Team:Washington/Protocols/PCR

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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

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