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Sporulation and Dissection From Kristina Schmidt, USF
 

Summary: Sporulation and Dissection
From Kristina Schmidt, USF
Sporulation
1. Patch out diploid from -80C stock on a fresh YPD plate. Incubate for 2 days at 30C.
2. Lightly inoculate 2.5 ml of YPD liquid medium. Grow 24 hours in drum rotator at 30C.
3. Transfer 250 l 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 l 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 l
2X Leucine 333X Leu 60 l
2X Uracil 100X Ura 200 l
50 g/ml Zinc Acetate 5 mg/ml ZnOAc 100 l
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 30C with shaking.

  

Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida

 

Collections: Biology and Medicine