Yeast Sporulation and Dissection



Method 1

  1. Grow up a 5 ml YPD culture at 30oC overnight.

  2. Count the cells the next day, and dilute the culture down to 1E6 cells/ml, again into a total volume of 5 ml YPD.

  3. Let the new culture grow at 30oC 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 distilled H2O.

  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 23oC.


Method 2

  1. 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).

  2. Wash once with 25 ml sterile distilled H2O.

  3. Resuspend in 25 ml SPM media.

  4. Leave 2-3 days and check under microscope for the formation of tetrads.

Dissection

  1. Spin down 1 ml of sporulation culture and wash with sterile distilled H2O three times.

  2. Resuspend in 50 ul of zymolyase solution (0.5 mg/ml zymolyase in 1M sorbitol).

  3. Incubate for 8-10 minutes at 30oC.

  4. Slowly add 0.8 ml sterile distilled H2O to the tube and place it on ice.

  5. Spread some on a plate and DISSECT!!!


PSP2 media

To make 1 L: 

  • 6.7 g YNB without amino acids
  • 1 g Yeast extract
  • 10 g KOAc

Fill to 1 L with 50 mM potassium phthalate pH5.0 buffer and autoclave to sterilize.

SPM media

To make 1 L: 

  • 3 g KOAc
  • O.2 g raffinose
Fill to 1 L with sterile distilled H2O.


Dissection of Tetrads
Pictures courtesy of Gottschling Lab.


Dissect1 Dissect2 Dissect3
Dissect4 Dissect5 Dissect6