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.
