Biol3140 Lab Manual

SC/BIO3140 (Fall 2017)

Laboratory Manual

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 Biol3140 Lab Manual

Laboratory Overview

Lab 1 - Lab techniques (5%)

Introduction, including pipetting and standard curve

Lab 2 - Protein analysis module (20%)

Protein Analysis for alpha Amylase:
Learn principles of SDS-PAGE and Western blotting.
Run SDS-PAGE and Western blot.
Interpret the results of SDS-PAGE and Western blot.
Understand applications of SDS-PAGE and Western blot.

Lab 3 - Restriction Mapping of -Amylase Plasmid DNA (5%)

Restriction enzyme digest of the amylase containing plasmid
DNA gel electrophoresis of cleavage products
Tutorial on assembly of restriction map

Lab 4 - DNA Cloning and analysis module (20%)

Learn the principle of PCR.
Set up a PCR reaction.
Genomic DNA and plasmid DNA extraction (difference and similarity).
Learn cloning strategy and procedures.
Learn advantages, limitations and applications of PCR based gene cloning.
Learn advantages, limitations and applications of gene cloning using a genomic library.
Verification of the cloned gene.

Lab 5 - Genetic mapping and Southern blot (15%)

Review the basic concept of genetic mapping.
Learn techniques used to do genetic mapping.
Learn applications of Southern Hybridization.
Analyze a DNA Sequencing gel.
Use bioinformatics technology to examine unknown DNA fragment.

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Laboratory Schedule
(Note! Lab content is subject to change)

Week 1: Introduction to Laboratory Techniques & Protein Analysis Module

DAY 1:
Learn to use a micropipettor by measuring the mass of water, ethanol and glycerol with a balance
Maltose Standard Curve

DAY 2:
TA Demo: SDS-PAGE gel preparation, followed by.....
SDS-PAGE set up

Week 2: Protein Analysis for -amylase

DAY 1:
SDS-PAGE sample preparation
Run SDS-PAGE gel #1
Transfer to PVDF membrane overnight for Western blot analysis

DAY 2:
Western Blot detection
Preparation of SDS-PAGE gel #2


Week 3 & 4: Coomassie Analysis of -amylase proteins and Chromosomal DNA extraction
and Restriction Mapping of -Amylase Plasmid DNA

DAY 1:
SDS-PAGE and Coomassie Blue staining

DAY 2:
Restriction enzyme digest of the amylase containing plasmid

Week 3 & 4: Coomassie Analysis of -amylase proteins and Chromosomal DNA extraction
and Restriction Mapping of -Amylase Plasmid DNA

DAY 1:
DNA gel electrophoresis of cleavage products

DAY 2:
Genomic DNA isolation from B.licheniformis

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Week 5: DNA electrophoresis of Bacillus genomic DNA, PCR of the -amylase gene &
restriction enzyme digest of the -amylase PCR product & B.licheniformis genomic DNA

DAY 1:
DNA gel electrophoresis of Bacillus genomic DNA
Spectrophotometric determination of Bacillus genomic DNA concentration

DAY 2:
PCR of the -amylase gene from B.licheniformis genomic DNA
-amylase PCR product cleanup
Restriction enzyme digest of PCR of the -amylase gene using genomic DNA extracted from
B.licheniformis


Week 6: Restriction enzyme digest of the -amylase PCR product and
B.licheniformis genomic DNA & Digestion reaction cleanup, ligation of the products

Day 1:
Restriction enzyme digest of the -amylase PCR product and pET15b with BamHI
Agarose gel electrophoresis of a) -amylase PCR product and pET15b double digest and b)
genomic DNA extracted from B.licheniformis and pRL498 digested with HindIII

DAY 2:
Digestion reaction cleanup for PCR product (double digest) and the genomic DNA digested with
HindIII
Ligation reaction for both PCR product and the genomic DNA digested with HindIII

Week 7: Study / writing time.

Week 8: Colony PCR

DAY 1:
Colony PCR and agarose gel electrophoresis for screening of positive clones
Staining of the LB-KAN+-Starch plates with Lugol's iodine solution for detection of -amylase
activity in bacterial colonies

DAY 2:
Free day for "catch-up" experiments as needed.


Week 9: Enzyme activity of -amylase clones

DAY 1:
Restriction enzyme digest of genomic DNA from B.licheniformis for southern hybridization
PCR labeling of probe DNA with Biotin

DAY 2:
Measure the enzyme activity of a culture of cells that express the -amylase enzyme

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Week 10: Southern Blot Hybridization

DAY 1:
Agarose gel electrophoresis
First part of Southern Blot Transfer
Read DNA sequence autoradiogram

DAY 2:
Dot Blot preparation
Finish Southern blot transfer


Week 11: Southern Blot Hybridization, continued

DAY 1:
Hybridization of the Southern Blot nylon membrane

DAY 2:
Washing of the Southern Blot nylon membrane and detection

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Laboratory Reports

Upon completion of each of the following laboratory sets a full and detailed report must be
submitted. Further details of how reports should be written will be given in class. TAs will
announce exact deadlines for handing in lab reports. Late reports will be deducted 25% for
up to 24 hours and a further 10% per day each subsequent day.

Lab 1 - Introduction to Laboratory Techniques (5%)

Lab 2 - Protein analysis module (20%)

Lab 3 - Restriction Mapping of -Amylase Plasmid DNA (5%)

Lab 4 - DNA Cloning and analysis module (20%)

Lab 5 - Genetic mapping & Southern blotting (15%)

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Laboratory #1
Week 1: Introduction to Laboratory Techniques:

Learn to use a micropipettor by measuring the mass of water, ethanol and glycerol with a
balance
Maltose Standard Curve

Brief overview:

A colorimetric enzyme assay will be used to measure the units of -amylase activity in samples
of human saliva and in commercial preparations of -amylase isolated from different species.
The students will also learn how to extrapolate information on unknown DNA size and DNA
concentration as well as how to use micropipettors properly by measuring the mass of different
chemicals with a balance.

Week 1 - Day 1: .

Students perform the following:

1. Learn to use a micropipettor by measuring the mass of water, ethanol and glycerol with a
balance
2. Maltose Standard Curve

Procedures:

Learn to use a micropipettor by measuring the mass of water, ethanol and glycerol
with a balance:
1. Label three 1.5 eppendorf tubes water, ethanol, and glycerol
2. Measure the weight of tubes.
3. Place 1mL of water, ethanol and glycerol into corresponding tubes.
4. Measure the weight of tubes.
5. Calculate the density of water, ethanol, and glycerol. Fill out the following table.
Water Ethanol Glycerol
Weight difference
(After Before)
Volume
Density

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MaltoseStandard Curve:

1. Label six conical bottom 15-ml tubes from 1 to 6 as indicated in the table below:
Tube 1 (Blank) 2 3 4 5 6
Water 2 ml 1.8 ml 1.6 ml 1.4 ml 1.2 ml 1 ml
Maltose 0 ml 0.2 ml 0.4 ml 0.6 ml 0.8 ml 1 ml
(2mg/ml)
Maltose 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml
Colour
Reagent

2. Add the appropriate volumes of water, maltose and maltose colour reagent into the tubes
3. Cap and vortex ALL tubes
4. Incubate the tubes at 100 0C for exactly 15 minutes.
5. Place the tubes on ice in order to achieve room temperature.
6. Add 9 ml of deionized water (dH2O) to each tube, tightly cap the tubes, and mix the tubes
by gently inverting several times. Aliquot 1 mL of mixture into standard cuvette.
7. Measure the absorbance at 540 nm wavelength using cuvettes in the spectrophotometer
and fill in the following table (first use tube 1 for the blank measurement):

Tube 1 (Blank) 2 3 4 5 6
A540nm
mg of 0 0.4 0.8 1.2 1.6 2
maltose
8. Calculate the mass (mg) of maltose in each sample and record the value in the table
9. Make the following plot:
X-axis: total mass (mg) of maltose
Y-Axis: A540nm absorbance
Generate a best fit line and determine the amount of maltose present in the
solutions on the basis of its absorbance

Lab 1 ends
Lab 2 begins

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Laboratory #2
Week 1 - Day 2: .

