Mating

1. Label 3 areas on a YPD plate: 1 for each parental strain, and 1 for a mating patch. 

2. Using a toothpick, patch part of a colony of 1 parental strain to the parental patch and part of the colony to the mating patch.

3. Patch part of a colony of the other parental strain to the parental patch, and part of the colony to the mating patch, mixing it well with the other strain already on the plate. 

4. Incubate at 30°C overnight.

Diploid selection and sporulation

1. Using plates that are selective for mated cells only (e.g. SC-HIS + G418), streak the two parental strains and the mating patch on to the plate for single colonies. 

2. Incubate at 30°C for 2 days.  

3. After the incubation, only diploids should be growing on the plate and neither parental strain should grow.  Take 4 diploid colonies and streak them on to YPD for single colonies. 

4. Incubate at 30°C for 2 days.

5. From each of the 4 diploids, patch large colonies on to SPO plates. 

6. Incubate at room temperature for 3-7 days.  Start checking for tetrads after 3 days, although they may not be visible until 4-5 days. 

Tetrad dissection

1. Resuspend a small section of cells from the SPO patch in 500mL of sterile water.

2. Spin at 10Krpm, 2 minutes.  Remove supernatant.

3. Wash in 1mL of sterile water.  Spin at 10Krpm, 2 minutes, and remove supernatant.

4. Resuspend cells in 19mL of sterile water.  Add 1mL of zymolyase (10mg/mL stock).

5. Incubate at 30°C for 10 minutes. 

6. Add 200mL of sterile water and place immediately on ice to stop the digestion. 

7. Drip 15mL of the solution down the center of a YPD plate.  Let dry for a few minutes prior to beginning tetrad dissection.