Filter Assay for the Detection of Yeast Colonies Beta-Galactosidase

[oBWhat]

Filter Assay for the Detection of Yeast Colonies Producing Beta-Galactosidase

Preparation of stock solutions
Z buffer (per litre)
Na2HPO4.7H2O
OR Na2HPO4 16.1 g
8.5 g
NaH2PO4.H2O 5.5 g
KCl 0.75 g
MgSO4.7H2O 0.246 g
Adjust pH to 7.0
X-gal

Dissolve 5-bromo-4-chloro-3-indolyl--D-galactoside (X-gal) in N,N-dimenthylformamide at a concentration of 20 mg/ml.
Procedure

1. Prepare a X-gal/Z buffer solution containing 100 ml of Z buffer, 0.27 ml of 2-mercaptoethanol, and 1.67 ml X-gal stock. To assay colonies on a 100 mm diameter dish, pipet 1.8 ml of this solution to a clean dish and soak a filter placed onto the solution. Use either Whatman 1 or VWR grade 413 paper filters.
2. After the colonies have grown, they are picked and transferred to a filter (or place a sterile filter onto a transformation plate and orient it).
3. The filter is placed into a pool of liquid nitrogen for 10 seconds, then removed and allowed to thaw at room temperature.
4. Carefully layer the thawed filter, colony side up, onto the presoaked with X-gal/Z buffer solution. Colonies producing b-galactosidase will turn blue any time from 30 min to 30 hours.

[rimanwan]

If the 10 seconds in nitrogen is too short? we usually place in nitrogen for 1 minite.