X-Gal / 5-Bromo-4-chloro-3-indolyl-b-D-galactopyranoside

X-Gal (5-Brom-4-chlor-3-indolyl-β-D-galactopyranosid) is a chromogenic substrate for β-galactosidase. It produces a blue color that can easily be detected visually. X-Gal is used in the identification of lacZ + bacteria, especially for the detection of ß-galactosidase, which is expressed by the recombinant vectors. X-Gal is often used in conjunction with IPTG > (for the induction of the lac promoter (lac operon)), for the screening of blue colonies. It is also used for the screening of ß-galactosidase reporter gene activity in transfections of eukaryotic cells.

X-Gal is a substrate of ß-galactosidase, which releases by cleavage of the substrate 5-bromo-4-chloro-3-indoxyl (X). The enzymatically cleaved indoxyl is oxidized to the insoluble indigo. As a stock solution, for example, a 20mg/mL X-Gal solution in dimethylformamide can be prepared. For the use in LB / Amp - plates this stock solution is to be diluted 1 : 1000 (final concentration 20μg/mL) or 1: 500 (final concentration 40μg/mL). The heated medium should thereby be cooled to about 50°C. The amount of X-Gal may be reduced if an X-Gal solution in DMF is sprayed to the agar plates (already with bacterial colonies!). X-Gal is the substrate of choice for blue-white selection of recombinant bacterial colonies with the lac⁺ genotype.