non-lethal ß-galactosidase assay
adapted from H. M. Duttweiler, TIG, 12(9), 340, 1996
- grow yeast to be tested for three days. Then overlay the plates with CHCl3 and incubate for 5-7 min.
- carefully decant the CHCl3 and maybe do the hole procedure two times for better evaporation.
- after that invert the plates! Let them dry for about 5 min (untill CHCl3 smell has vanished).
- overlay the plate with 10 ml agarose solution pouring it slowly from one edge of the plate to the other (do not rock the plate).
- invert the plate and incubate at RT or 30°C untill blue colour appears (takes several hours, but staining might be already visible after 15 min).
| Kpi pH=7
|
100 ml
|
| 1 M K2HPO4 |
61.5 ml
|
| 1 M KH2PO4 |
38.5 ml
|
mix solutions and adjust pH
| Agarose Solution
|
concentration
|
100 ml
|
| 20 mg/ml X-ß-Gal (in DMF) |
1 mg/ml |
5 ml
|
| 1 M Phosphate Buffer |
100 mM |
10 ml
|
| Low melting agarose |
0.3% |
500 mg
|
melt 300 mg Agarose in 85 ml water and cool it down to 45°C. Then add the 1M Phosphate Buffer and immediately before use the X-Gal.
|
