EXPERIMENT 5 - Cloning Clone JET PCR Cloning

CBB40203 DNA TECHNOLOGY
LAB 3: CloneJET PCR Cloning Kit
Introduction
The Thermo Scientific CloneJET PCR Cloning Kit can be used with PCR products
generated by any DNA polymerase, therefore supporting both blunt – and sticky-end fragments
from 6 bp to 10 kb long.
Recommendations
1. Thoroughly mix every vial before use.
2. Gel-analyze the PCR product for specificity and yield before cloning.
 Specific PCR products of <1 kb appearing as one discrete band on the gel can be used for
ligation directly from the PCR reaction mixture without any purification.
 Gel purification is recommended for PCR products longer than 1 kb, PCR products
contaminated with nonspecific sequences or primer-dimers, and PCR templates
containing the β-lactamase (ampicillin resistance) gene.
 For efficient cloning of gel-purified DNA fragments, it is important to avoid DNA
damage by ethidium bromide and UV light.
 The optimal insert: vector molar ratio is 3:1. Refer to Table 1 or use dedicated software
(thermoscientific.com/reviewer) to calculate the optimal amount of PCR product to use.
 For PCR products >3 kb, ligation can be prolonged to 30 min.
Note: All components of this kit should be stored at 20°C.
1
Table 1. The recommended amount of PCR product for the ligation reaction.
Length of PCR product Optimal quantity of PCR product for

ligation reaction (0.15 pmol ends)
100 bp 5 ng
300 bp 15 ng
500 bp 25 ng
1000 bp 50 ng
2000 bp 100 ng
3000 bp 150 ng
4000 bp 200 ng
5000 bp 250 ng
2
PRELAB PREPARATION
Preparation of X-GAL and IPTG
1) IPTG
Dissolve 1.2g IPTG (isopropyl-β-D-thiogalactopyranoside) in deionized water to a final volume
of 50ml. Filter sterilize (0.2µm), and store in 5ml aliquots at –20°C. The final concentration of
IPTG is 0.1M. The stock solution of IPTG is stable at this temperature for 2–4 months.
2) X-GAL
Dissolve 100mg X-Gal in N, N'-dimethylformamide to a final volume of 2ml. Dispense into
500µl aliquots, and store protected from light at –20°C. The final concentration of X-Gal is
50mg/ml. The stock solution of X-Gal should be stable for 2–4 months at this temperature
3) Ampicillin stock solution

Dissolve 50 mg of ampicillin (sodium salt) in 1 ml of cool sterile distilled water.
For the final volume of reagent from stock solution is as following:
1. Autoclave 500 ml of LB medium

2. Cool down to around 55°C
3. At this temperature, add ampicillin at a final concentration of 50µg/ml (Pipette 0.5 ml/500 µl
of ampicillin stock solution of 50 mg/ ml), add 500 µl of 0.1M IPTG, add 600 µl of 50 mg/
ml of X-gal, mix well and keep at 4°C
3
LAB A - Option 1: Protocol for blunt end cloning
 For cloning blunt-end PCR products generated by proofreading DNA polymerases,

if the supplier of a DNA polymerase does not specify the end structure of the PCR
products it generates, follow the “Sticky-end cloning” protocol.
 For cloning blunt-end DNA fragments generated by restriction enzyme digestion,

gel-purify the DNA fragment before ligation and use in a 3:1 molar ratio with
pJET.2/blunt, the positive-selection vector.
STEPS:
1. Set up the ligation reaction on ice.
2. Set up the following items as described in Table 2:
Table 2: Mixing recipe for ligation of inserts

Component Volume
2X reaction buffer 10 ul
Unpurified PCR product, purified PCR product,
1 µL (0.15 pmol ends)
or other blunt-end DNA fragment
pJET1.2/blunt (50 ng/µL) 1 µL (0.05 pmol ends)
Water, nuclease-free To 19 µL
T4 DNA ligase 1 µL
TOTAL VOLUME 20 μl
3. Vortex briefly and centrifuge for 3–5 sec.

