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