Yeast Colony PCR
1. Pipette 10 ul NaOH (0.02 M) to a pcr tube
2. Take a single colony from your plate and resuspend it in the tube.
3. Boil it in a pcr machine for 10 min at 99 C.
4. centrifuge the tube for few seconds to precipitate cell debries
5. Use 2-3 ul of the supernatant as template in 20 ul pcr reaction.
* For yeast cPCR, I have got better results with Kappa (robust) polymerase in compare to Phusion or Taq specially if fragment size is larger than 500 bp.
*Kappa polymerase reaction mix (*20 μl rxn)
5x KAPA2G Buffer B 4.0 μl
dNTP mix 10 mM 0.40 μl
KAPA2G Robust 0.10 μl
Template genomic DNA (see above) 2-3 ul
Primer forward & reverse
add water to final volume of 20 μl