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.
modified September 2000.