Phusion PCR

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GroseLab:Protocols

PCR Protocol for Phusion Polymerase

Reference: Adapted from New England BioLabs Phusion Hot Start High-Fidelity DNA Polymerase documentation.

Phusion Polymerase Reaction
Components Final Concentration 1x Volume Volume for Master
H2O N/A 33.1 ul __
5x Phusion HF Buffer 1x 10 ul __
25 mM DNTPs 200 uM 0.4 ul __
DMSO, Betaine, etc. N/A N/A __
Phusion Polymerase 0.02 U/ul 1 U = 0.5 ul __
Primers 0.5 uM 2.5 ul each = 5 ul __
Template DNA N/A 1 ul __
  • For difficult templates replace HF Buffer with GC Buffer and add DMSO or Betaine as required.
  • Do not exceed 2 U polymerase/50 ul (0.04 U/ul), especially for amplicons > 5 kb.

98 x 0:30, x (98 x 0:10, ___ x 0:20, 72 x ___), 72 x 7:00, 4 x Infinity

Primers and Tms

Forward:

Reverse:

Amplicons


TM Calculator for Phusion

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