Agar Plates for Selection of Clones in Bacteria
Cloning of PCR products
Stocks:
LB Agar: Luria Broth after Lennox:
per Liter
Tryptone | 10 g |
Yeast Extract | 5 g |
Sodium Chloride | 5 g |
Bact. Agar | 15 g |
pH 7.0
Autoclave and store sterile at room temperature
Stock solutions:
Store all frozen in aliquots at -20C (can be stored in dark at 4C but -20 is usually as simple!)
X-Gal: 20 mg/ml in Dimethylformamide (DMF)
IPTG: 100 mM
Ampicillin: 100 ug/ml (micrograms)
(see also page on BAC isolation with BACs including chloramphenicol resistance.)
Filter sterilize
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.
IPTC is isopropylthio-beta-D-galactoside. It is a false substrate (not cleaved) and inducer of beta-galactosidase
Ampicillin is an antibiotic which requires the resistance plasmid in the bacteria
Final concentrations on plates
X-gal of 80ug/ml (1:250 dilution of the stock solution, can use half this)
IPTG 0.05 mM /L (1:200 dilution of stock, can use twice 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).
