Contributor: Namjin Chung
Date: June 18, 1996
1. An average-size yeast colony (0.5-2mm) or a cell pellet from a liquid culture is touched with a
sterile pipette tip.
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.
2. Rinse the tip with 10uL Zymolyase Solution* by pipetting up and down 3-5 times.
3. Incubate for 5 min at 37C.
4. Use 2uL spheroplasted yeast cells for 50uL PCR reaction.** Overlay one drop of mineral oil.
Run PCR with as follows:
Initial denaturation at least for 3min at 94C;
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.
5. After removing mineral oil, load 10uL PCR reactants on 1% agarose gel.
* Zymolyase Solution
2.5mg/mL Zymolyase (ICN)
1.2M sorbitol
0.1M Na phosphate, pH 7.4
Aliquots of Zymolyase Solution can be stored at -20C for at least 6
months.
** PCR reaction (per 50uL)
1x PCR buffer
200uM dNTPs
1uM each primer
0.5 uL Pfu polymerase (Stratagene) or other DNA polymerase
Reference
1. NAR (1995) 23, 4924-5.
2. Biotechniques (1995) 19, 745-7.
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