In relative standard curve and comparative CT (ΔΔCT) experiments, the sample used as
the basis for relative quantitation results. Also called the calibrator.

refSNP ID Identifies the reference SNP (refSNP) cluster ID. Generated by the Single Nucleotide
Polymorphism Database of Nucleotide Sequence Variation (dbSNP) at the National
Center for Biotechnology Information (NCBI). The refSNP ID can be used to search the
Thermo Fisher Scientific Store for an Thermo Fisher Scientific SNP Genotyping Assay.
Also called an rs number.

regression Values calculated from the regression line in standard curves, including the R2 value,
coefficients slope, and y-intercept. You can use the regression coefficients to evaluate the quality of
results from the standards. See also standard curve.

regression line In standard curve and relative standard curve experiments, the best-fit line from the
standard curve. Regression line formula:
CT = m [log (Qty)] + b
where m is the slope, b is the y-intercept, and Qty is the standard quantity.
See also regression coefficients.

reject well An action that the software performs during analysis to remove one or more wells from
further analysis if a specific flag is applied to the well. Rejected wells contain results
calculated up to the point of rejection.

relative standard Method for determining relative target quantity in samples. With the relative standard
curve method curve method, the StepOne™ software measures amplification of the target and of the
endogenous control in samples, in a reference sample, and in a standard dilution series.
Measurements are normalized using the endogenous control. Data from the standard
dilution series are used to generate the standard curve. Using the standard curve, the
software interpolates target quantity in the samples and in the reference sample. The
software determines the relative quantity of target in each sample by comparing target
quantity in each sample to target quantity in the reference sample.

Remote Monitor A feature in the StepOne™ software that allows you to monitor a StepOne™ or
StepOnePlus™ instrument over the network. With the Remote Monitor, you can monitor
the instrument status, send an experiment to the instrument, monitor amplification plots
and temperature plots in real time, and download the results to your computer. You cannot
operate the StepOne™ or StepOnePlus™ instrument using the Remote Monitor.

replicate group A set of identical reactions in an experiment.

replicates Total number of identical reactions containing identical components and identical
volumes.

reporter Fluorescent dye used to detect amplification. If you are using TaqMan® reagents, the
reporter dye is attached to the 5′ end. If you are using SYBR® Green reagents, the reporter
dye is SYBR® Green dye.

reverse primer An oligonucleotide that flanks the 3′ end of the amplicon. The reverse primer and the
forward primer are used together in PCR reactions to amplify the target.

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reverse An enzyme that converts RNA to cDNA. Reverse transcriptase is added to the PCR
transcriptase reaction to perform 1-step RT-PCR.

Rn See normalized reporter (Rn).

ROX™ dye A dye supplied by Thermo Fisher Scientific and precalibrated on the StepOne™ and
StepOnePlus™ systems. ROX dye is used as the passive reference.

rs number See refSNP ID.

run method Definition of the reaction volume and the thermal profile for the StepOne™ or
StepOnePlus™ instrument run.

sample The template that you are testing.

Sample DNA (10✕) In the StepOne™ software, a reaction component displayed on the Reaction Mix
Calculations tab of the Reaction Setup screen. The software assumes the sample DNA is
added to the reaction mix at a 10✕ concentration. For example, if the reaction volume is
20 μL, the calculated volume of sample for 1 reaction is 2 μL.

Sample Library In the StepOne™ software, a collection of samples. The Sample Library contains the
sample name and the sample color.

