Corum:Whole Plasmid PCR  From OpenWetWare  Revision as of 22:19,
25 August 2012 by Sean P Corum (Talk | contribs) Jump to:
navigation, search    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.   For a 50 μL whole plasmid PCR PCR reaction:    • 36
μL H2O   • 5 μL 10X PFU Ultra PCR buffer   • 5 μL 100 μM
sense/antisense primer mix (10 μM final)   • 1 μL 5 nM plasmid
template (0.1 nM final)   • 1 μL PfuUltra II fusion HS DNA
polymerase   • 1 μL PFU ligase     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: Ta °C = 20 s, where Ta = Tm - 5 °C     4.
Elongation: 72 °C 2 min / kb template     5. Ligatio