PCR protocol

We use the Thermopol PCR conditions described by New England Biolabs.

Digestion protocol (biopsies or ES cells)
100µl of 50mM NaOH.
Tap or spin down quickly.
Place tubes in a 100°C heating block for 30 min.
Add 30µl 1M Tris, pH 7.4 to neutralize, mix well.
Use 1µl of the lysate as DNA template for PCR or scale appropriately.

10x PCR Thermopol buffer
0.2M Tris pH 8.8
0.1M KCl
0.1M (NH4)2SO4
20mM MgSO4>
1% Triton

PCR Reactions
10x ThPol buffer            2.5
dNTP (25mM)                0.2
Primer-F (10µM)            0.5
Primer-R (10µM)            0.5
H2O                             17.8
10x Rediload                2.5
(DNA)                            (1)


add 0.12 µl Taq per reaction

DNA is added last.
Cover with 1 drop of paraffin oil.

If preparing a master mix, keep reactions on ice until ready for the thermocycler.
Start step 1, pause at 93°C, place samples in the machine.
Most of our PCRs work with a 60°C annealing temperature:

1. 93°C for 3 min
2. 93°C for 15 sec
3. 60°C for 30 sec
4. 68°C for 30 sec
5. Go to step 2, 34 times
6. 68°C for 2 min
7. End.