Alternate Sporulation Method for BY4743
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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 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.