Sample or Standard In the StepOne™ software, a reaction component displayed on the Reaction Mix
(10✕) Calculations tab of the Reaction Setup screen. The software assumes the sample or
standard is added to the reaction mix at a 10✕ concentration. For example, if the reaction
volume is 20 μL, the calculated volume of sample or standard for 1 reaction is 2 μL.

sample/SNP assay In genotyping experiments, the combination of which sample to test and which SNP assay
reaction to perform in one PCR reaction. Each PCR reaction can contain only one sample and one
SNP assay.

sample/target In quantitation experiments, the combination of which sample to test and which target to
reaction detect and quantify in one PCR reaction. In the Design Wizard, you can detect and
quantify only one target in one PCR reaction. Use Advanced Setup to detect and quantify
more than one target in one PCR reaction.

serial factor In the StepOne™ software, a numerical value that defines the sequence of quantities in the
standard curve. The serial factor and the starting quantity are used to calculate the
standard quantity for each point in the standard curve. For example, if the standard curve
is defined with a serial factor of 1:10 or 10✕, the difference between any 2 adjacent points
in the curve is 10-fold.

series See standard dilution series.

slope Regression coefficient calculated from the regression line in the standard curve. The slope
indicates the PCR amplification efficiency for the assay. A slope of −3.32 indicates 100%
amplification efficiency. See also amplification efficiency (EFF%) and regression line.

SNP Abbreviation for single nucleotide polymorphism. The SNP can consist of a base
difference or an insertion or deletion of one base.

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SNP assay Used in genotyping experiments, a PCR reaction that contains primers to amplify the SNP
and two probes to detect different alleles.

SNP Assay Library In the StepOne™ software, a collection of SNP assays to add to genotyping experiments.
The SNP assays in the library contain the SNP assay name, SNP assay color, and for each
allele, the allele name or base(s), reporter, quencher, and allele colors. The SNP assays in
the library may also contain the assay ID and comments about the SNP assay.

spatial calibration Type of StepOne™ and StepOnePlus™ system calibration in which the system maps the
positions of the wells in the sample block(s). Spatial calibration data are used so that the
software can associate increases in fluorescence during a run with specific wells in the
reaction plate.

stage In the thermal profile, a group of one or more steps. There are three types of stages:
holding stage (including pre-PCR read and post-PCR read), cycling stage (also called
amplification stage), and melt curve stage.

standalone layout A system layout in which the StepOne™ or StepOnePlus™ instrument is not connected to
a computer by the yellow cable. In this layout, you control the instrument only with the
instrument touchscreen, and you use a USB drive or network connection to transfer data
between the instrument and computer.

standard Sample that contains known standard quantities. Standard reactions are used in
quantitation experiments to generate standard curves. See also standard curve and
standard dilution series.

standard curve In standard curve and relative standard curve experiments:

• The best-fit line in a plot of the CT values from the standard reactions plotted
against standard quantities. See also regression line.
• A set of standards containing a range of known quantities. Results from the
standard curve reactions are used to generate the standard curve. The standard curve
is defined by the number of points in the dilution series, the number of standard
replicates, the starting quantity, and the serial factor. See also standard dilution
series.

standard curve Method for determining absolute target quantity in samples. With the standard curve
method method, the StepOne™ software measures amplification of the target in samples and in a
standard dilution series. Data from the standard dilution series are used to generate the
standard curve. Using the standard curve, the software interpolates the absolute quantity
of target in the samples. See also standard and standard curve.

standard dilution In standard curve and relative standard curve experiments, a set of standards containing a
series range of known quantities. The standard dilution series is prepared by serially diluting
standards. For example, the standard stock is used to prepare the first dilution point, the
first dilution point is used to prepare the second dilution point, and so on. In the StepOne™
software, the volumes needed to prepare a standard dilution series are calculated by the
number of dilution points, the number of standard replicates, the starting quantity, the
serial factor, and the standard concentration in the stock. See also standard curve.

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standard quantity A known quantity in the PCR reaction.

