X-Gal也称5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside或5-Bromo-4-chloro-3-indolyl β-D-galactoside，X-Gal是β-半乳糖苷酶(β-galactosidase)的显色底物，在β-半乳糖苷酶的催化下会产生蓝色产物。常用于β-半乳糖苷酶的原位染色检测以及蓝白斑筛选。

分子式为C14H15BrClNO6，分子量为408.63，CAS Number 7240-90-6。 X–Gal是β–半乳糖苷酶（β–galactosidase）的底物，水解后呈蓝色。基于这个特点，pUC系列载体DNA（或其他带有lacZ 基因载体DNA）以lacZ 缺失细胞为宿主进行转化时、或用M13噬菌体载体DNA进行转染时，如果在平板培养基中加入X–Gal和IPTG，由于β–半乳糖苷酶的α–互补性，可以根据是否呈现白色菌落（或噬菌斑）而方便地挑选出基因重组体。

X-gal is a galactoside and indole.

Uses：

cloning：

In gene cloning, X-gal is used to indicate whether a bacterium expresses the β-galactosidase enzyme, which is encoded by the lacZ gene, in a technique called blue/white screening.

X-gal is cleaved by β-galactosidase yielding galactose and 5-bromo-4-chloro-3-hydroxyindole. 5-bromo-4-chloro-3-hydroxyindole then is oxidized into 5,5'-dibromo-4,4'-dichloro-indigo, an insoluble blue product. Thus, if X-gal and an inducer of β-galactosidase (usually IPTG) is contained within an agar medium on a culture plate, colonies which have a functional lacZ gene can easily be distinguished.

This is used when cloning genes as a technique to see whether plasmids have acquired foreign genetic material. E. coli bacteria which do not produce β-galactosidase are transformed with a plasmid, some of which contain an insert in the lacZ gene. For bacteria harboring plasmids with the insert in the lacZ, this gene is disrupted and they are unable to make beta-galactosidase. For bacteria without the insert, β-galactosidase is produced, resulting in a blue colony. This is the case with many commercially available cloning vectors, such as Promega's pGem-T Vectors, which carry lacZα, a truncated form of β-galactosidase, and require specific E. coli hosts strains (such as DH5α) to achieve α-complementation.

Reporter

The lacZ gene may be used as a reporter in combination with growth media containing X-gal. In two-hybrid analysis for example, it is necessary to distinguish between those bacteria in which there is a successful interaction, leading to the binding of an activation domain to a promoter, and those in which there is not. If the promoter is linked to a lacZ gene, the production of β-galactosidase will be indicated by the production of blue pigment by colonies that host a successful interaction. [1] Due to its manual nature, this technique is limited to situations in which the number of colonies that must be distinguished is less than around 106. [1]

Water testing

In addition to use in molecular biology, X-Gal is used to determine E. coli and coliform content in drinking water samples.

ReadyCult® [2] is one such product that is a USEPA approved test for the Presence/Absence of total coliforms and E. coli in drinking water. This test exploits the fact that naturally occurring E. coli will have the β-Galactosidase necessary to cleave X-gal and produce an observable blue product. A positive result indicates that the water may be unsafe to drink.