X-gal (5-Bromo-4-chloro-3-indolyl-ß-D-galactopyranoside) is a synthetic analog of lactose that mimics lactose as an inert chromogenic substrate for ß-galactosidase. In combination with, IPTG (needed for the induction of the expression of ß-galactosidase), X-Gal can be used for blue/white screening of the lac phenotype of E.coli in cloning experiments and discriminate between bacterial colonies transformed with recombinant plasmid containing the DNA fragment of interest (white) versus non-recombinant plasmid (blue), as ß-galactosidase hydrolyzes X-Gal into galactose and a blue precipitate.
Appearance: White powder.
Storage: 2ºC – 8ºC for at least 1 years, at -20ºC at least 3 years
Preparation: Prepare a stock solution of 20mg/ml in DMF (N,N-dimethylformamide) or DMSO (dimethylsulfoxide) and store in glass containers at -20ºC for 6-12 months. There is no need for sterilization. If the solution becomes pinkish, it should be discarded.
Usage: For incorporation in agar plates, add 5 ml of X-Gal stock solution (together with IPTG (e.g. 0,5ml of a 1M stock solution) and appropriate antibiotics) to 1 liter of autoclaved media agar, just prior to pouring the plates. Medium temperature should be below 55ºC. Alternatively, in order to minimize the required amount of X-Gal, X-Gal can be applied directly on top of prepared plates: add 40 μl of the X-Gal stock solution (and 8 μl of a 100mg/ml IPTG solution, if not already present in the medium) and spread the solution over the entire surface of the plate. Ensure that the whole area is covered to prevent false positives. Allow the solution to be absorbed by incubation of the plates for 1-2 hours at 37ºC before proceeding with cell plating.
MW: 408,6 g/mol
Purity: >99% (HPLC, on dry basis)
Purity: single spot (TLC)
Water content: <1% (Karl Fischer)
Solubility: DMF or DMSO (see preparation)