Overview
Colony PCR can be used to identify colonies where your favorite gene yfg1 has been replaced with a marker gene by homologous recombination, and to distinguish homologous recombination events from non-homologous. It can also be used to identify colonies from a tetrad that carry a particular gene replacement. This is not a substitute for Southern blotting, but an adjunct. See references below.
Protocol
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 4 °C for up to 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 cells off the tip with 10 uL Zymolyase Solution by pipetting up and down; this spheroplasts them.
3. Incubate for 5-15 min at 37 °C.
4. Use 2 uL spheroplasted yeast cells for 50 uL PCR reaction. (we use AmpliTaq)
Spheroplasted cells should be made fresh; if not PCR won't be as efficient.
Zymolyase Solution
· 2.5mg/mL Zymolyase (20T)
· 1.2M sorbitol
· 0.1M Na phosphate, pH 7.4
Aliquots of Zymolyase Solution can be stored at -20 °C for at least 6 months.
Colony PCR can be used to identify colonies where your favorite gene yfg1 has been replaced with a marker gene by homologous recombination, and to distinguish homologous recombination events from non-homologous. It can also be used to identify colonies from a tetrad that carry a particular gene replacement. This is not a substitute for Southern blotting, but an adjunct. See references below.
Protocol
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 4 °C for up to 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 cells off the tip with 10 uL Zymolyase Solution by pipetting up and down; this spheroplasts them.
3. Incubate for 5-15 min at 37 °C.
4. Use 2 uL spheroplasted yeast cells for 50 uL PCR reaction. (we use AmpliTaq)
Spheroplasted cells should be made fresh; if not PCR won't be as efficient.
Zymolyase Solution
· 2.5mg/mL Zymolyase (20T)
· 1.2M sorbitol
· 0.1M Na phosphate, pH 7.4
Aliquots of Zymolyase Solution can be stored at -20 °C for at least 6 months.
