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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 30C.
Repatch to another freshly prepared GNA pre-sporulation plate for one more day at 30C.

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 25C followed by 3 days at 30C.
The sporulated cultures can be stored at 4C 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.

modified September 2000.