Sporulation Protocol#1

1. Grow up a small (5mL) overnight culture in YEPD (rich glucose media), 30°C.
2. Count the cells the next day, and dilute the culture down to 1E6 cells/mL, again into a total volume of 5 mL of rich media.
3. Let the new culture grow at 30 deg C until it reaches the end of log phase, which is 8-9E7 cells/mL.
4. Spin down the cells and wash them once with sterile water.
5. Resuspend the cells in sporulation media (2% potassium acetate, pH 7.0) supplemented with necessary nutrients. Set up this culture so that the cells are at a concentration of 5E7 cells/ mL, and a total volume of 2.5 mL.
6. Let the cultures sporulate 5-7 days at 23 deg C.

Sporulation Protocol #2

SPM
3 g potassium acetate
0.2 g raffinose
Mix in 1 L of sterile water

1. Grow cells overnight in YEPD.
2. Remove 200 µl, add to 5 ml sterile water.
3. Spin 5 min.
4. Resuspend pellet in 2.5 ml SPM.
5. Incubate at 23°C with good aeration for 2-4+ days and check under microscope for the formation of tetrads.

Yeast Vivisection

1. Spin down 0.5 mL of sporulation culture and resuspend in 1 ml water.
2. Wash with sterile water.
3. Resuspend in 50 µL of zymolyase solution (0.5 mg/mL in 1M sorbitol).
4. Incubate for 8-10 minutes at 30°C.
5. Slowly add 0.8 mL sterile water to the tube and place it on ice.
6. Spread some on a plate and dissect.