Please find the following protocol:

Sporulation and Dissection of Yeast a/α Diploids


1. Starting with diploid Saccharomyces cerevisiaecells, inoculate 2 ml of YPD/Ade Medium with a single, large colony of diploid cells and grow the culture overnight at 30°C with constant agitation and aeration.

2. Dilute 200 μl of the overnight culture in 5 ml of ddH2O in a conical centrifuge tube.

3. Centrifuge the cells for 2 min at approximately 1,000 X g.

4. Discard the supernatant.

5. Resuspend the cells in 5 ml of ddH2O, and pellet the cells again. Discard the supernatant.

6. Resuspend the cells in 2 ml of 0.5% Potassium Acetate. Add 0.5X nutrients for auxotrophic markers. Remember to omit the nutrients required to select genes harbored on plasmids.

7. Incubate cells with agitation and aeration at room temperature for 3 to 7 days. Aeration is important. Also make sure all solutions are sterile, as growth of culture for this many days may encourage contaminants to grow.

8. Check for sporulation after 3 days by visual inspection with the light microscope. If there are tetrads present (diploid cells have sporulated) you will see four tight cells together in a "diamond" shape surrounded by a thick cell wall.

9. Pellet the tetrads in a glass tube with centrifugation at 1000 X g for 2 min and discard the supernatant.

10. Resuspend the tetrads in 5 ml ddH2O, pellet the tetrads again, and discard the supernatant. Repeat the washes with ddH2O two more times.

11. Resuspend the tetrads in 2 ml of ddH2O.

12. Transfer 180 μl of resuspended tetrads in a microcentrifuge tube

13. Centrifuge in a microcentrifuge at maximum speed for 10 seconds, discard the supernatant, and resuspend the cells in 90 μl of Zymolyase Solution.

9. Incubate the microcentrifuge tube at 30°C for 5 to 10 min.

10. Slowly add 0.3 ml of ddH2O on ice to stop the digestion of tetrad cell walls.

11. Pipette 30 μl of the digested tetrads directly on a YPD plate and dissect the tetrads.


Solutions:

1 M Sorbitol........................................................Filter sterilize
Zymolyase Solution............................0.5 mg/ml in 1 M Sorbitol
0.5% (w/v) Potassium Acetate, pH 7.0........................in ddH2O
100X Nutrients(dilute to working solution directly in media): ..........................................3 mg/ml Lysine
..........................................2 mg/ml Methionine
..........................................3 mg/ml Leucine
..........................................2 mg/ml Histidine
..........................................2 mg/ml Adenine
..........................................4 mg/ml Tryptophan
..........................................2 mg/ml Uracil
YPD/Ade Medium.....................................20 g Dextrose
......................................20 g Peptone
.......................................2 mg/ml Adenine
......................................10 g Yeast Extract


Bioreagents and Chemicals:

Adenine
Potassium Acetate
Uracil
Tryptophan
Lysine
Sorbitol
Zymolyase
Dextrose
Methionine
Histidine
Leucine
Yeast Extract
Peptone


Reference Link:

http://www.bio.com/protocolstools/protocol.jhtml?id=p1149