• In standard curve experiments, the quantity of target in the standard. In the
StepOne™ software, the units for standard quantity can be for mass, copy number,
viral load, or other units for measuring the quantity of target.
• In relative standard curve experiments, a known quantity in the standard. Standard
quantity can refer to the quantity of cDNA or the quantity of standard stock in the
PCR reaction. The units are not relevant for relative standard curve experiments
because they cancel out in the calculations.

starting quantity When defining a standard curve in the StepOne™ software, corresponds to the highest or
lowest quantity.

step A component of the thermal profile. For each step in the thermal profile, you can set the
ramp rate (ramp increment for melt curve steps), hold temperature, hold time (duration),
and you can turn data collection on or off for the ramp or the hold parts of the step. For
cycling stages, a step is also defined by the AutoDelta status. With StepOnePlus™
systems, which contain the VeriFlex™ blocks, each step contains 6 temperatures (1 for
each VeriFlex block).

study name Entered during study setup, the name that is used to identify the study. Study names
cannot exceed 100 characters and cannot include any of the following characters: forward
slash (/), backslash (\), greater than sign (>), less than sign (<), asterisk (*), question mark
(?), quotation mark ("), vertical line (|), colon (:), semicolon (;), and sign (&), percent sign
(%), dollar sign ($), at sign (@), circumflex (^), left parenthesis ( ( ),
right parenthesis ( ) ), or exclamation point (!).

SYBR® Green PCR reaction components that consist of two primers designed to amplify the target and
reagents SYBR® Green dye to detect double-stranded DNA.

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system dye Dye supplied by Thermo Fisher Scientific and precalibrated on the StepOne™ or
StepOnePlus™ system. Before you use system dyes in your experiments, make sure the
system dye calibration is current in the Instrument Maintenance Manager.
System dyes on the StepOne™ system:
• FAM™ dye
• JOE™ dye
• ROX™ dye
• SYBR® Green dye
• VIC® dye
System dyes on the StepOnePlus™ system:
• FAM™ dye
• JOE™ dye
• NED™ dye
• ROX™ dye
• SYBR® Green dye
• TAMRA™ dye
• VIC® dye

IMPORTANT! We do not recommend the use of TAMRA™ dye as reporter or quencher

with the StepOne™ system. TAMRA dye may be used as a reporter or quencher with the
StepOnePlus™ system.

TaqMan® reagents PCR reaction components that consist of primers designed to amplify the target and a
TaqMan® probe designed to detect amplification of the target.

target The nucleic acid sequence that you want to amplify and detect.

target color In the StepOne™ software, a color assigned to a target to identify the target in the plate
layout and analysis plots.

Target Library In the StepOne™ software, a collection of targets to add to experiments. The targets in the
library contain the target name, reporter, quencher, and target color. The target in the
library may also contain comments about the target.

task In the StepOne™ software, the type of reaction performed in the well for the target or SNP
assay. Available tasks:
• Unknown
• Negative Control
• Standard (standard curve and relative standard curve experiments)
• Positive control (genotyping experiments)
• IPC (presence/absence experiments)
• Blocked IPC (presence/absence experiments)

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technical replicate Reactions that contain identical components and volumes, and that evaluate the same
sample.

temperature plot In the StepOne™ software, a display of temperatures for the sample, instrument cover, and
instrument block during the StepOne™ or StepOnePlus™ instrument run.

template In the Design Wizard of the StepOne™ software (and in QuickStart for quantitation
experiments), the type of nucleic acid to add to the PCR reaction. The recommended
template varies according to experiment type:
• Quantitation experiments (standard curve, relative standard curve, and comparative
CT) – cDNA (complementary cDNA), RNA, or gDNA (genomic DNA)
For quantitation experiments, the template type selection affects the run method,
reaction setup, and materials list.
• Genotyping experiments – Wet DNA (gDNA or cDNA) or dry DNA (gDNA or
cDNA)
For genotyping experiments, the template type selection affects the reaction setup.
• Presence/absence experiments - DNA
For presence/absence experiments, we recommend adding DNA templates to the
PCR reactions.

thermal profile Part of the run method that specifies the temperature, time, ramp, and data collection
points for all steps and stages of the StepOne™ or StepOnePlus™ instrument run.

threshold 1. In amplification plots, the level of fluorescence above the baseline and within the
exponential growth region The threshold can be determined automatically (see
automatic CT) or can be set manually (see manual CT).

