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Alternate Sporulation Method for BY4743

Protocol:

1. Patch cells that have been freshly grown on YPD to freshly prepared GNA pre-sporulation plates for 1 day at 30°C.
Repatch to another freshly prepared GNA pre-sporulation plate for one more day at 30°C.

2. Transfer to liquid sporulation medium by resuspending a small amount of cells int 2mls of supplemented liquid sporulation medium.
Don't put in too many cells!

3. Incubate on a roller wheel for 5 days at 25°C followed by 3 days at 30°C.
The sporulated cultures can be stored at 4°C for up to one week before dissection; older cultures lose viability.

Sporulation efficiencies should be around 30%.

Materials and Reagents:

GNA Presporualtion Plates

Final Concentrations	recipe for 2 plates	recipe for 10 plates
5% D-glucose	5 gm	25 gm
3% Difco nutrient broth	3 gm	15 gm
1% Difco yeast extract	1 gm	5 gm
2% Bacto agar	2 gm	10 gm
water to the final volume	100 ml	500 ml

Autoclave for 15 minutes. Plates should always be freshly made and no more than 1-2 days old.

Supplemented Liquid Sporulation Medium

Final Concentrations	Stock solutions	recipe for 200 mls
1% Potassium Acetate	10% Potassium Acetate (20g/200ml)	20 mls
0.005% Zinc Acetate	0.5% Zinc acetate (1g/200ml)	2 mls
1 x ura supplement	100 x ura supplement solution	2 mls
1 x his supplement	200 x his supplement solution	1 ml
1 x leu supplement	200 x leu supplement solution	1 ml
		water to final volume

This is an updated sporulation from Linda Riles and Matt Curtiss.

modified September 2000.