PCR reactions (generic)
O. Gjoerup 1-14-2000
100 µl total volume – use tiny tubes, mini-rack – keep on ice.
1. Spin down lyophilized primer – resuspend in sterile water at 50 picomoles/ µl (mix
thoroughly to dissolve). Store frozen.
2. Add 1 µl (50 pmoles) of upstream and 1 µl (50 pmoles) of downstream primer to the
tube.
3. Add 82 µl of sterile, ‘good quality’ water.
4. Add 10 µl of 10xThermopol. Buffer (for Vent polymerase, NEB)
5. Add 4 µl 10 mM dNTP mix (contains 10 mM of each dATP, dTTP, dCTP, dGTP,
e.g. make from 100 mM Pharmacia stock – store frozen). Final dNTP conc. is 400
µM.
6. Add appr. 10 ng of template DNA (in 1 µl)
7. Add 2 U Vent polymerase (1 µl) –start program.
8. (For most machines mineral oil is not required)
Cycling:
96 ˚C 4 min 1 cycle (initial denaturation)
96 ˚C 1 min
|
55 ˚C 1 min
| 30 cycles
72 ˚C 30 s-4 min extension time, roughly 1 min/ kb product
|
Obvious variables:
Annealing temp., Enzyme (e.g. try Pfu, Taq), Mg2+ conc., primer design.