Whole Plasmid PCR-PDF

Overview

It is imperative to use 5′-phosphorylated primers if the nicked DNA is going to be repaired downstream with ligase. PCR should be limited to templates of about 5 kb, but the protocol could probably be pushed up to 10 kb. See the PfuUltra II fusion manual. Primers should be sense-antisense pairs. Optimal amplification occurs with 30-35 b primers at 0.5 μM final concentration. At higher concentrations, the primers bind to each other, inhibiting amplification. For longer primers, the annealing temperature will need to be adjusted from the one mentioned in this protocol.

Materials

For a 50 μL WP-PCR reaction:

• 37 μL H₂O
• 5 μL 10X PFU Ultra PCR buffer
• 5 μL 10 μM sense/antisense primer mix (0.5 μM final, each)
• 1 μL 12.5 mM (each) dNTP mix (0.25 mM final)
• 1 μL 5 nM plasmid template (0.1 nM final)
• 1 μL PfuUltra II fusion HS DNA polymerase

Procedure

1. In a PCR tube, add 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 30 times
   6. Final elongation: 72 °C 30 min
   7. 12-16 °C hold