TA Demo: SDS-PAGE gel preparation, followed by;

Students perform the following:

1. SDS-PAGE set up

SDS-PAGE set up:

You will learn and cast Polyacrylamide gel in order to use it later (Week 2, Day 1).The type of
Polyacrylamide Gel Electrophoresis you will perform during Week 2 and Week 3 activities
employs the most popular discontinuous buffer system (SDS-PAGE buffer system by Laemmli),
therefore you need to cast a gel which consists of two portions: 10% Separating (or Resolving, or
Running) SDS-Polyacrylamide Gel and 4% Stacking SDS-Polyacrylamide Gel.

Your TA will provide a demonstration of how to make SDS-PA gel. Pay close attention to TA
demo, and all hints and notes in the following protocol to ensure a trouble-free gel casting. The
temperature of ingredient solutions, the order of mixing and the timing and way of dealing with
the monomer solution while pouring are all important factors for success.

Warning!
Acrylamide is a NEUROTOXIN. It can be absorbed through unbroken skin. Thus, exercise
caution when handling unpolymerized acrylamide: wear gloves during the entire process of
casting your gel.
Also, do NOT pour unpolimerized acrylamide down the sink! Any liquid leftovers must be
collected in the Acrylamide Waste container located in the Fume Hood.

Procedure:

1. First, assemble your gel cassette using glass plates (Short plate and 1.5 mm Spacer plate),
Casting Frame and Casting Stand according to your TAs instructions. Also, you may find
useful a copy of BIO-RAD Assembly guide provided to you.
Note: we use two (gray and green) gaskets instead of just one gray.
Note: make sure both glass plates (Short plate and 1.5 mm Spacer plate) are absolutely
clean. If not, use Windex glass cleaning solution provided in Spray bottles and
KimtechWipes.

2. Next, check your gel cassette for leaks (optional?) and mark the level to which Separating gel
is poured.
Can be done by several ways:
a) Fill the assembled gel cassette with 6.5 mL of dH2O to test whether it is sealed properly
and also to mark the level of water with a marker. Then drain the water by carefully tilting
the entire assembly on its side or even inverting it up-side down over your liquid waste

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container at your workstation. Use KimtechWipes to pad dry any remaining water. Let
your gel cassette dry.
Note: 6.5 mL is the volume of Separating gel to be poured into gel cassette with 1.5 mm
Spacer Plate. It was determined for you by the Laboratory Technician.
Note: With a leak occurring, you have to disassemble the gel cassette, pad dry plates
and start again.
b-c) Insert a comb into the gel cassette. Mark the Short plate 1 cm below the comb teeth.
Remove the comb. Now check for leaks with water (no need for measuring exact volume)
or take a risk and proceed without leak proofing (not recommended though).

1. It is time to get ingredients needed to make a gel (stored at 40C) and place them on ice. Each
group is provided with 5 aliquots of solutions.
Note: Volumes of reagents aliquoted for you are designed to be enough to make one gel.
If you need to re-do gel casting, use spare aliquots.
Note: TEMED is located in the Fume Hood. Do not take it out, simply add TEMED into
your solution over there.

2. Gel Casting.
A)-Prepare the Separating gel monomer solution by combining all reagents except 10%APS
and TEMED.
-Add them into a 125 mL Separating gel flask in an order corresponding to the order of
ingredients listed in the Table below.

Table SDS-PAGE Formulations for casting one gel (1.5 mm thickness).

Ingredients 10 % Separating gel 4% Stacking gel

dH2O *(adjust accordingly) 2.95 mL 2.7 mL

1.5M Tris-HCl, pH 8.8 1.9 mL N/A

1.0M Tris-HCl, pH 6.8 N/A 0.47 mL

30% Acrylamide/Bis 2.5 mL 0.5 mL

10% SDS 75 L 37.5 L

10% APS 75 L (or 37.5 L?) 37.5 L (or 18.75 L?)

TEMED** 3.75 L 3.75 L

Total Volume ~7.5 mL ~3.75 mL

*Volume of dH2O is 2.9875mL = ~2.988mL, if your TA recommends to use 37.5 L of 10% A*PS.

**In Total Volume calculation the volume of TEMED is ignored.

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-Mix the contents of the flask by careful swirling few times.

-Immediately prior to pouring the gel, add 10% APS (the initiator of polymerization) and
TEMED (the catalyst).
-Swirl GENTLY (no more than three short times!) to mix and initiate polymerization.
Note: Remember, vigorous/energetic mixing will introduce not wanted air bubbles with
oxygen (inhibitor of polymerization) into your solution.
-Immediately pour the solution into gel cassette to fill it up to the marked level using
disposable transfer pipette. Do it carefully and smoothly to prevent it from mixing with air.
-Overlay this layer with Isopropanol. Add it VERY SLOWLY and EVENLY to prevent mixing.
Isopropanol will exclude oxygen and ensure a flat interface between the Separating and
Stacking gels.
-Allow the gel to polymerize for 35 to 45 minutes. A line will become visible at the top of the
gel as it polymerizes.
-Pour off the alcohol overlay into your liquid waste container at your workstation. Rinse the
gel surface with dH2O and drain the water by inverting the gel.
Note: At this point, you have a choice (well, your TA will let you know) to postpone
making the Stacking gel until the next week. If you need to stop, just add 2 ml of diluted
1:4 1.5M Tris-HCl, pH 8.8 on top of the Separating gel to keep it hydrated, cover it with
comb, put in a few damp brown paper towels, place in a Zipper bag and store at 4 0 C
(upright in a pegged rack).

B)-Prepare the Stacking gel monomer solution.

-Add required ingredients (except 10% APS and TEMED) into 25 mL Stacking gel flask in
a-proper order.
-Swirl gently to mix.
-Insert a piece of filter paper to dry the area in between the glass plates above the Stacking
gel completely. Pay attention not touch the surface of the gel. (Optional)
-Add 10% APS and TEMED, swirl GENTLY to mix and immediately pour the solution
between glass plates (on top of the Separating gel) until the rim of the Short plate is
reached.
-Insert the comb into the gel cassette. Take care not to splash out the Stacking gel
monomer solution. It is easiest to insert a comb between spacers starting at an angle and
insert well 1, then well 2 and so on until it is completely inserted. Do it slowly, carefully and
try to avoid trapping air bubbles under the comb. Prepare Kimtech Wipes pads to collect
spills and pad dry at the bottom of your gel cassette.
-Let the Stacking gel to polymerize for 25 to 35 min.
-Cover your gel in a few damp brown paper towels to keep it hydrated and place in a Zipper
bag. Store at 4 0 C (upright in a pegged rack) to use it next week (Week 2, Day 1)
Note: Obviously, the last step is omitted, if you are making the Stacking portion of your
gel on the day of its use.
Note: At the day running SDS-PAGE, gently remove the comb and rinse wells thoroughly
with running buffer.

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Week 2: Protein Analysis for -amylase

SDS-PAGE sample preparation

Western Blotting and transfer
Western Blot detection
Coomassie Blue stain

Brief overview:

During this week students will use SDS-PAGE in order to analyze the protein composition of
several -amylase containing samples, i.e., crude protein lysates from Bacillus cells, human
saliva, and several preparations of commercially purified -amylases. Students will run identical
samples on two SDS-PAGE gels (prepared previously) and use one for Coomassie Blue staining
while the second will be used for Western blotting detection of the -amylase protein in crude
preparations.

DAY 1: Students perform the following:

1. SDS-PAGE sample preparation

2. Run SDS-PAGE gel #1 (prepared by students on Week 1 Day 2)
3. Transfer to PVDF membrane overnight for western blot analysis

DAY 2: Students perform the following:

1. Western Blot detection

2. Preparation of SDS-PAGE gel #2 for use during Week 3 Day 1 (students will follow TA
instruction whether or not to only prepare the separating gel and keep this portion in the
fridge for subsequence usage)

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Week 2 - Day 1 .