2. In presence/absence experiments, the level of fluorescence above which the

StepOne™ software assigns a presence call.

threshold cycle (CT) The PCR cycle number at which the fluorescence meets the threshold in the amplification
plot.

Tm See melting temperature (Tm).

touchscreen Instrument display that you touch to control the StepOne™ or StepOnePlus™ instrument.

unknown In the StepOne™ software, the task for the target or SNP assay in wells that contain the
sample you are testing:
• In quantitation experiments, the task for the target in wells that contain a sample
with unknown target quantities.
• In genotyping experiments, the task for the SNP assay in wells that contain a
sample with an unknown genotype.
• In presence/absence experiments, the task for the target in wells that contain a
sample in which the presence of the target is not known.

unknown-IPC wells In presence/absence experiments, wells that contain a sample and internal positive control
(IPC).

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VeriFlex™ The StepOnePlus™ instrument contains six independently thermally regulated VeriFlex™
Technology blocks, creating up to six different zones for the 96 sample wells. After you enable the
VeriFlex blocks in the StepOne™ software, you can set a different temperature for one or
more of the VeriFlex blocks.

y-intercept In the standard curve, the value of y where the regression line crosses the y-axis. The y-
intercept indicates the expected threshold cycle (CT) for a sample with quantity equal to 1.

zone One of up to six sample temperatures among the 96 wells formed by independently
thermally regulated VeriFlex™ blocks during the StepOnePlus™ instrument run. You can
set a different temperature for one or more of the VeriFlex blocks, or you can set the same
temperature for each of the VeriFlex blocks.

Note: For melt curve steps, you need to set the same temperature for each of the
VeriFlex blocks.

zone boundary The edge of a zone for samples formed by the six independently thermally regulated
VeriFlex™ blocks. In the StepOne™ software, the zone boundaries are displayed in the
plate layout as thick red lines.

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A backup storage devices 31

baseline 170
advanced setup 169
baseline-corrected normalized reporter (ΔRn) 171
AIF. See assay information file.
biohazardous waste, handling 21
allele 169
bleach solution, usage 145
allelic discrimination plot 169
blocked IPC 171
amplicon 169
bold text, use of 12
amplification 169
plot 169
stage 170 C
amplification efficiency (EFF%) 169 calibration
analyze background 122–127
background calibration 126 dye (custom) 139–141
dye calibration 135–138 dye (system) 128–137
RNase P experiment 70–72, 96–98 schedule 116
spatial calibration 121 spatial 118–121
antivirus software 32, 74, 99 calibrator 171
APIPA. See Automatic Private IP Addressing. CAUTION, description 15
Applied Biosystems chemical
contacting 14 safety 19
Technical Support 14 waste safety 21
temperature verification service 117 chemistry 171
Applied Biosystems StepOne and StepOnePlus Real- chemistry installation kit 42
Time PCR System. See instrument or system.
clearance requirement, instrument 39
archiving files 142
colocated layout 33, 171
assay 170 connect system components 61–63
ID 170 connect to a network 102
mix 170 example network layout 103
assay information file (AIF) 170 install computer 52–59
AutoDelta 170 installation 50–74
automatic baseline 170 installation workflow 50
perform RNase P experiment 65–73
automatic CT 170
when to install 50
Automatic Private IP Addressing compatibility 106
comparative CT (ΔΔCT) method 172
computer
B connect to instrument 61
back up experiments 142 default user name and password 55, 80
background calibration 122–127 monitor instrument 110
analyze the data 126 name, define 58, 83
guidelines 122 placement 53, 79
perform 124 prepare operating system 56–58, 81–83
prepare 123–124 remote monitoring set up 108
purpose 122 requirements 30
site preparation 53, 79
background component, about 122
Computer Port
background plate
about 104
about 122
connect the computer and instrument 54
create 127
number of reuses 123 connect, instrument to computer 61
contamination, removal 145–147

