Corum:Whole Plasmid PCR

Overview

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.

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: T_a °C = 20 s, where T_a = T_m - 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

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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

• Sean P Corum
• SC 18:46, 23 July 2012 (EDT):

or instead, discuss this protocol.