Corum:Whole Plasmid PCR

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Whole plasmid PCR protocol using PFU Ultra DNA polymerase.


For a 50 μL whole plasmid PCR PCR reaction:

  • 35 μL H2O
  • 5 μL 10X PFU Ultra PCR buffer
  • 5 μL 2mM (each) dNTP mix
  • 1 μL 5nM plasmid template (0.1 nM final)
  • 1 μL 10μM sense primer (200nM final)
  • 1 μL 10μM antisense primer (200nM final)
  • 1 μL PFU Ultra DNA polyermerase
  • 1 μL PFU ligase


  1. In a PCR tube, mix the components on ice in the order they are listed above.
  2. Perform the following thermocycling program:
    1. 95 °C 1 min
    2. 95 °C 30 s
    3. TH = 55 °C 30 s
    4. 72 °C 2 min for each 1 kb PCR product
    5. Repeat steps 2-4 a total of 12-36 times (24 is standard).
    6. 72 °C 20 min
    7. 12 °C hold


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

  1. List troubleshooting tips here.
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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


or instead, discuss this protocol.

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