1. Make 8 to 10 ml stock solution per plate to be assayed.
2. Add 5 mg/ml agarose to the solution and microwave on high to bring the temperature of the solution to 60°C to 70°C (for 50 ml of solution, about 1 min).
3. Add 0.1 to 0.5 mg/ml X-Gal and 1 drop (about 50 μl/100 ml) 2-mercaptoethanol.
4. Using a plastic pipette, cover the surface of each plate of cells with 8 to 10 ml of the warm solution. The Dimethylformamide and SDS will permeabilize the cells and the buffer plus 2-mercaptoethanol will keep the beta-galactosidase active.
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, the blue color may be seen in 1 to 2 hr at 25°C. For a weak inducer, the color becomes visible in 6 to 8 hr at 25°C.
6. Colonies may be picked through the top agar as long as 5 days after the overlay assay. 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 step, the cells will grow.
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