Sporulation and Tetrad Dissection

1) Grow cells at 25ºC overnight in 25ml PSP2 (premade above centrifuge) supplemented with 25% of the recommended concentration of amino acids (liquid stocks in chemical cabinet) that are missing in both of the haploid strains.

2) Spin down cells at 3000rpm for 3min at room temperature. Dump supernatant.

3) Wash with sterile water.

4) Resuspend in 25ml SPM (premade above centrifuge).

5) Incubate at 25ºC 1-2 days. Check for spores under microscope.

6) When spores are present, transfer 1ml to an eppendorf tube. Spin down and aspirate supernatant.

7) Resuspend in 950_l P solution.

8) Add 50_l of 10mg/ml zymolyase.

9) Let stand at room temperature for ~10min.

10) While cells are digesting, cut a YEPD plate to accommodate the dissection arm.

11) Put cells on ice. Check under microscope to see if the cell wall is digested. If not digested, incubate for 10min more.

12) If the cell wall is digested, dilute the cells 1:10 in P solution. Drip ~20_l of diluted cells in a line near the top of the plate. Let dry (10min-3hours).

13) Tetrad dissect.