Procedures:

a) Preparation of the samples:

1. Each group is given a 1.5 ml tube of an overnight culture of Bacillus amyloliquefaciens

and a 1.5 ml tube of an overnight culture of Bacillus licheniformis. Centrifuge the cultures
at 13,000 rpm for 1-2 minutes to pellet the bacterial cells.
2. Discard the supernatant and add 100l of TE buffer (10mM Tris-HCl, pH 8, 1mM EDTA).
Vortex vigorously to resuspend the bacterial cells.
3. Pulverize the Bacillus cells with the glass beads by vortexing for 1 minute. Subsequently
place the tube on ice for 1 minute. Repeat this process 5 more times. Now that the
bacterial cells have been lysed proceed to the next step.
4. Spin down the lysate (13,000 rpm for 3 minutes) to ensure that the beads are in the
bottom of the tube and transfer the bacterial lysate into a new 1.5 ml eppendorf tube (only
the soluble component. Avoid the pellet at the bottom).
5. Label the new tubes as follows: B.amyloliquefaciens and B.licheniformis.
6. Transfer 15 l of bacterial lysate to a new tube and pipette another 15 l of SDS-PAGE
loading buffer. Mix well by pipetting and briefly vortex the sample for 15 seconds.
7. Collect some saliva (one from each partner labelled A and B) in the weighing cups
provided. Transfer your own saliva to a 1.5 ml eppendorf tube by using a 1 ml tip that has
a broad end (use the scissors provided to cut off the fine end of the tip-ask your TA for
help if you do not understand this step !!!)
8. Mix 15 l of the saliva with 15 l of SDS-PAGE loading buffer and vortex the sample for
15 seconds.
9. Add 15 l of the following samples to new, appropriately labeled 1.5 ml eppendorf tubes:
a) Commercial -amylase from Bacillus licheniformis
b) Commercial -amylase from Aspergillus oryzae
c) Commercial -amylase from porcine pancreas
d) Unknown sample #1
e) Unknown sample #2
10. Add 15 l of SDS-PAGE loading buffer and vortex the samples for 15 seconds.
11. Heat all the samples and the two protein molecular weight markers for 5 minutes at 95-
100C.
12. Spin down all the tubes @13,000 rpm for 1 minute to ensure that all the samples settle to
the bottom of the tubes (some of the sample evaporates due to the boiling!!!)

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b) Loading and running of the protein samples:

1. Wear gloves when handling polyacrylamide gels

2. Assemble the gel cassettes in the electrophoresis devices provided as depicted below:

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SDS-PAGE apparatus assembly instructions:

Step one: Fit gel into the inner frame. The long gel plate is facing towards you,
and the shorter one is facing away from you. It is important to get a tight seal
between the green gasket and the white frame on the gel. The gel and the buffer
dam on the other side will form the inner buffer chamber.
Step two: The buffer dam and the gel form the inner buffer chamber. The frame is
then slid into the support. Note that the gel is held in place to get a tight seal.
Once the frame is inserted, the levers are closed to LOCK the frame in place. The
levers must be closed at the same time. You should get some resistance.
Step three: Buffer is added to the inner chamber and students should check for a
tight seal. If there is no buffer leakage in the inner chamber proceed and add more
buffer (500 ml) (SDS-PAGE buffer, made in week 1) to the outer chamber. You
are now ready to proceed.
3. Remove any leftover gel and air bubbles from the wells by gently flushing the wells with
running buffer using your pipette tip or a Pasteur pipette.
4. Load 15 l of each sample using your micropipette in the following order:

Lane number (from left Contents

to right)
1 B.amyloliquefaciens lysate
2 B.licheniformis lysate
3 Commercial -amylase from Bacillus licheniformis

4 Commercial -amylase from Aspergillus oryzae

5 Commercial -amylase from porcine pancreas

6 Saliva sample A
7 Saliva sample B
8 Unknown sample #1

9 Unknown sample #2

10 Protein molecular weight marker

5. Run the gel at 125 Volts for 1 hour until the bromophenol blue dye is at the bottom of the
gel
6. While the gel is running prepare and activate your PVDF membrane. Typically the PVDF
membrane is soaked in methanol for 30 seconds. Then the PVDF membrane is soaked in
deionized water for 2 minutes. Lastly the membrane is place in cold transfer buffer for at
least 5 minutes prior to the transfer.

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c) Transferring of the proteins from the polyacrylamide gel to the PVDF membrane:

1. Soak the PVDF membrane in 50 ml of methanol (this will activate the membrane) for 30
seconds.
2. Collect the methanol and place the PVDF membrane in 100 ml of deionized water to rinse
off the methanol. Let the membrane sit in the water for exactly 2 minutes.
3. Place the PVDF membrane in 100 ml of ice cold transfer buffer for 5 minutes.
4. Now you are ready to proceed to the next step.
5. Assemble transfer sandwich by orientating cathode, fiber pad, filter paper, gel,
membrane, filter paper, fiber pad and anode so protein transfer goes in the direction of
cathode to anode (see illustrated diagram). Pay special attention to prevent or remove air
bubbles during assembly, as bubbles will cause interruptions in protein transfer (Anode is
typically color-coded in red, while cathode is typically color-coded in black. Place the
transfer sandwich into the transfer unit and place into the gel box. The transfer process
will generate heat, so to avoid band smearing, use a chilling unit, pre-chilled transfer
buffer, or carry out the transfer in a cold room.
6. Fill the unit with sufficient transfer buffer (see recipe) to cover the entire membrane(s).
7. Transfer for 1.5 hours at 110 V (the transfer can proceed at 30 V overnight (usually do
this in the cold room)).

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Week 2 - Day 2 .

d) Immunological detection of -amylase proteins:

1. When your transfer is finished, turn off and disconnect the power supply. Carefully
disassemble the sandwich and verify the presence of the pre-stained molecular weight
marker on the membrane. Do not allow the PVDF membrane to air dry.
2. Place the PVDF membrane in 25 ml of TBS buffer and let it equilibrate for 5 minutes.
3. Discard the TBS and add 30 mls of western blocking buffer (5% dry milk in TBST) (we do
this in order to minimize background) for 30 minutes hour at room temperature.
4. Discard the blocking buffer and incubate the membrane with 30 ml of primary antibody (1
Ab-primary antibody) solution. Add the primary antibody to membrane and incubate 1
hour at room temperature with shaking using the shakers provided in the laboratory.
5. Remove the primary antibody solution and wash membrane three times for 5 minutes.
6. Discard the TBST and incubate the membrane with the secondary antibody (2 Ab-
secondary antibody) solution for 1 hour at room temperature with shaking. Secondary
antibodies conjugated with horseradish peroxidase (HRPO) allow for rapid, cost efficient
and convenient detection.
7. Remove the 2 antibody solution and wash membrane three times for 5 minutes each in
TBST buffer.
8. Add 50 mls of AP reaction buffer to the PDVF membrane and GENTLY rock the
membrane for 2 minutes at room temperature.
9. Discard the AP reaction buffer and add 20 ml of AP substrate solution (BCIP/NBT) and
allow the reaction to proceed for 15-30 minutes in the dark (place your dish in your drawer
so that there is little light exposure) until you can visualize the bands on the gel.
10. To stop the reaction, discard the AP substrate solution (dH2O) and add 50 ml of AP stop
buffer.
11. Discard the AP stop solution and allow the membrane to air dry. Take an image of your
membrane for your laboratory report results section.

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Week 3 & 4: Coomassie Analysis of -amylase proteins and Chromosomal DNA

extraction:

Coomassie Blue stain of SDS-PAGE gel

DNA extraction from B.licheniformis

Brief overview:

Genes are segments composed of DNA and are located on chromosomes. The -amylase gene
is part of the genomic DNA in many different strains of Bacillus. The size of the Bacillus genome
is approximately 4000 kb. The -amylase gene is 1.6 kb in size. The isolation of genomic DNA is
the first step in the cloning of this gene and the subsequent laboratory exercises. In addition, this
week covers the analysis of -amylase proteins by use of Coomassie Blue staining which allows
for the visualization of all of the polypeptide bands in an SDS-polyacrylamide gel.

DAY 1: Students perform the following:

1. SDS-PAGE and Coomassie Blue staining

DAY 2: Students perform the following:

1. Genomic DNA isolation from B.licheniformis

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Week 3 - Day 1 .

Procedure:

Coomassie Blue staining of the SDS-PAGE gel:

1. Assemble and run your SDS-PAGE gels exactly as outlined above (section (d) b-week 2:
Loading and running of the protein samples). The only difference is that after completing
the electrophoresis part, the gel is not transferred to a PVDF membrane but taken out of
the plates and used immediately for the staining with Coomassie Blue stain.
2. Add 100 ml of Coomassie Blue stain to the shallow plastic container provided.
3. Open the gel cassette by gently prying the plates apart with the small plastic spatula
provided. Please ask your TA for help if you feel uncomfortable doing this part (the plates
are extremely fragile so in order to avoid injuries ask for help!!!)
4. Submerge the plate. Gel-side down, into the stain solution and gently pry the gel off the
plate by inserting the spatula under one corner of the gel.
5. DO NOT MICROVAWE THE GEL!!!!!
6. Place the container on the gel rocker and let it stain 20 minutes.
7. The next day discard the staining solution in the disposal vessel provided (in the fume
hood) and briefly wash the gel with 200 ml of deionized water.
8. Add 100 ml of Coomassie destain solution and place a few Kim wipes into the container
to absorb the dye.
9. Rock the gel on the gel rocker for 15 minutes. Protein bands should become visible within
this time frame. The gel can be stored in destain solution indefinitely.
10. Photograph your gel after you place it on plastic wrap. Use the image for your laboratory
report.
11. Discard of the destain solution in the disposal container provided (in the fume hood).

