Intact cells, a colony on a plate or liquid cultures which are stored at 4C for upto 3 months could still be used for this method.
Wooden toothpicks should be avoided because they may interfere either release of DNA from yeast cells or PCR reaction itself.
Initial denaturation at least for 3min at 94C;5. After removing mineral oil, load 10uL PCR reactants on 1% agarose gel.
30 cycles of;
30sec at 94C;
30sec at an appropriate annealing temperature;
> 1min/kb at 72C for extenstion;
7min at 72C for final extension.
The remaining spheroplast samples can be stored at -20C for repeated use.
* Zymolyase Solution
2.5mg/mL Zymolyase (ICN)
0.1M Na phosphate, pH 7.4
Aliquots of Zymolyase Solution can be stored at -20C for at least 6