Yeast Sporulation and Dissection

• Sporulation Method 1
• Sporulation Method 2
• Yeast Dissection
• Pictures of Yeast Dissection

Method 1

1. Grow up a 5 ml YPD culture at 30^oC 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 30^oC 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 23^oC.
   
   

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 30^oC.
   
   
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!!!
   
   

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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.

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Dissection of Tetrads
Pictures courtesy of Gottschling Lab.