LAB 2 ends

LAB 3 begins

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Laboratory #3

Week 3 & 4: Restriction Mapping of -Amylase Plasmid DNA

Brief overview:

The goal this week is to learn the process of restriction mapping: the cleaving of DNA with a set
of restriction enzymes, both singly and in combination, followed by the separation of these
fragments by gel electrophoresis in order to calculate fragment size and ultimately deduce the
relative location of the of the cleavage sites on the original strand of DNA. In the last two labs of
the course, you will use numerous restriction enzymes to create a restriction map of the -
amylase gene you inserted into the pET15b vector.

DAY 1: Students perform the following:

1. Restriction enzyme digest of the amylase containing plasmid

DAY 2: Students perform the following:

1. DNA gel electrophoresis of cleavage products

2. Tutorial on assembly of restriction map

Week 3 - Day 2 .

Procedure:

a) Assembly of the restriction enzyme digestion reactions:

The restriction digests are as follows:

Single cut:
Hind III alone
EcoR I alone
Pst I alone
Bgl II alone

Double cut:
Hind III and EcoR I together
Hind III and Pst I together
Hind III and Bgl II together
EcoR I and Pst I together
EcoR I and Bgl II together
Pst I and Bgl II together

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1. Set up the above enzymatic reactions as follows, adjusting the amount of H2O as
necessary, and using the correct buffer for each reaction as indicated:
Component One Enzyme Two Enzymes
dH2O
~150 ng amylase-pET15b plasmid
10X buffer 2.0 l 2.0 l
Restriction enzyme 1 2.0 l 2.0 l
Restriction enzyme 2 0 2.0 l
Total 20.0 l 20.0 l

2. Place the 1.5 ml Eppendorf tubes in a 37oC water bath and leave them for 1 2 hrs.
3. Store the enzyme digests in the -20oC until next week.

b) Casting 1% agarose gels:

Please refer to Appendix A for details on how to prepare the agarose gel.
Once prepared, gels in running trays are to be wrapped with wet paper towels, placed in
zip-lock bags and kept in the fridge for next week.

Please note, that this also can be done on Week 4 Day 1. The decision when exactly
to cast gels will be made by your TA and Lab Technician.

Week 4 - Day 1 .

c) Agarose gel electrophoresis of the DNA digest:

Please note, that the Running Buffer for this experiment is 0.25 X TAE.
1. Prepare 0.5 L of Running Buffer (0.25 X TAE) from 50 X TAE stock provided. Place it on
ice or into the fridge to keep it cold.
2. If it is not done on Week 3 Day 2, prepare 2 x 1% agarose gels (please refer to Appendix
A for details on how to prepare the agarose gel). Important: Use 1 X TAE to cast gels.
3. Prepare the samples by mixing 15 ul enzyme digest with 3 ul 6X DNA loading dye.
4. Place each tray with the gel in the electrophoresis tank, fill the tank with pre-chilled
Running Buffer until the buffer is ~1-2 mm deep over your gel.
5. Load your samples in the following order:
Gel 1: Gel 2:
DNA Hind III digest DNA Hind III digest
Hind III alone Pst I alone
EcoR I alone Bgl II alone
100 bp (or 50 bp) DNA Ladder 100 bp (or 50 bp) DNA Ladder

Hind III and EcoR I together EcoR I and Pst I together

Hind III and Pst I together EcoR I and Bgl II together
Hind III and Bgl II together Pst I and Bgl II together
1 Kb DNA ladder 1 Kb DNA ladder

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6. Run the gels at 200 Volts for approximately 30 minutes or less. BE AWARE of high
voltage.
7. Photograph your gel using the imager in the laboratory and save this image for your
laboratory report .

d) Assembly of the restriction map:

1. Using the 1 Kb DNA ladder, calculate the lengths of the restriction fragments
2. List the single and double digest fragment lengths in two separate tables
3. Follow TA instructions for assembly of restriction map. This will be submitted in your
laboratory report.

LAB 3 ends

LAB 4 begins

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Laboratory #4

Week 4 - Day 2 .

Isolation of genomic DNA from B.licheniformis:

1. You are given 26 ml of an overnight culture of B.licheniformis. Transfer 25 ml the

overnight culture to a 50 ml falcon tube provided and centrifuge the tube @ 5500 rpm for
10 minutes to pellet the bacterial cells.
2. Pour off the supernatant in the disposal container labeled bacterial disposal containing a
dilute bleach solution.
3. Add 2 ml of TE buffer to the pellet and disperse the cells by vortexing vigorously (20
seconds). Make sure that there are no visible cell clumps before you proceed to the next
step.
4. Add 20 ml of TE and vortex the cells (this is an extra washing step) for 10 seconds.
5. Centrifuge the cells @ 5500 rpm for 5 minutes in order to obtain a firm pellet.
6. Pour off the supernatant in the disposal container labeled bacterial disposal and add 2
ml of TE buffer. Vortex the cells vigorously to ensure complete dissociation of the
bacterial pellet and place the cells on ice.
7. Transfer 1 ml of glass beads to the 15 ml tube provided and transfer your 2 ml bacterial
suspension to the same tube.
8. Add 1 ml of phenol: chloroform: isoamyl alcohol (25:24:1) to the 15 ml tube containing
your bacterial lysate and the glass beads, close the tube and vortex for 1 minute. (BE
CAREFUL TO AVOID PHENOL BURNS)!
9. Place the tube on ice for 1 minute.
10. Repeat this sequence of steps three more times to give a total time of 4 minutes.
Vortexing in the presence of glass beads and organic solvents causes rupture of the
bacterial cells and the subsequent release of DNA, proteins and other cellular
components.
11. Centrifuge the lysed cell suspension @ 6000 g for 5 minutes @ 4C TO SEPARATE THE
PHASES
12. There will be three separate phases. Only remove the upper clear layer since this is the
one that contains the DNA (look at diagram below)

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13. Carefully transfer 2 ml of the upper phase (aqueous phase) to a new 15 ml tube and
carefully add 2 ml of phenol: chloroform: isoamyl alcohol (25:24:1) to the tube.
14. Centrifuge the tube @ 10,000 g for 5 minutes. Carefully remove the upper phase and
transfer it to another 15 ml tube.
15. Add another 2 ml of phenol: chloroform: isoamyl alcohol (25:24:1) to the tube and gently
invert the tube to mix the contents.
16. Centrifuge the tube @ 10,000 g for 5 minutes.Carefully remove the upper phase and
transfer it to another 15 ml tube.
17. Add another 2 ml of chloroform: isoamyl alcohol (24:1) to the tube and gently invert the
tube to mix the contents.
18. Centrifuge the tube @ 10,000 g for 5 minutes.Carefully remove the upper phase and
transfer it to a new 15 ml tube.
19. You should have about 1.5-2 ml of aqueous phase by now which contains all the genomic
DNA from Bacillus. Transfer this phase to a new 15 ml tube.
20. Add 150 l of 3 M sodium acetate to the aqueous phase and mix the contents by gently
inverting the tube.
21. Add 3.3 ml of ice-cold 95% ethanol and centrifuge the tube at 13,000 rpm for 10 minutes.
22. Carefully discard the ethanol ensuring that the DNA pellet at the bottom of the tube is not
disturbed.
23. Wash the pellet with 70% ethanol once (vortex the preparation for 20 seconds) and spin
down the tube at 13,000 rpm for 10 minutes
24. Carefully remove all the ethanol and let the pellet air dry for 5 minutes.
25. Resuspend the pellet in 120 l of TE buffer containing RNAse A (1g/ml). The RNAse will
digest away any RNA in the preparation. Carefully pipette a few times to ensure that the
pellet is dissolved in TE buffer.
26. Now you are ready to check your genomic DNA by gel electrophoresis.

