Guidelines for Using Phusion Flash PCR Master Mix

Protocol

1. Basic reaction conditions for PCR amplifications
   Carefully mix and spin down the Phusion Flash PCR Master Mix tube before opening to improve recovery. The PCR setup can be performed at room temperature.
   
   Pipetting instructions

   Component	50 µl reaction	20 µl reaction	Final Conc.
   H2O	add to 50 µl	add to 20 µl
   2x Phusion Flash PCR Master Mix	25 µl	10 µl	1X
   Primer A	x µl	x µl	0.5 µM
   Primer B	x µl	x µl	0.5 µM
   Template DNA	x µl	xµl

2. Cycling conditions:
   
   Cycling instructions

   Cycle step	2-step protocol	3-step protocol	Cycles
   Temp.	Time	Temp.	Time
   Initial denaturation	98°C	10 s	98°C	10 s	1
   Denaturation Annealing Extension	98°C - 72°C	0 or 1 s - 15 s/kb	98°C X°C 72°C	0 or 1 s 5 s 15 s/kb	30
   Final extension	72°C 4°C	1 min hold	70°C 4°C	1 min hold	1