Zymolyase Solution:
- Regular stock of zymolyase is 10 mg/ml diluted in water, mix by inverting, not all will go into solution (filter it) store aliquots at -20°C (minimize freeze-thawing if possible)
- Make 2.5 mg/ml zymolyase solution in 0.1 M sodium phosphate buffer pH 7.5 immediately before the PCR experiment
Lysis of Yeast:
- Aliquot 15 µl 2.5 mg/ml enzyme solution (in 0.1 M sodium phosphate buffer pH 7.5) into thin-walled PCR tubes
- Scrape a small amount of yeast colony and resuspend in a PCR tube (one lysis reaction when dilute will yield ~15 PCR reactions, only 5 µl is used for a template in each PCR reaction)
- Incubate on the bench (RT) for 20 min
- Place in PCR block and heat to 37°C for 5 min and then 95°C for 5 min
PCR Step:
- Dilute the lysis solution 1:5 by adding 60 ul ddH2O to each PCR tube
- Place 5 ul of this dilution into a fresh PCR tube
- Add PCR mix (usually 45 ul total per reaction):
- 2.5 µl 5’ Primer (10 µM)
- 2.5 µl 3’ Primer (10 µM)
- 5 µl Thermopol Buffer
- 1 µl dNTPs (10 uM)
- 33.5 µl ddH2O
- 0.5 µl AmpliTaq or Vent DNA Polymerase
- Run the Lyse Program (LysAn50), which takes about 5 hours
- Check product by running 10 µl PCR sample + 2 µl 6x DNA dye on 1% agarose gel
