Phusion PCR

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 --- 33.1 ul 5x Phusion HF Buffer 1x 10 ul 25 mM DNTPs 200 uM 0.4 ul DMSO, Betaine, etc. --- --- Phusion Polymerase 0.02 U/ul 1 U = 0.5 ul Primers 0.5 uM 2.5 ul each = 5 ul Template DNA --- 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 [1]