Summary: Sporulation and Dissection
From Kristina Schmidt, USF
1. Patch out diploid from -80°C stock on a fresh YPD plate. Incubate for 2 days at 30°C.
2. Lightly inoculate 2.5 ml of YPD liquid medium. Grow 24 hours in drum rotator at 30°C.
3. Transfer 250 µl of culture to a sterile 1.5 ml tube. Pellet at top speed for 30 seconds.
4. Remove supernatant and resuspend pellet in 0.5 ml of sterile dH2O (to wash).
5. Pellet cells at top speed for 30 seconds. Remove supernatant.
6. Resuspend cells in 250 µl of freshly prepared sporulation medium.
Final Concentration Stock (Sherman) 10 ml
1% Potassium Acetate 0.1 g
dH2O 9.5 ml
2X Histidine 500X His 40 µl
2X Leucine 333X Leu 60 µl
2X Uracil 100X Ura 200 µl
50 µg/ml Zinc Acetate 5 mg/ml ZnOAc 100 µl
Prepare sporulation medium just before use. Mix and syringe filter sterilize. Freshly prepare
ZnOAc stock (5 mg/ml = 0.1 g in 20 ml). Do not store ZnOAc stock for later use.
7. Transfer to sterile GLASS test tube containing 4.75 ml of sporulation medium.
8. Incubate 3-5 days at 30°C with shaking.