Sporulation Method for BY4743.v1
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Efficient sporulation of BY4743 requires growth as a patch on freshly prepared GNA presporulation plates at 30°C overnight prior to transfer to liquid sporulation medium. (Note: if GNA is inoculated with an old culture, transfer to another GNA plate for an additional day.) Resuspend one colony equivalent of cells into 2mls of liquid sporulation medium in a 17x150mm glass test tube. Sporulation cultures should be incubated on a roller wheel for 3-5 days at 25°C. The cultures should be ready to dissect and can be stored at 4°C for a couple of weeks. However, dissection within two weeks is the best. Sporulation efficiencies should be around 20-30%. If you prespore on YPD instead of GNA the sporulation efficiency is much lower.
	
	GNA Presporulation 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 last only for 1-2 weeks at 4°C for optimum sporulation.
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 | 
Resuspend the stocks in dH2O and filter sterilize. The stocks are mixed in sterile water just before sporulation.









