Yeast colony PCR
- Resuspend a swath of yeast (about the size of a glass bead) in 12µl lysis buffer.
- Incubate at 37°C for 5 min, then 95°C for 5 min.
- Dilute the DNA 10 times with water, and use 3µl for 50µl PCR.
- 0.1 M NaPO4 buffer of pH 7.4
- 2.5 mg/ml Zymolase 20T (21100 U/g, Seikagaku Corporation)
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.