X-Gal Stock Solution Recipe

X-Gal = 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside.

Dissolve X-Gal at 20 mg/ml in 100% dimethylformamide (DMF) or DMSO (dimethyl sulfoxide). Use a glass or plastic vial and store at −20 °C in the dark. Discard when the color of the stock solution changes significantly. It is not necessary to sterilize X-Gal solutions.

Working concentration: use at a final concentration of 20 µg/ml in the plate.

Reference: Sambrook, J. and Russell, D.W. 2001. Molecular Cloning: A Laboratory Manual, 3rd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. p. 1.123–1.125.

What does X-Gal do?

X-Gal is a color-producing substrate for the enzyme β-galactosidase, which is encoded by the lacZ gene. When β-galactosidase cleaves X-Gal, the reaction ultimately produces an insoluble blue compound. This visible color change makes X-Gal useful for detecting β-galactosidase activity.

In blue-white screening, colonies with functional β-galactosidase activity turn blue, while colonies in which the relevant lacZ function has been disrupted typically remain white. This allows colonies carrying recombinant plasmids to be distinguished visually from many non-recombinant colonies.

X-Gal itself is the chromogenic substrate. It should not be confused with IPTG, which is often used alongside X-Gal. IPTG induces expression of the lac system, while X-Gal provides the substrate that produces the blue color.