Yeast colony PCR

Use a fresh yeast colony! Old colonies don't work as well, even if they've been refrigerated.

Steps

1. Aliquot 40 ul of 20 mM NaOH into a tube
2. Lightly touch a colony with a pipette tip, re-suspend in the NaOH. Solution should be slightly cloudy but still transparent. If it gets too opaque, you have too many cells and you'll have too much DNA for the PCR
3. Incubate 20 min at 95C
4. Spin down until you can see the cell pellet (can just use a small tabletop spinner, or a microcentrifuge)
5. Use 1 ul of supernatant as a template in a PCR, 35 cycles
6. Take 1 ul of PCR product, use as template for another PCR, 35 cycles
7. Run gel to confirm band size

PCR recipe

Reagent	Volume (µl)
Q5 2X master mix	10
template	1
FWD primer, 10 µM	1
REV primer, 10 µM	1
ddH2O	7
TOTAL	20

PCR thermal cycler protocol

• 98C, 3 m
• Repeat 35x:
  • 98C, 30s
  • Ta, 30s
  • 72C, 60s
  • 72C, 2m