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Herman Lab

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 
Phusion0.2 µl 
Primer F0.75 µl 
Primer R0.75 µl 
DNA1-2 µl(test yourself)
dNTPs2 µl  
ddH20to 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 °C2 min 
72 °C5 min 
10 °C Hold