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.
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 |
|
|