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 || |
|3% Difco nutrient broth || |
|1% Difco yeast extract || |
|2% Bacto agar || |
water to the final volume
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) || |
|0.005% Zinc Acetate ||0.5% Zinc acetate (1g/200ml) || |
Resuspend the stocks in dH2O and filter sterilize. The stocks are mixed in sterile water just before sporulation.
New alternate protocol