Blue-White Screening
Today we’re going to shine a spotlight on a widely used technique in the field of molecular biology,...
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.
Store at -20°C. Protect from Light.
How do you make X-Gal stock solution?
How much X-Gal do you use for blue-white screening?
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.
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.
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.
Catalog ID | X4281 |
---|---|
Name(s) |
X-Gal; 5-Bromo-4-chloro-3-indoxyl-beta-D-galactopyranoside |
CAS # | 7240-90-6 |
Grade | MOLECULAR BIOLOGY GRADE |
Formula | C14H15BrClNO6 |
MW | 408.63 g/mol |
Biological Activity | Blue color with Lambda, pUC, and M13 |
PubChem Chemical ID |
619018 |
Storage/Handling | Store at -20°C. Protect from light. |
I2481C In stock | 1 g |
$ 20.00 |
|
I2481C5 In stock | 5 g |
$ 28.00 |
|
I2481C25 In stock | 25 g |
$ 116.00 |
|
I2481C50 In stock | 50 g |
$ 180.00 |
|
I2481C100 In stock | 100 g |
$ 332.00 |
|
I2481C200 In stock | 200 g |
$ 636.00 |
|
I2481C300 In stock | 300 g | $ 847.00 | |
I2481C400 In stock | 400 g |
$ 1,100.00 |
|
I2481C500 In stock | 500 g |
$ 1,324.00 |
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