Difference between revisions of "Corum:Whole Plasmid PCR"
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PCR protocol using DNA polymerase. PFU ligaseis used in to repair the nicked plasmid products as PCR proceeds. It is imperative to use 5'-phosphorylated primers for the ligase to repair the nicks.
Revision as of 20:14, 25 August 2012
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
- 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:
- Initial melting: 95 °C 2 min
- Melting: 95 °C 20 s
- Annealing: Ta °C = 20 s, where Ta = Tm - 5 °C
- Elongation: 72 °C 2 min / kb template
- Repeat steps 2-4 a total of 12-20 times (20 is standard).
- Final elongation: 72 °C 30 min
- 12 °C hold
- Verify product with gel electrophoresis.
- Quantify product with quantifluore DNA quantification.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
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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.