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        Alternate Sporulation Method for BY4743

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        621

         

        Alternate Sporulation Method for BY4743

        Protocol:

         

        1. Patch cells that have been freshly grown on YPD to freshly prepared GNA pre-sporulation plates for 1 day at 30°C.
        Repatch to another freshly prepared GNA pre-sporulation plate for one more day at 30°C.

        2. Transfer to liquid sporulation medium by resuspending a small amount of cells int 2mls of supplemented liquid sporulation medium.
        Don't put in too many cells!

        3. Incubate on a roller wheel for 5 days at 25°C followed by 3 days at 30°C.
        The sporulated cultures can be stored at 4°C for up to one week before dissection; older cultures lose viability.

        Sporulation efficiencies should be around 30%.

         

        Materials and Reagents:

        GNA Presporualtion 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 no more than 1-2 days old.

        Supplemented 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

        1 x ura supplement 100 x ura supplement solution
        2 mls
        1 x his supplement 200 x his supplement solution
        1 ml
        1 x leu supplement 200 x leu supplement solution
        1 ml
           
        water to final volume

        This is an updated sporulation from Linda Riles and Matt Curtiss.

         

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