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 Biol3140 Lab Manual

Week 5: DNA electrophoresis of Bacillus genomic DNA and Cloning of the -amylase gene
& Restriction enzyme digest of the -amylase PCR product and B.licheniformis genomic
DNA

DNA gel electrophoresis of Bacillus genomic DNA

PCR of the -amylase gene using genomic DNA extracted from B.licheniformis
-amylase PCR product cleaning up and restriction enzyme digestion

Brief overview:

The goal this week is to clone the -amylase gene from B.licheniformis into E.coli. The next few
weeks will employ two separate strategies: 1) cloning the -amylase gene using PCR, 2) creating
a B.licheniformis genomic DNA library (pRL498) using the HindIII restriction enzyme, and 3)
inserting cloned -amylase genes into the plasmid vector (pET15b). Successful restriction
digests will be verified using agarose gel electrophoresis.

DAY 1: Students perform the following:

1. DNA gel electrophoresis of Bacillus genomic DNA

2. Spectrophotometric determination of Bacillus genomic DNA concentration
3. PCR of the -amylase gene from B.licheniformis genomic DNA

DAY 2: Students perform the following:

1. Agarose gel electrophoresis of the -amylase PCR product

2. Reaction cleanup of the -amylase PCR product
3. Restriction enzyme digest of the -amylase PCR product and pET15b with NdeI
overnight
4. Restriction enzyme digest of genomic DNA extracted from B.licheniformis and pRL498
with HindIII overnight

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 Biol3140 Lab Manual

Week 5 - Day 1 .

Procedures:

a) Agarose gel electrophoresis of genomic DNA from B.licheniformis:

1. Prepare a 0.7 % agarose gel (see Appendix A!)

2. In order to assess the quality of your genomic DNA you must make a few dilutions to run
on the agarose gel. Add 45 l of TE buffer to each of two 1.5 ml eppendorf tubes. Label
the first as 10-1dilution and the second as 10-2dilution. To the first tube add 5 l of your
DNA and mix it really well by pipetting up and down 5 times. Then transfer 5 l of DNA
and TE buffer from the first dilution over to the second tube and mix well by pipetting
carefully another 5 times.
3. Transfer 5 l of each dilution to a new 1.5 ml eppendorf tube and add 5 l of 2X DNA Gel
loading dye to each tube and mix well by pipetting up and down a few times. Centrifuge
the tube to collect all the sample at the bottom of the tube.
4. Load the entire sample into a separate well. Load 10 l of the 1Kb DNA molecular weight
marker and run the gel at 100 volts for 1 hr.
5. Photograph your gel and keep this photograph for your laboratory report. Your DNA
preparation should look like the DNA in the figure below:

Figure: Agarose gel electrophoresis of genomic DNA isolated from different orgamisms:
M: 1 kb ladder marker, line 1: Chicken whole blood, line 2: Human whole blood, line 3: E. coli,
line 4: L. brevis, line 5: Streptomyces hygroscopicus subsp

b) Quantitation of genomic DNA from B.licheniformis:

1. Firstly, use 1 ml of TE buffer as the BLANK measurement in the Quartz cuvette provided.
2. After you are finished with the BLANK measurement proceed to the next step
3. Mix 2 l of your genomic DNA preparation plus 998 l of TE buffer in a 1.5 ml eppenforf
tube. Mix well by pipetting up and down at least 5 times.
4. Transfer all the solution (approximately 1 ml) to the Quartz cuvette and measure the
absorbance at 260 and 280 nm of wavelength
5. Now you are ready to quantify your DNA concentration
a) The absorbance at 260nm is used to calculate the concentration of nucleic acids. At a
concentration of 1 g/ml and a 1 cm path length* dsDNA has A260 = 50. The
absorbance value is also dependent on the amount of secondary structure in the DNA
due to hypochromicity.

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b) To ensure the concentration reading is accurate, the absorbance reading should be within
the linear range of the spectrophotometer. The Lambert-Beer law relates the absorption
of light to the properties of the material through which the light is travelling. The law states
that there is a logarithmic dependence between the transmission of light through a
substance and the product of the absorption coefficient of the substance and the distance
the light travels through the material (i.e. the path length). The law tends to break down at
very high concentrations, especially if the material is highly scattering. So for
concentrated solutions the absorbance value and therefore the concentration can be
inaccurate. It is often useful to prepare and measure a series of dilutions to check not
only the concentration but the accuracy of the dilutions as well. Inaccurate dilutions can
occur if the DNA is not homogeneously re-suspended. For reliable spectrophotometric
DNA quantification A260 readings should lie between 0.1 and 1.0.
c) To improve the accuracy of DNA concentration determination allowance should be made
for any impurities in the solution. This can be estimated by adjusting the A260
measurement for turbidity which is measured at an absorbance of A320. The equation
below can be used:
d) Concentration (g/ml) = (A260 reading A320 reading) dilution factor 50g/ml
e) Total yield is obtained by multiplying the DNA concentration by the final total purified
sample volume.
f) DNA Yield (g) = DNA Concentration Total Sample Volume (ml)

c) Cloning of the -amylase gene from Bacillus by PCR:

1. Prepare a diluted genomic DNA sample with a final concentration of 0.5 g/l
2. Set up the PCR reactions as indicate in the table below:

Component Negative control pAMY8 positive B.licheniformis

control
2X PCR master Mix 25 l 25 l 25 l
Forward Primer 2 l 2 l 2 l
0.2ug/ul
Reverse Primer 2 l 2 l 2 l
0.2/ug/ul
Template DNA N/A 6 l 1 l
dH20 21 l 15 l 20 l
Total Volume 50 l 50 l 50 l

3. Mix the samples well and briefly spin the tubes down (1000 rpm for 1 minutes)
4. Now you are ready to proceed with the PCR reaction using the thermal cycler ( program
3140 PCR1)

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Week 5 - Day 2 .

Procedure:

a) Agarose gel electrophoresis of the unpurified -amylase PCR product :

1. Prepare a 1% agarose gel

2. Prepare three samples by mixing 5 l of the unpurified PCR product with (1) 5 l 2X DNA
loading dye.
3. Load the three PCR samples on the gel and load one lane with the 1 Kb DNA molecular
weight marker.
4. Run the gel at 100 volts for 20 minutes.
5. Photograph your gel using the imager in the laboratory and save this image for your
laboratory report.

b) PCR reaction cleanup:

1. Add 5 volumes of Buffer PB to 1 volume of the PCR reaction and mix. For example, add
250 l of Buffer PB to 50 l PCR reaction.
2. To bind DNA, load the samples into the MinElute centrifuge at 10,000 rpm for 1 min (The
maximum loading volume of the column is 800 l. For sample volumes greaterthan 800 l
simply load again).
3. Discard flow-through. Place the MinElute column back into the same tube.
4. To wash, add 750 l Buffer PE to the MinElute column and centrifuge for 1 min.
5. Discard flow-through and place the MinElute column back in the same tube.
6. Centrifuge the column for an additional 1 min at maximum speed.
7. Place the MinElute column in a clean 1.5 ml microcentrifuge tube.

To elute DNA, add 44 l Buffer EB (10 mM TrisCl, pH 8.5) to the center of the membrane, let the
column stand for 1 min, and then centrifuge for 1 min at maximum speed. Label the side of the
tube with your name.

c) Restriction enzyme digest setup (NdeI digest) for the PCR product and pET15b
vector:

1. Set up the restriction enzyme digestion in a 1.5 ml eppendorf tube as indicated in the
tables below:

Component Volume
Purified PCR product (DNA) 40 l
10 X NEB buffer 3.1 5 l
100x BSA 0.5 l
NdeI enzyme 1 l
dH20 3.5 l
Total 50 l

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 Biol3140 Lab Manual

Component Volume
pET15b vector (DNA) 5 g 5 l
10 X NEB buffer 3.1? 5 l
100x BSA 0.5 l
NdeI enzyme 1 l
dH20 (adjust accordingly) 37.5 l
Calf intestinal phosphatase 1ul
Total 50 l

2. Place the 1.5 ml eppendorf tubes in a 37 C water bath and leave them overnight. Next
morning the Laboratory Technician will place tubes at -20 0 C.

d) Restriction enzyme digest setup (HindIII digest) for the B.L genomic DNA and
pRL498 vector:

1. Set up restriction enzyme digestion in a 1.5 ml eppendorf tube as indicated in the tables
below:

Component Volume
pRL498 vector (DNA) 1-2g 1-2 l
10 X NEB buffer 2.1 5 l
100x BSA 0.5 l
HindIII enzyme 1 l
dH20 (adjust accordingly) 40.5 - 41.5 l
Calf intestinal phosphatase 1ul
Total 50 l

Component Volume
B.L Genomic DNA (DNA) 5g 1-2 l
10 X NEB buffer 2.1 5 l
100x BSA 0.5 l
HindIII enzyme 1 l
dH20 (adjust accordingly) 41.5 42.5 l
Total 50 l

3. Place the 1.5 ml eppenforf tubes in a 37 C water bath and leave them overnight. Next
morning your Laboratory Technician will place tubes at -20 0 C.