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conventions dyes
bold text 12 custom 130, 172
for describing menu commands 12 system 28, 129, 182
IMPORTANTS! 12
in this guide 12
italic text 12
E
Notes 12 EFF%. See amplification efficiency.
safety 15 electrical protective devices 31
user attention words 12 electrical safety 22
create electromagnetic compatibility standards.
background plate 127 See EMC standards
custom dye plate 139–141
EMC standards 24
CT. See threshold cycle. encryption software 32, 74, 99
custom dye
endpoint read 173
about 172
calibration 139–141 ergonomics, safety 23
plate, how to create 139–141 experiment 173
cycle threshold 172 download from instrument 112
RNase P. See RNase P experiment.
cycling stage 172 send to instrument 112
type 174
D experiment workflow
DANGER, description 15 colocated layout 33
standalone layout 34
data
about collection 27, 172
backup/storage devices 31 F
date and time settings FAM™ system dye 28, 129
define for the instrument 45 spectral profile 137, 138
define for the software 57, 82
file compression software 32, 74, 99
delta Rn (ΔRn) 172
firewall software 32, 74, 99
derivative reporter (- Rn′) 172
forward primer 174
Design Wizard 172
diluent 172
diluted sample concentration 172
G
dilution factor 173 guidelines
background calibration 122
dimensions
bleach solution, usage 145
computer 53, 79
chemical safety 19
instrument 39 chemical waste disposal 21
disable 'Local Area Connection' messages 63 chemical waste safety 21
disk space, checking 142 lifting and moving 40, 53, 79
dissociation curve 173 networking 105
documentation remote monitoring 110
obtain 14
related 13 H
shipped with the system 43
hardware, optional 31
dye calibration 128–137
analyzing data 135 hazard icons. See safety symbols, on instruments.
analyzing the data 135–138 hazard symbols. See safety symbols, on instruments
perform 134–135 hazards. See safety
prepare 132–133 Help system, accessing 13
purpose 128 holding stage 174
spectra, evaluating 131
housekeeping gene 174
dye plate
humidity requirement
about 128
computer 53, 79
create 139
instrument 39
custom dye 128

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I IPC 174
IPC blocking agent 174
IMPORTANT, description 15
IPC+ 174
incompatibilities, testing third-party software 32
IPv4 link-local (IPV4LL) 104
installation
category, overvoltage rating 22 italic text, when to use 12
colocated layout 50–74
instrument 38–47 J
network 102–114
operating system updates 154 JOE™ system dye 28, 129
preparation 29 spectral profile 137, 138
required materials 30
service pack updates 155 K
site preparation 39, 53, 79
kits provided
software 56–59, 81–84
installation chemistry kit 42
software, administrator rights requirement 59, 84
packing kit 41–42
software, third-party 32, 74, 99
software and documentation kit 43
standalone layout 76–99
third-party software 32
using this guide 29 L
Windows service pack updates 155
LAN Port
workflow 35, 38
about 104
installation kit define IP settings 107
chemistry kit 42 layout. See colocated layout or standalone layout.
packing kit 41–42
software and documentation kit 43 lifting guidelines 40, 53, 79
installation specification 66, 71, 89, 97 line conditioner, requirements 31
instrument load, instrument 68, 91
Automatic Private IP Addressing 106 local area connection messages, disable 63
background calibration 122–127
Computer Port 104 M
connect to a network 106
connect to computer 61 MAC address, instrument 106
define settings 45 made-to-order assays 174
download experiment from instrument 112 maintenance
dye calibration 128–137 archiving files 142
dye calibration (custom) 139–141 back up, experiments 142
dye spectra 137, 138 decontamination 145–147
installation 38–47 disk space, checking 142
IPV4LL 104 infrequent procedures 144
LAN Port 104 notifications 116
load 68, 91 optics verification 117
MAC address 106 required connection 116
maintenance data 117 run cycle performance test 117
maintenance schedule 116 schedule 116
mDNS/DNS compatibility 104 statistics, instrument 117
placement 40 temperature verification service 117
send experiment to instrument 112 using this guide 29
set up 44 view information 117
site preparation 39 managed data network service. See mDNS.
spatial calibration 118–121 manual baseline 174
temperature verification service 117
unload 73, 93 manual CT 174
instrument name, define 45 materials required, to
analyze the RNase P experiment 93
instrument operation, safety 18 create a background plate 127
internal positive control (IPC) 174 decontaminate the sample block(s) 145
inventoried assays 174 install the system 30
IP settings, LAN Port 107 move the instrument 148