4. Incubate the ligation mixture at room temperature (20°C) for 5 min. For PCR products >3
kb, ligation can be prolonged to 30 min.
5. Use the ligation mixture directly for transformation.
LAB A - Option 2 – Protocols For Sticky-end Cloning

• For cloning PCR products with 3´ A overhangs generated by Taq DNA polymerase.
• For cloning PCR products when the supplier of a DNA polymerase does not specify the end
structure of the PCR products it generates.
4
• For cloning DNA fragments with 5´ or 3´ overhangs generated by restriction enzyme

digestion; gel-purify the DNA fragment before ligation and use in a 3:1 molar ratio with
pJET.2/blunt, the positive-selection vector.
STEPS:
1. Set up the blunting reaction on ice:

2. Set up the following items as described in Table 2:
Table 2: Mixing recipe for ligation of inserts.
Component Volume
2X reaction buffer 10 ul
Unpurified PCR product, purified PCR product,
or other blunt-end DNA fragment
Water, nuclease-free To 17 µL
DNA blunting enzyme 1 µL
TOTAL VOLUME 18 μl

4. Incubate the ligation mixture at 70°C for 5 min. Then, chill on ice.
5. Set up the ligation reaction on ice. Add the following to the blunting reaction mixture:
Component Volume
pJET1.2/blunt (50 ng/µL)
T4 DNA ligase
1 µL
Total volume 20 µL

7 Incubate the ligation mixture at room temperature (20°C) for 5 min. For PCR products >3
kb, ligation can be prolonged to 30 min.
8. Use the ligation mixture directly for transformation.
Note: Keep the ligation mixture at –20°C if transformation is postponed. Thaw on ice and
mix carefully before transformation.
5
Lab B: One Minute Transformation Protocol
Heat-shock Cold Plating
1. Thaw competent cells (1 vial) under the running tap water for ~20 sec until 1/3 is thawed.
2. Add DNA (pre-chilled on ice, the volume should be ≤ 5% of competent cells) immediately.
3. Vortex for 1 sec or tap the tube with a finger to mix well.
4. Keep tubes on ice for 1~5 min.
5. Heat-shock the cells in the pre-warmed 42oC water bath for 45 sec.
6. Plate the cells using Platting Beads onto a pre-chilled (4oC) and dried LB agar plate
7. Incubate the plates at 37oC for 24 hr.
8. Count the number of white and blue colonies produced on the agar.
9. Store the result at 4oC for the next lab (Plasmid extraction).
10. Record and discuss the results.
6
APPENDIX A
Vector map of yT&A cloning vector
Figure 1: Map and sequence reference points of the yT&A cloning vector.

EXPERIMENT 5 - Cloning Clone JET PCR Cloning

Uploaded by

Copyright:

Available Formats

EXPERIMENT 5 - Cloning Clone JET PCR Cloning

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

EXPERIMENT 5 - Cloning Clone JET PCR Cloning

Uploaded by

Copyright:

Available Formats

CBB40203 DNA TECHNOLOGY

LAB 3: CloneJET PCR Cloning Kit

1. Thoroughly mix every vial before use.

Note: All components of this kit should be stored at 20°C.

Length of PCR product Optimal quantity of PCR product for

3) Ampicillin stock solution

For the final volume of reagent from stock solution is as following:

1. Autoclave 500 ml of LB medium

LAB A - Option 1: Protocol for blunt end cloning

 For cloning blunt-end PCR products generated by proofreading DNA polymerases,

 For cloning blunt-end DNA fragments generated by restriction enzyme digestion,

1. Set up the ligation reaction on ice.

2. Set up the following items as described in Table 2:

Table 2: Mixing recipe for ligation of inserts

3. Vortex briefly and centrifuge for 3–5 sec.

LAB A - Option 2 – Protocols For Sticky-end Cloning

• For cloning DNA fragments with 5´ or 3´ overhangs generated by restriction enzyme

1. Set up the blunting reaction on ice:

3. Vortex briefly and centrifuge for 3–5 sec.

6. Vortex briefly and centrifuge for 3–5 sec.

Lab B: One Minute Transformation Protocol

Heat-shock Cold Plating

Vector map of yT&A cloning vector

You might also like

Pfad - The Proxy pFad of © 2024 Garber Painting. All rights reserved.