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 Biol3140 Lab Manual

Week 6: Restriction enzyme digest of the -amylase PCR product and

B.licheniformis genomic DNA & Digestion reaction cleanup, ligation of the products

Second restriction enzyme digestion of -amylase PCR product and pET15b vector
Cleanup of enzymatic digestion products and agarose gel electrophoresis
Ligation of products.

Breif overview:

In this week, Ndel digested -amylase PCR product and pET15b vector will be further digested
by BamHl. After cleanup, the DNA fragments will be inserted into plasmid vectors by ligation.

DAY 1: Students perform the following:

1. BamHl digest for the PCR product and pET15b vector

2. Cleanup of enzymatic digestion prodcuts.

DAY 2: Students perform the following:

1. Agarose gel electrophoresis of cleaned digestion products.

2. A ligation is performed so that the PCR product is ligated into pET15b vector and the
digested genomic DNA is ligated into the pRL498 vector.

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Week 6 - Day 1 .

a) Restriction enzyme digest setup (BamHI digest) for the PCR product and pET15b
vector:

1. Set up restriction enzyme digestion in a 1.5 ml eppendorf tube as indicated in the tables
below:

Component Volume
NdeI digested PCR product (DNA) 50 l
10 X NEBuffer 3.1 2.5 l
10x BSA 7.5 l
BamHI enzyme 1 l
NaCl 5M 1 l
dH20 12 l
Total 75 l

Component Volume
NdeI pET15b digestedvector product (DNA) 50 l
10 X NEBuffer 3.1 2.5 l
10x BSA 7.5 l
BamHI enzyme 1 l
NaCl 5M 1 l
dH20 12 l
Total 75 l

2. Place the 1.5 ml eppendorf tubes in a 37 C water bath and leave them for 1-2 hrs.

b) Reaction cleanup for the digestion products.

1. Add 300 l of Buffer ERC to the enzymatic reaction and mix. The maximum volume of
enzymatic reaction that can be processed per MinElute column is 100 l. If the enzymatic
reaction is in a volume of <20 l, adjust the volume to 20 l. If the enzymatic reaction
exceeds 100 l, split your reaction, add 300 l of BufferERC to each aliquot of the split
reaction and use the appropriate number of MinElute columns.
2. Check that the color of the mixture is yellow (similar to Buffer ERC without the enzymatic
reaction).If the color of the mixture is orange or violet, add 10 l of 3 M sodium acetate, pH
5.0, and mix. The color of the mixture will turn to yellow.
3. Place a MinElute column in a 2 ml collection tube in a suitable rack.
4. To bind DNA, apply the sample to the MinElute column and centrifuge for 1 min.
To obtain maximal recovery, transfer all traces of sample to the spin column.
5. Discard the flow-through and place the MinElute column back into the same tube.
6. To wash, add 750 l Buffer PE to the MinElute column and centrifuge for 1 min.
7. Discard the flow-through and place the MinElute column back in the same tube.
Centrifuge the column for an additional 1 min at maximum speed.
8. Place the MinElute column in a clean 1.5 ml microcentrifuge tube.
9. To elute DNA, add 20 l Buffer EB (10 mM TrisCl, pH 8.5) to the center of
the membrane, let the column stand for 1 min, and then centrifuge for 1 min.

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Week 6 - Day 2 .

a) Agarose gel electrophoresis of the DNA digest:

1. Prepare a 1% agarose gel

2. Prepare four samples by mixing 5 l of the PCR product with 5 l 2X DNA loading dye.
3. Load the PCR samples on the gel and load one lane with the 1 Kb DNA molecular weight
marker.
4. Run the gel at 100 volts for 20 minutes.
5. Photograph your gel using the imager in the laboratory and save this image for your
laboratory report.

b) Ligation reaction set up for the PCR product and genomic DNA:

1. Keep all the reaction components on ice!!!!

2. Set up the 2 ligation reactions as indicated in the tables below:

Component Volume
Cleaved PCR product 10 l
Vector (pET15b) 1 l
10X T4 ligase buffer 2 l
T4 DNA ligase 1 l
dH20 6 l
Total 20 l

Component Volume
Genomic DNA library 10 l
Vector (pRL498) 1 l
10X T4 ligase buffer 2 l
T4 DNA ligase 1 l
dH20 6 l
Total 20 l

3. Place the sample in the thermal cycler and incubate overnight at 16 C. The program is
called 16 and is programmed into the thermal cycler. The laboratory technician will keep
your ligation products in the -20 C for you to use next day.

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Week 8: Colony PCR

After the ligation products are transformed into chemically competent E.coli cells --->
Colonies are selected after the transformation and screened by PCR for positive
clones.

Brief overview

The goal this week is to proceed with introduction of ligation products (foreign (DNA)) into
chemically competent E.coli cells. This process is known as bacterial transformation and is a
technique routinely used in Molecular Biology laboratories. Following the transformation the
bacteria are plated on LB agar plates containing an antibiotic. Only bacteria that contain the
proper antibiotic resistance plasmids will grow and proliferate. However, not all growing colonies
will contain your gene of interest (-amylase). For this reason you will have to screen a few
colonies in an attempt to identify positive clones.

DAY 1 & 2: Students perform the following:

1. Colony PCR and agarose gel electrophoresis for screening of positive clones
2. Staining of the LB-KAN+-Starch plates with Lugol's iodine solution for detection of -
amylase activity in bacterial colonies.( run PCR products on gel.)

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Week 8 - Day 1 .

Procedure:

a) Bacterial transformation protocol:

1. You will find SOC broth in the 42C water bath for use as outgrowth medium (step 10).
2. Thaw two aliquots of SoloPack Gold cells on ice (one tube for each of PCR product,
genomic DNA).
3. When the cells have thawed, swirl the tubes to gently mix the cells.
4. Add 5l of the experimental DNA to each of the tubes of cells.
5. Swirl the tubes gently, then incubate the tubes on ice for 30 minutes.
6. Heat-pulse the tubes in a 42C water bath for 30 seconds. The temperature and duration of
the heat pulse are critical for maximum efficiency. For consistent results, remove any ice
trapped around the outside bottom of the tube.
7. Incubate the tubes on ice for 2 minutes.
8. Add 250 l of preheated (37C) SOC broth and incubate the tubes at 37C for 1 hour with
shaking at 225-250 rpm.
9. Plate 200 l of the transformation mixture on LB agar plates containing the appropriate
antibiotic (the PCR product and pET15b transformation reactions will be plated on LB-
AMP+ plates whereas the genomic DNA library ligation will be plated on LB-KAN+-
Starch plates).
10. Incubate the plates at 37C overnight.
*** Your TA will transform a separate tube of bacteria with the pUC18 control plasmid
to test for transformation efficiency***

Procedures:

b) Colony PCR protocol:

1. Pick 4 colonies from the LB- AMP+ transformed with your PCR product and re-suspend
them in 20 l of dH20. You will use this mixture for the PCR outlined below.
2. Set up the following six PCR reactions as depicted below:

Component Negative control pET15b positive Colony Samples

control (X4)
2X PCR master Mix 10 l 10 l 10 l
T7 Forward Primer 2 l 2 l 2 l
T7 Reverse Primer 2 l 2 l 2 l
Template DNA N/A 2 l 2 l
dH20 6 l 4 l 4 l
Total Volume 20 l 20 l 20 l

3. Mix well all the ingredients in the PCR tube and place them in the thermal cycler. The
program for this PCR is called 3140PCR2.
4. While waiting the reaction to go to completion prepare a 1% agarose gel.

