X-Gal

5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside is most commonly known as X-Gal. X-gal is a histochemical substrate for β-galactosidase which cleaves X-Gal to yield an insoluble blue precipitate.

X-Gal is a mainstay in molecular biology cloning applications in which it is used to detect the activity of β-galactosidase. X-Gal is used to detect the insertion of foreign DNA into the lacZ region of a plasmid DNA. Insertion of DNA into the lacZ region results in the loss of β-galactosidase activity. Bacteria cells that retain active β-galactosidase will result in characteristic blue colonies. Successful disruption of the lacZα gene disrupts the α-complementation of the β-galactosidase gene and the precipitate does not form, resulting in white colonies.

Many other applications also use X-Gal as a substrate to detect β-galactosidase activity. These include β-galactosidase -antibody linked immunoassays and immunohistochemistry, coliphage detection based on β-galactosidase induction and the detection of micrometastasis formation during tumor progession.

X-gal is commonly used in conjunction with IPTG (GoldBio #I2481) for blue-white screening.

Storage/Handling

Store at -20°C. Protect from Light.

X-Gal Quick Answers

What is X-Gal?

How do you make X-Gal stock solution?

How much X-Gal do you use for blue-white screening?

How do you store X-Gal?

What is X-Gal?

X-gal is the chromogenic substrate for the enzyme β-galactosidase. When X-gal is cleaved by

β-galactosidase, it forms galactose and 5-bromo-4-chloro-3-hydroxyindole.

5-bromo-4-chloro-3-hydroxyindole can dimerize and then is oxidized, forming 5,5'-dibromo-4,4'-dichloro-indigo, an insoluble blue product.

The blue product suggests the presence of β-galactosidase. Therefore, this substrate is often used as a reporter in molecular biology.

How do you make X-Gal stock solution?

Instructions to prepare a 20 mg/ml X-gal stock solution are as follows

1. Weigh 200 mg of X-Gal into a 15 ml polypropylene centrifuge tube.

2. Add 10 mL of Dimethylformamide (DMF). Vortex to dissolve the X-Gal.

3. Store at -20°C. Wrap the centrifuge tube in aluminum foil to protect from light.

Note: The X-Gal solution is stable for 6-12 months unless improperly handled. We recommend making 1 ml aliquot portions of the solution to minimize degradation if there will be frequent small scale use of the X-Gal solution.

How much X-Gal do you use for blue-white screening?

For blue-white screening, use 10 µl per 1 mL of media if you’re using a 20 mg/ml stock solution. If you’re using a 100 mg/ml stock solution, which is only stable for 1 week, use 2 µl.

How do you store X-Gal?

• Store powder X-gal at -20°C. Protect from Light.
• Store X-gal stock solution at -20°C. It’s important to protect your stored stock solution from light. You can do this by wrapping the tube in aluminum foil.