PCR using Phusion Taq
WHEN DO YOU USE PHUSION?
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.
For a 40µl reaction. (Do not try PCR for the first time at 40ul- that is a waste!)
|Phusion buffer (Mg added)||4 µl|
|Primer F||0.75 µl|
|Primer R||0.75 µl|
|DNA||1-2 µl||(test yourself)|
|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|