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Sporulation Method for BY4743.v1
click here for v.2

 

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.

 

New alternate protocol