Corum:Whole Plasmid PCR

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==Procedure==
==Procedure==
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# In a PCR tube, mix the components on ice in the order they are listed above.
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# In a PCR tube, mix the components on ice in the order they are listed above. Mix gently and spin.
# Perform the following thermocycling program:
# Perform the following thermocycling program:
## Initial melting: 95 °C 2 min
## Initial melting: 95 °C 2 min

Revision as of 23:11, 25 August 2012

Contents

Overview

Whole plasmid PCR protocol using PFU Ultra DNA polymerase. PFU ligase is used in the reaction to repair the nicked plasmid products as PCR proceeds. It is imperative to use 5'-phosphorylated primers for the ligase 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 PFU Ultra II high-fidelity DNA polyermerase

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. Repeat steps 2-4 a total of 12-20 times (20 is standard).
    6. Final elongation: 72 °C 30 min
    7. 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!

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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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