PCR using Phusion Taq


When you want to PCR a long fragment and you need the amplicon to be perfect. For example when building a construct, or PCRing a gene.

Don’t use Phusion for any ‘normal’ PCR, just because your PCR isn’t working

NOTE: Phusion does not add A’s to the end of the amplicon. It has a proof reading capability which removes any A’s added to the end. This means if you want to then sub-clone you will have to add the A’s yourself.

General MIX
For a 40µl reaction. (Do not try PCR for the first time at 40ul- that is a waste!)

Phusion buffer (Mg added) 4 µl  
Phusion 0.2 µl  
Primer F 0.75 µl  
Primer R 0.75 µl  
DNA 1-2 µl (test yourself)
dNTPs 2 µl  
ddH20 to 40 µl  

General program to start with- optimize as needed

98 °C 1 min  
98 °C 10 sec  
58-65 °C 30 sec
Repeat for 33-36 cycles
72 °C 2 min  
72 °C 5 min  
10 °C Hold