Corum:Whole Plasmid PCR

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* 1 μL 12.5 mM (each) dNTP mix (0.25 mM final)
* 1 μL 12.5 mM (each) dNTP mix (0.25 mM final)
* 1 μL 5 nM plasmid template (0.1 nM final)
* 1 μL 5 nM plasmid template (0.1 nM final)
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* 1 μL PFU Ultra II high-fidelity DNA polyermerase
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* 1 μL [http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1095 PfuUltra II fusion HS DNA polymerase]
==Procedure==
==Procedure==
Line 20: Line 20:
## Annealing: T<sub>a</sub> °C = 20 s, where T<sub>a</sub> = T<sub>m</sub> - 5 °C
## Annealing: T<sub>a</sub> °C = 20 s, where T<sub>a</sub> = T<sub>m</sub> - 5 °C
## Elongation: 72 °C 2 min / kb template
## Elongation: 72 °C 2 min / kb template
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## Ligation: 55 °C 30 s (time?)
## Repeat steps 2-4 a total of 12-20 times (20 is standard).
## Repeat steps 2-4 a total of 12-20 times (20 is standard).
## Final elongation: 72 °C 30 min
## Final elongation: 72 °C 30 min
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## Final ligation: 55 °C 1 hr
## 12 °C hold
## 12 °C hold
# Verify product with [http://openwetware.org/wiki/Corum:Gel_Electrophoresis gel electrophoresis].
# Verify product with [http://openwetware.org/wiki/Corum:Gel_Electrophoresis gel electrophoresis].

Revision as of 22:17, 25 August 2012

Contents

Overview

Whole plasmid polymerase chain reaction (WP-PCR) protocol using PfuUltra II fusion HS DNA polymerase. PFU ligase is used in parallel to repair the nicked plasmid products as PCR proceeds. It is imperative to use 5'-phosphorylated primers for the ligase to be able to repair the nicks.

Materials

For a 50 μL whole plasmid PCR PCR reaction:

  • 37 μL H2O
  • 5 μL 10X PFU Ultra PCR buffer
  • 5 μL 100 μM sense/antisense primer mix (10 μM final)
  • 1 μL 12.5 mM (each) dNTP mix (0.25 mM final)
  • 1 μL 5 nM plasmid template (0.1 nM final)
  • 1 μL PfuUltra II fusion HS DNA polymerase

Procedure

  1. In a PCR tube, mix the components on ice in the order they are listed above. Mix gently and spin.
  2. Perform the following thermocycling program:
    1. Initial melting: 95 °C 2 min
    2. Melting: 95 °C 20 s
    3. Annealing: Ta °C = 20 s, where Ta = Tm - 5 °C
    4. Elongation: 72 °C 2 min / kb template
    5. Ligation: 55 °C 30 s (time?)
    6. Repeat steps 2-4 a total of 12-20 times (20 is standard).
    7. Final elongation: 72 °C 30 min
    8. Final ligation: 55 °C 1 hr
    9. 12 °C hold
  3. Verify product with gel electrophoresis.
  4. Quantify product with quantifluore DNA quantification.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

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References

Relevant papers and books

  1. Sambrook, J and Russell, DW (2001) Molecular Cloning: A Laboratory Manual (Volume II) - Cold Spring Harbor Laboratory Press ISBN 0879695773

Contact

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