Herskowitz Lab
Protocol
From : Greg Petsko 7/96
X-Gal Agarose Overlay Assay
This assay is semiquantitative. Cells can even be recovered from underneath the overlaid agar. The method could be used for any enzyme with a colorimetric assay.
Cells should be grown up for 1-3 days as either patches or single colonies.
Stock solution:
0.5 M Potassium
Phosphate Buffer, pH 7.0
Mix 61 ml of 1 M K2HPO4 (228 g per liter H2O) and 39 ml of 1 M KH2PO4
(136 g per liter H2O) and add H2O to a final volume of 200 ml;
filter sterilize and store 6% Dimethyl Formamide (DMF) 0.1% SDS
(I make up the stock solution in 100 ml quantities by mixing 93 ml of phosphate
buffer, 6 ml of DMF and 1 ml of 10% SDS. The stock solution will be foamy
but this is no problem.)
To run the assay:
| 1. Mix: 8-10 ml stock solution per plate to be assayed 5 mg/ml low-melting agarose (I use GIBCO BRL ULTRAPURE LMP AGAROSE, from Life Technologies Cat.# 15517-022) |
| 2. Microwave this mixture on HIGH to bring the temperature to 60-70°C (For 50 ml of solution this is approx. 1 minute) |
| 3. Add to the warm solution: 0.1 to 0.5 mg/ml X-Gal 1 drop beta-mercaptoethanol (i.e., about 50 microliters per 100 ml) |
| 4. Using a plastic pipette, cover the surface of each plate of cells with 8-10 ml of the warm solution. The DMF and SDS will permeabilize the cells and the buffer plus mercaptoethanol will keep the beta- galactosidase happy. |
| 5. After the agar cools and solidifies, the plates may be incubated at either 25°C or 30°C. The blue color develops in a few hours, depending on the strength of the inducer. For a strong inducer such as a multiple STRE element linked to lac-Z, the blue color may be seen in 1-2 hours at 25°C. For a weak inducer such as a single HSE linked to lac-Z, the color becomes visible in 6-8 hours at 25°C. |
| 6. Colonies may be picked through the top agar even 5 days later. Just take a sterile Pasteur pipette and poke it through the agar into the desired colony or patch. Then use the tip of the Pasteur pipette to streak a fresh plate and, despite the permeabilization, the cells will grow up. |
Related protocols include a quantitative liquid assay and a semi-quantitative filter assay
