Yeast colony PCR
(analytic)
* take cells from a fresh plate (it doesn't work as well if the plate comes out of the
fridge)
* with a p2 pipet tip resuspend less than a microliter of the yeast colony in 25 µl PCR
mastermix (Too many cells will hamper the reaction)
* run PCR with the following parameters:
1. 95 °C, 10 min
2. 95 °C, 30 sec.
3. 50-55 °C, 30 sec.
4. 72 °C, 1min/kb
5. 72 °C, 3 min
run 35 cycles (steps 2 to 4)
* load the whole reaction on agarose gel
Buffers:
PCR mastermix, per reaction:
0.5 µl Taq-polymerase (5 U/µl); 2.5 µl 10 x buffer; 1 µl 25 x dNTPs (5 mM); 0.5 µl each
primer (100 pmol/µl);
20 µl H2O