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network the instrument 106 P

perform a background calibration 122
perform a custom dye calibration 139 packing kit, contents 41–42
perform a dye calibration 128 packing material, removal 45
perform a spatial calibration 118 passive reference 176
perform an RNase P experiment 65, 88 password, default for computer 55, 80
replace the instrument fuses 150 perform
set up remote monitoring 108 background calibration 124
set up the computer 52, 78 dye calibration 134–135
ship the instrument 152 RNase P experiment 68, 91
materials, shipped with the system 41 spatial calibration 120
mDNS/DNS compatibility 104 placement
Media Access Code address. See MAC address. computer 53, 79
melt curve stage 175 instrument 40
melt curve 175 plate layout 176
melting temperature (Tm) 175 plates
menu commands, conventions for describing 12 background plate 122
background plate, custom 127
monitor the instrument. See remote monitoring.
MicroAmp plates 41, 42
moving and lifting, safety 18 number of reuses 123
moving guidelines 40, 53, 79 RNase P plate 42, 65, 88
multicomponent plot 175 spectral calibration plate 118, 128
point 176
N positive control 176
post-PCR read 176
NED® system dye 28, 129
spectral profile 138 power line regulator 31, 52, 78
negative control (NC) 175 power options settings, for the software 56, 81
negative control-blocked IPC wells 175 power requirement
negative control-IPC wells 175 computer 53, 79
instrument 39
network 102–114
preparation, system installation 29
example layouts 103
guidelines 105 prepare
set up workflow 102 background calibration 123–124
setup 102 dye calibration 132–133
using this guide 29 RNase P experiment 67, 90
spatial calibration 118–119
no amplification control (NAC) 175
no template control (NTC) 175 pre-PCR read 176
nonfluorescent quencher-minor groove binder (NFQ- primer mix 176
MGB) 175 primer/probe mix 176
normalized quantity 175 protective devices, electrical 31
normalized reporter (Rn) 175 pure dye calibration. See dye calibration.
notification settings, enable 113 pure dyes 176

O Q
omit well 70, 96, 175 quantitation method 176
online Help. See Help system quantity 176
operating system quencher 177
prepare 56–58, 81–83 QuickStart 177
update 154
optics verification 117 R
optimization software 32
R2 value 72, 98, 177
outlier 175
radioactive waste, handling 21
outliers 70, 96
ramp 177
overvoltage category (rating) 22

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raw data plot 177 labels, on instruments 17

