Corum:Whole Plasmid PCR

Overview

Whole plasmid PCR protocol using PFU Ultra DNA polymerase. PFU ligase is used in the reaction to repair the nicked plasmid products as PCR proceeds. It is imperative to use 5'-phosphorylated primers for the ligase to repair the nicks.

Materials

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

Procedure

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 (initial melting)
   2. 95 °C 30 s (melting)
   3. T_H = 55 °C 60 s (annealing, ligation)
   4. 72 °C 2 min for each 1 kb PCR product (elongation)
   5. Repeat steps 2-4 a total of 12-20 times (20 is standard).
   6. 72 °C 20 min (final elongation)
   7. 55 °C 60 min (final ligation)
   8. 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.