Quint Lab:non-lethal ß-galactosidase assay

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non-lethal ß-galactosidase assay

adapted from H. M. Duttweiler, TIG, 12(9), 340, 1996

  1. grow yeast to be tested for three days. Then overlay the plates with CHCl3 and incubate for 5-7 min.
  2. carefully decant the CHCl3 and maybe do the hole procedure two times for better evaporation.
  3. after that invert the plates! Let them dry for about 5 min (untill CHCl3 smell has vanished).
  4. 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).
  5. 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.