Corum:Whole Plasmid PCR

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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.


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


  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.



  1. List troubleshooting tips here.



Relevant papers and books



or instead, discuss this protocol.

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