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Week 8 - Day 2 .

c) Agarose gel electrophoresis of the colony PCR products:

1. Prepare a 1% agarose gel (please refer to earlier text for details on how to make the
agarose gel)
2. Prepare three samples by mixing 10 l of the PCR product with 10 l 2X DNA loading
dye.
3. Load the three PCR samples on the gel and load one lane with the 1 Kb DNA molecular
weight marker.
4. Run the gel at 100 volts for 20 minutes.
5. Photograph your gel using the imager in the laboratory and save this image for your
laboratory report.

d) Analysis of -amylase activity using starch agar and Lugol's iodine staining:

1. Use 4-5 ml of Lugol solution to cover the surface of the LB-KAN+-Starch plate containing
bacterial colonies. Wait for 2-3 minutes until the plate is stained deep blue. Colonies
exhibiting amylase activity will appear as having a white halo around them. The image
below is representative of amylase activity.
2. Take a photograph of your plates and save these images for your lab report.

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Week 9: Enzyme activity of -amylase clones

The goal of this lab is to measure -amylase activity of E.coli strains carrying the
cloned amyE gene

Brief overview:

The goal this week is to use a quantitative -amylase assay to measure -amylase activity of the
recombinant strain of E.coli carrying the amyE plasmid, negative E.coli cells and B.licheniformis.
In addition, biotin-labeled -amylase probes will be generated to detect the -amylase gene in
restriction fragments of the B.lichenformis genome using the Southern Hybridization technique.

DAY 1: Students perform the following:

1. Measure the enzyme activity of a culture of cells that express the -amylase enzyme

DAY 2: Students perform the following:

1. Restriction enzyme digest of genomic DNA from B.licheniformis for southern hybridization
2. PCR labeling of probe DNA with Biotin

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Week 9 - Day 1 .

Procedure:

a) Enzyme activity of -amylase clones:

1. label three ml culture tubes according to the chart that follows:

Tube # Strain
1 AmyE E.coli
2 Negative E.coli
3 B.licheniformis

2. Spin down 1.5 ml of each of the cultures provided to you at 13,000 rpm for 2 minutes in a
1.5 ml eppendorf tube.
3. Keep the supernatant on ice as it will serve as the culture medium in the assay.
4. Add 0.1 ml of 1x TBS to each tube of cells. Vortex for 30 seconds to fully resuspend the
pellet of cells.
5. Transfer re-suspended cells into 2mL tubes containing 0.1 ml of glass beads and vortex
the mixture for 1 minute.
6. Place the tube on ice for 1 minute.
7. Repeat the cycles of vortexing for 1 minute and placing the mixture on ice for four more
times to ensure complete lysis of the bacteria.
8. Add 1.3 ml of 1X TBS to each tube and vortex to mix.
9. Spin the tubes at 13,000 rpm for 2 minutes. The glass beads should be firmly adhered to
the bottom of the tube. Carefully transfer the bacterial lysate to another fresh tube and
keep these tubes on ice while you set up the assay tubes.
10. Label eight 15 ml assay tubes , according to the table below:

Sample Condition LB broth Culture 1X TBS Lysate

medium
1 Culture medium blank 1 ml 0 ml
2 amyE E.coli medium 0.9 ml 0.1 ml
3 Negative E.coli 0 ml 1 ml
medium
4 B.licheniformis 0 ml 1 ml
medium
5 Cell lysate blank 1 ml 0 ml
6 amyE E.coli lysate 0.9 ml 0.1 ml
7 Negative E.coli lysate 0 ml 1 ml
8 B.licheniformis lysate 1 ml

11. Add 1 ml of 1% starch solution to each of the assay tubes including the blank tubes
12. Vortex the tubes and place them in the boiling water bath (100C) for exactly 12 minutes.

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13. In the same order that the starch solution was added, add 1 ml of the maltose color
reagent to each tube. Vortex the tubes and place them in the boiling water bath (100C)
for exactly 15 minutes.
14. Place the tubes on ice until they reach room temperature. Add 9 ml of deionized water
and invert the tubes several times to mix their contents.
15. Use the appropriate blank to zero the spectrophotometer at 540 nm and measure the
absorbance of the samples at 540 nm.
16. Record your absorption readings in the following table:

amyE Negative B.licheniformis Amy E Negative B.licheniformis

E.coli E.coli medium E.coli E.coli lysate
medium medium lysate lysate
A540 nm
mg of maltose
produced during
assay
Calculated mg of
maltose produced
by 1ml of test
solution

Lab 4 ends
Lab 5 begins

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Laboratory #5
Week 9 - Day 2 .

b) Assembly of restriction enzyme digestion reactions:

1. Set up the following two enzymatic digests using genomic DNA from B.licheniformis
Component Volume
Genomic DNA (10 g) ~ 8-10 l
10 X NEB buffer 2.1 5 l
HindIII enzyme 4 l
dH20 31 l
Total 50 l

Component Volume
Genomic DNA (10 g) ~ 8-10 l
10 X NEB buffer 3.1 5 l
EcoRV enzyme 4 l
dH20 31 l
Total 50 l

2. Place the 1.5 ml eppendorf tubes in a 37 C water bath and leave them overnight.
3. Next morning your Laboratory Technician will place tubes at -20 0 C.

c) PCR labeling of probe DNA with Biotin:

1. Set up your PCR reactions as follows:

Component Negative control pAMY8 positive B.licheniformis

control
2X PCR master Mix 25 l 25 l 25 l
Forward BIO-Primer 5 l 5 l 5 l
Reverse BIO-Primer 5 l 5 l 5 l
Template DNA 1 l ( 0.1 g) 2 l ( 1 g)
dH20 15 l 14 l 13 l
Total Volume 50 l 50 l 50 l
2. Run your PCR reaction using the thermocycler (BIO PROBE program).
3. When competed (or next morning) your Laboratory Technician will place tubes at -
20 0 C.

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 Biol3140 Lab Manual

Week 10: Southern Blot Hybridization

Part 1: Electrophoresis and Southern Blot transfer:

The goal of this lab is to carry out electrophoresis and Southern Blot transfer
Dot Blot preparation

Brief overview:

A dot blot (or slot blot) is a technique in molecular biology used to detect biomolecules, and for
detecting, analyzing, and identifying proteins. It represents a simplification of the northern blot,
Southern blot, or western blot methods. In a dot blot the biomolecules to be detected are not first
separated by electrophoresis. Instead, a mixture containing the molecule to be detected is
applied directly on a membrane as a dot, and then is spotted through circular templates directly
onto the membrane or paper substrate. This differs from the western blot because protein
samples are not separated electrophoretically. This is then followed by detection by either
nucleotide probes (for a northern blot and southern blot) or antibodies (for a western blot).

DAY 1: Students perform the following:

1. Agarose gel electrophoresis

2. First part of Southern Blot Transfer

DAY 2: Students perform the following:

1. Dot Blot preparation

2. Finish Southern Blot Transfer

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Week 10 - Day 1 .

Procedures:

a) Agarose Gel Electrophoresis:

1. Prepare a 0.8% agarose gel

2. Load your samples in the following order:
*Boil the biotinylated marker at 90C for 5 mins before loading
Add the appropriate amount of loading dye to make the sample 1x.

volume 6X DNA loading Total Volume

Dye
Biotinylated marker 5 l
HindIII digested DNA 20 l

EcoRV digested DNA 20 l

pAMY8 PCR product 5 l
B.licheniformis PCR 5 l
product
Water PCR product 5 ul
B.licheninformis genomic 10ul
DNA (5ug)
pAMY8 plasmid 1ul
(1ug)
3. Run the gel @ 100 Volts until the bromophenol blue band is (was) down on the gel.
4. Photograph your gel using the imager in the laboratory and save this image for your
laboratory report to compare against your final southern blot exposure.

b) Southern Blot transfer protocol:

1. Transfer gel to a tray containing ~100 mL of Southern denaturing solution. Gel needs to
be covered by the solution. Shake gently for 25-30 minutes.
2. Decant denaturing solution carefully and add 100 ml of Southern neutralizing buffer.
3. Soak the following while gel is shaking in Southern neutralizing buffer:

a) transfer membrane in dH2O

b) thin blotting papers in Southern transfer buffer
c) thick blotting paper in Southern transfer buffer

-place blotting paper into tray containing 20xssc buffer to wick solution for transfer.
-Place gel face down onto filter paper
-place nylon membrane on top of gel. Make sure that the membrane is cut exactly the size of the
gel.
-Place 5 sheets of thin filter paper on top of the nylon membrane. Make sure that paper is cut
exactly the size of the gel
-place papertowels on top of filter paper. Make sure that paper is cut exactly the size of the gel.
-Place large plate and weight on top of the entire set-up to ensure constant contact of gel to
nylon and even movement of buffer through the paper towel.

