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Yeast colony PCR

  1. Resuspend a swath of yeast (about the size of a glass bead) in 12µl lysis buffer.
  2. Incubate at 37°C for 5 min, then 95°C for 5 min.
  3. Dilute the DNA 10 times with water, and use 3µl for 50µl PCR.

lysis buffer:

  • 0.1 M NaPO4 buffer of pH 7.4
  • 2.5 mg/ml Zymolase 20T (21100 U/g, Seikagaku Corporation)

Trouble shooting

Yeast lysis can often be problematic. If the PCRs fail, here are some troubleshooting tips:

  • Use yeast freshly grown O/N on a YEPD plate.
  • Use a long extension time for the PCR (we use 5 min for KOD at 68°C).
  • Do not pause the process. Right after lysis, set up the PCR.