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Latest revision as of 02:13, 4 October 2012

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 XX ^oC)*
   5. 0.5uL Reverse Primer (Tm YY ^oC)*
   6. 0.5uL Phusion polymerase
   7. 36.5uL diH2O
2. Amplification PCR Reaction
   1. 98 ^oC - 30s
   2. 98 ^oC - 10s
   3. ZZ ^oC - 10s*
   4. 72 ^oC - 30s/kb target gene
   5. Repeat 2-4 29x
   6. 72 ^oC - 5min
   7. 10 ^oC - forever

• Tms of all primers must first be calculated after primer design. Annealing temperature (represented by ZZ above) should be 2 ^oC higher than XX or YY, whichever is lower.

Refer for further details to NEB's online protocol for Phusion:

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

Equipment Necessary

1. DNTPs
2. Phusion DNA Polymerase
3. Forward/Reverse Primers
4. Phusion Buffer

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