Corum:Whole Plasmid PCR
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
- In a PCR tube, mix the components on ice in the order they are listed above.
- Perform the following thermocycling program:
- 95 °C 1 min (initial melting)
- 95 °C 30 s (melting)
- TH = 55 °C 60 s (annealing, ligation)
- 72 °C 2 min for each 1 kb PCR product (elongation)
- Repeat steps 2-4 a total of 12-20 times (20 is standard).
- 72 °C 20 min (final elongation)
- 55 °C 60 min (final ligation)
- 12 °C hold
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
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Relevant papers and books
- Sambrook, J and Russell, DW (2001) Molecular Cloning: A Laboratory Manual (Volume II) - Cold Spring Harbor Laboratory Press ISBN 0879695773
- Sean P Corum
- SC 18:46, 23 July 2012 (EDT):
or instead, discuss this protocol.