These pages are the sole responsibility of Pat Heslop-Harrison.
Department of Biology, University of Leicester, LE1 7RH UK
Phone: +44/0 116 252 5079 / 3381
FAX: +44/0 116 252 2791
Agar Plates for Selection of Clones in Bacteria
Cloning of PCR products
LB Agar: Luria Broth after Lennox:
Autoclave and store sterile at room temperature
X-Gal: 20 mg/ml in Dimethylformamide (DMF)
X-Gal is 5-bromo-4-chloro-3-indolyl-beta-D-galactoside. It is hydolysed by. beta galactosidase to form an indigo-type blue-green precipitate. Bacteria with the B-galactosidase gene give blue colonies.
Final concentrations on plates
X-gal of 80ug/ml (1:250 dilution of the stock solution, can use half this)
so to 100 ml LB agar (molten but < 50C, ie you can hold the bottle in your hand without burning), add 100 ul Ampicillin stock, 400 ul X-Gal stock and 500 ul IPTG stock
Use 20 ml per Petri dish (cover bottom then move once side-to-side to distribute).