reaction mix 177 moving and lifting instrument 18
reagents 177 moving/lifting 18
repetitive motion 23
real-time PCR 177 standards 24
reference sample 178 symbols, on instruments 16
refSNP ID 178 workstation 23
regression sample 179
coefficients 178 sample block(s), decontaminate 145–147
line 178
Sample DNA (105) 179
regulator, power line 31, 52, 78
Sample Library 179
reject well 178 Sample or Standard 179
relative standard curve method 178 sample/SNP assay reaction 179
remote monitoring 102, 178
sample/target reaction 179
enable notification settings 113
guidelines 110 screen resolution, for the software 57, 82
monitor instrument 110 screen saver, for the software 56, 81
set up (computer) 108 SDSs
removing outliers from RNase P runs 70, 96 description 19
repetitive motion, safety 23 obtaining 14, 20
replicate group 178 security software 32, 74, 99
replicates 178 serial factor 179
reporter 178 series 179
requirements service pack, updates 154
computer 30, 53, 79 set up
instrument 39 computer 53, 78
See also materials required. instrument 44
reverse primer 178 site requirements
reverse transcriptase 179 computer 53, 79
instrument 39
Rn. See normalized reporter.
slope 179
RNase P experiment 65, 88
analyze 70–72, 96–98 SNP 179
materials required 65, 88 SNP assay 180
perform 68, 91 SNP Assay Library 180
prepare 67, 90 software
purpose 65, 88 administrator rights requirement 59, 84
R2 value 72, 98, 177 antivirus 32, 74, 99
transfer data 94 encryption 32, 74, 99
when to perform 65, 88 file compression 32, 74, 99
ROX™ system dye 28, 129, 179 firewall 32, 74, 99
spectral profile 137, 138 installation 56–59, 81–84
rs number 179 installation (third-party) 32
run cycle performance test 117 installation, administrator rights requirement 59, 84
optional 31
run method 179
prepare for use 86
prepare operating system 56–58, 81–83
S shipped with the system 43
system optimization 32
safety
before operating the instrument 18 software, third-party 32
biological hazards 23 spatial calibration 118–121, 180
chemical 19 analyzing the data 121
chemical waste 21 perform 120
conventions 15 prepare 118–119
electrical 22 purpose 118
ergonomic 23 specification, installation 66, 71, 89, 97
guidelines 19, 21 spectrum, system dyes 137, 138
instrument operation 18

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stage 180 test

standalone layout 180 optics verification 117
connect to a network 102 run cycle performance 117
description 34 third-party software for incompatibilities 32
example network layout 103 text conventions 12
installation 76–99 thermal profile 183
installation workflow 76 third-party software 32
when to install 76
threshold 183
standard
threshold cycle CT 183
curve 180
curve method 180 Tm. See melting temperature.
standard dilution series 180 Tools menu, maintenance tests 117
standard quantity 181 touchscreen 183
standard 180 training, information on 14
standards transfer data 94
EMC 24 download experiment from instrument 112
safety 24 send experiment to instrument 112
starting quantity 181
statistics, instrument 117 U
step 181 uninterruptable power supply, requirements 31, 44, 52,
storage devices, backup 31 78
surge protector, requirements 31, 44, 52, 78 unknown 183
SYBR® Green I dsDNA Binding Dye 28, 129 unknown-IPC wells 183
reagents 181 unload, instrument 73, 93
spectral profile 137, 138 update
symbols, safety 16 operating system 154
system service packs 154
chemistry kit 42 UPS, requirements 31, 44, 52, 78
components 41 USB drive, transfer data 34, 94
data collection 27
user attention words, described 12
documentation 43
packing kit 41–42 user name, default for computer 55, 80
software 43 using this guide
system dyes 129, 137, 138, 182 installation 29
maintenance 29
networking 29
T
TAMRA® dye 28, 129 V
spectral profile 138
VIC® system dye 28, 129
TaqMan® Fast Universal PCR Master Mix, No
spectral profile 137, 138
AmpErase® UNG 65, 88
®
TaqMan RNase P Instrument Verification Plate 42, 65,
88 W
See also RNase P experiment. WARNING, description 15
TaqMan™ reagents 182 waste disposal, guidelines 21
target 182 waste profiles, description 21
color 182 weight
library 182 computer 53, 79
task 182 instrument 39
Technical Support, contacting 14 Windows operating system
temperature operating system upgrades, installation 155
plot 183 service pack updates, installing 155
requirement, computer 53, 79 workflow
requirement, instrument 39 install the instrument 38
verification service 117 install the system 35
template 183 workstation safety 23

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Y
y-intercept 184

Z
zone 184
zone boundary 184

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems 191

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192 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems

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For support visit thermofisher.com/support or email techsupport@lifetech.com
thermofisher.com

08 March 2016