Yeast Sporulation and Dissection
Grow up a 5 ml YPD culture at 30oC overnight.
Count the cells the next day, and dilute the culture down to 1E6 cells/ml, again into a total volume of 5 ml YPD.
Let the new culture grow at 30oC until it reaches the end of log phase, which is 8-9E7 cells/ml.
Spin down the cells and wash them once with sterile distilled H2O.
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.
Let the cultures sporulate 5-7 days at 23oC.
Grow cells in 25 ml of PSP2 (in 250 ml flask) supplemented with 25% of the recommended concentration of amino acids until they are about 1E7 cells/ml (~1 day).
Wash once with 25 ml sterile distilled H2O.
Resuspend in 25 ml SPM media.
Leave 2-3 days and check under microscope for the formation of tetrads.
Spin down 1 ml of sporulation culture and wash with sterile distilled H2O three times.
Resuspend in 50 ul of zymolyase solution (0.5 mg/ml zymolyase in 1M sorbitol).
Incubate for 8-10 minutes at 30oC.
Slowly add 0.8 ml sterile distilled H2O to the tube and place it on ice.
Spread some on a plate and DISSECT!!!
To make 1 L:
Fill to 1 L with 50 mM potassium phthalate pH5.0 buffer and autoclave to sterilize.
- 6.7 g YNB without amino acids
- 1 g Yeast extract
- 10 g KOAc
To make 1 L:
Fill to 1 L with sterile distilled H2O.
Dissection of Tetrads
Pictures courtesy of Gottschling Lab.