From : Greg Petsko 7/96
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.
be grown up for 1-3 days as either patches or single colonies.
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.)
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)
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)
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.
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.
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.
include a quantitative liquid
assay and a semi-quantitative filter