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4. Remove and invert the gel on saran wrap so that the bottom of the gel is facing up.
5. Remove the bubbles between the gel and saran wrap.
6. Cover the gel with the transfer membrane that was soaked in dH2O. Remove ALL of the
bubbles between the gel and membrane. Look at the following diagram for assistance
with setting up your southern blot:

7. Cover with the 2 thin blotting papers soaked in Southern transfer buffer.
8. Cover with thick blotting paper soaked in Southern transfer buffer.
9. Cover with a stack of dry blotting paper, tray, and bottles to provide weight. Let it stand
overnight.
**procedure 10-15 must be done next day

Week 10 - Day 2 .

10. Disassemble the blot after the transfer is complete and discard everything except for the
nylon membrane that carries your DNA.
11. Place the membrane in a dish containing 20 ml of 6X SCC buffer for 5 minutes.
12. Air dry the membrane.
13. Place the membrane between 2 filter papers and bake @ 80C for 30 minutes.
14. Keep your membrane in the freezer until next week.
15. Mark the edge in contact with the gel with a pencil. This side contains the bound DNA

b) Dot Blot preparation:

1. On a separate nylon membrane, spot the following samples:

***no pre-wet

Sample Volume
HindIII digested genomic DNA 20 l
EcoRV digested genomic DNA 20 l
Undigested B.lichenformis DNA 20 l
pAMY8 PCR product 5 l
gDNA pcr product 5 L
Water pcr product 5 L
Water only 20 L

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a. NOTE: To concentrate the dot blot, it is preferable to spot your sample 5 l at a

time onto the same spot, dry the blot in the oven for 5 minutes in between each
loading interval until all the sample is completely loaded.
b. With a PENCIL, draw a circle around each dot and label clearly
2. Air dry your membrane
3. Place the membrane between two filter papers and bake it @ 80C for 30 minutes
4. Keep your membrane in the freezer until next week.

*** This image represents a typical Dot Blot (in this case spotting protein on membrane) ***

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Week 11: Southern Blot Hybridization

Part 2: Hybridization, washing and detection

DAY 1: Students perform the following:

Hybridization of the Southern Blot nylon membrane

DAY 2: Students perform the following:

Washing of the Southern Blot nylon membrane and detection

Week 11 - Day 1 .

Procedures:

a) Hybridization of the probe:

1. Pair up with a group and place 4 blots into one glass hybridization tube with the DNA side
facing the inside of the tube.
2. Add ~20ml 2x SSC and mix at room temperature for 5 minutes. Make sure that the buffer
covers the top of the membranes and adjust the volume to make up for this.
3. Denature 1 ml of 10 mg/ml sheared herring sperm DNA by heating the DNA in a boiling water
bath at 100C for 10 minutes. Chill on ice. Add 500 l of the denatured herring sperm DNA to
9.5ml of Southern Prehybridization Solution (50% formamide, 5 x SSC, 5 x Denhardts reagent,
20 mM sodium
phosphate, pH 6.5).
4. Discard the 2 x SSC solution from the hybridization tube(s) and add the Southern
Prehybridization solution containing the herring sperm DNA to your membranes.
5. Incubate both membranes at 42C for at least 1 hour.
6.Near the end of the one hour, add 200 l of your heat denatured sperm DNA (10 mg/ml) to 9.8
ml of the Southern Hybridization Solution(45% formamide, 5 x SSC, 1 x Denhardts reagent, 20
mM sodium phosphate, pH 6.5)
7. Denature the rest (~40 l) of your BIO-PCR probe (i.e. the PCR reaction that produced a band
at ~433bp like your B. licheniformis DNA BIO-PCR product) by heating at 100C for 10 minutes.
Quick cool your probe on ice.
8. Add all of your denatured BIO-PCR probe to the Southern Hybridization
solution containing the denatured herring sperm DNA which was made in step 6.
9. Discard the Prehybridization solution from the hybridization tube and replace it with the
Southern Hybridization Solution/herring sperm DNA mixture containing your DNA probe.
10. Incubate the membranes at 42C overnight.

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Week 11 - Day 2 .

b) Washing of the nylon membrane:

1. Discard the Hybridization Solution

2. Add 25ml Southern Wash Solution 1(2 x SSC, 0.1% SDS), rotate gently for 2 minutes at room
temperature.
3. Discard wash and repeat step 2 with Wash Solution 1.
4. Discard Wash Solution1and add 25ml of Southern Wash Solution 2 (0.2 x SSC, 0.1% SDS),
rotate gently at room temperature for 2minutes.
5. Discard wash and repeat step 4 with Wash Solution 2.
6. Discard Wash Solution 2 and add 25ml of Southern Wash Solution 3 (0.16x SSC, 0.1% SDS)
which has been prewarmed to 65C. Incubate in an oven for 10 minutes at 65C.
7. Discard wash and repeat step 6 with Wash Solution 3.

c) Detection of the probe using enhanced Chemiluminescence:

1. Rinse the membrane in a tube in 20ml of Tris-NaCl (100 mM Tris-HCl, pH 7.5, 150 mM NaCl)
for 30 seconds.
2. Discard the rinse solution and add 30ml of Biotin Blocking Buffer (3% BSA in Tris-NaCl).
Incubate in an oven at 65C for 1 hour.
3. Discard the Biotin Blocking Solution and add 15ml of Biotin Blocking Solution containing
1:20,000 dilution of HRP-Streptavidin to each blot.
4. Gently rock at room temperature for 10 minutes.
5. Transfer the membrane to a new container and add 20 ml of Tris-NaCl.
6. Gently rock at room temperature for 5 minutes.
7. Repeat the Tris-NaCl wash three more times.
*Prepare 3 ml of ECL solution by mixing 1.5 ml of each solution provided in the kit
9. Discard the Tris-NaCl solution and incubate your membranes with ECL solution for 2 minutes.
DO not rinse the blot after this step.
10. Place the blots in an imaging cassette and expose signals on imaging film in the dark room
(TA is to do this).

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Appendix A

Preparation of Agarose Gel

1% agarose gel :
Use the 125 ml flask that is provided to you.
Measure 30 ml of 1X TAE buffer using a graduated 50 ml cylinder and pour into the flask
Weigh the proper amount of agarose in order to prepare the desired gel by weight per
volume (w/v).
Use the table below as a guide:

Gel Percentage 0.7 % 0.8% 1% 1.2 %

Agarose (g) 0.21 0.24 0.3 0.36
1 X TAE (ml) 30 30 30 30

Swirl the agarose in the flask to disperse evenly

Microwave the flask on high for 30-60 seconds until the agarose is melted. Remove the
flask using gloves (Be careful!!! Hot liquid!!!!) and allow the agarose solution to cool off
When the solution is relatively cool then add 2 l of 5 mg/ml ethidium bromide solution
and make sure that you swirl carefully so that the ethidium bromide is evenly distributed.
(CAUTION: ETHIDIUM BROMIDE is a potent MUTAGEN!!!!)

DNA electrophoresis:

a) Place 10 l of 2X DNA Loading dye into 5 1.5 ml eppendorf tubes and label them 1
through 5
b) Prepare serial dilutions by transferring 10 l of the HindIII digested -phage DNA to the
first tube. Mix well by pipetting up and down 10 times and subsequently spin the tube
down @ 3000 rpm for 30 seconds
c) Repeat step 2 , transferring from tube 1 to tube 2, tube 2 to tube 3 etc. all the way up to
tube 5
d) Prepare the agarose gel as outlined above
e) Load 10 l of each of these dilutions ( in sequence with the most concentrated sample [1
to 5])
f) Load 10 l of unknown DNA
g) Load 10 l of the 1Kb DNA ladder
h) Run your gel at 100 Volts for 30-40 minutes until the Loading dye is 2/3 of the way down
and regularly check to monitor progress during running time.
i) Photograph your gel using the imager in the laboratory (bring a memory stick as a printer
is not available).
j) Using the photo prepare a standard curve for the HIND III digested -phage DNA. Based
on the information supplied by your standard curve, estimate the size(s) of the unknown
DNA fragment(s). Based on your serial dilutions, provide an estimate of the starting
concentration of DNA in the unknown sample and comment on the validity of your
estimate

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