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.
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 |