Corum:Whole Plasmid PCR

Overview

Whole plasmid PCR protocol using PFU Ultra DNA polymerase.

Materials

For a 50 μL whole plasmid PCR PCR reaction:

• 36 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

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
   2. 95 °C 30 s
   3. T_H = 55 °C 30 s
   4. 72 °C 2 min for each 1 kb PCR product
   5. Repeat steps 2-4 a total of 12-36 times (24 is standard).
   6. 72 °C 20 min
   7. 12 °C hold

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

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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.