From 2011.igem.org

Revision as of 22:15, 13 September 2011

General PCR Protocol

1. Setup Amplification PCR Reaction
   1. 1uL Template
   2. 1uL 25mM dNTP's
   3. 10uL Phusion HF Buffer
   4. 0.5uL Forward Primer (Tm 65)
   5. 0.5uL Reverse Primer (Tm 65)
   6. 0.5uL Phusion polymerase
   7. 36.5uL diH2O
2. Amplification PCR Reaction
   1. 98C - 30s
   2. 98C - 10s
   3. 63C - 10s
   4. 72C - 30s/kb target gene
   5. Repeat 2-4 29x
   6. 72C - 5min
   7. 10C - forever

Refer to NEB's online protocol for Phusion:

http://www.neb.com/nebecomm/products/protocol87.asp