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        Sporulation Method for BY4743.v1

        互联网

        882

         

         

        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.

         

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