What is the specific function of X-gal in the LB agar?

What is the specific function of X-gal in the LB agar?

For screening the clones containing recombinant DNA, a chromogenic substrate known as X-gal is added to the agar plate. If β-galactosidase is produced, X-gal is hydrolyzed to form 5-bromo-4-chloro-indoxyl, which spontaneously dimerizes to produce an insoluble blue pigment called 5,5′-dibromo-4,4′-dichloro-indigo.

What is X-gal solution?

X-Gal Solution, ready-to-use, is stable, 0.22 µm membrane filtered solution formulated for direct use in conjunction with IPTG for blue/white colony screening. Applications. • Blue/white colony screening to distinguish recombinant (white) from non-recombinant (blue) colonies.

Why were the colonies on the X-gal control plate white?

This is called α-complementation. Those colonies containing plasmids with an insert can be differentiated from those without an insert by the color of the colony (white versus blue). The insert disrupted the β-galactosidase gene, and therefore these colonies remain white.

How do you dissolve a BLUO gal?

IPTG can be reconstituted in water. Make a stock of 100 mM in water and store working aliquots at -20°C. X-gal can be reconstituted in DMSO, or in a 50:50 mix of DMSO and water. To do the latter, you must dissolve in DMSO first, and then add water to bring up to final volume.

How do you dilute Iptg?

Stock Solution Preparation of 1 M IPTG (1 M IPTG Recipe)

1. Dissolve 2.38 g of IPTG in 8 mL of distilled H2O.
2. Bring to a final volume of 10 mL with molecular biology grade H2O.
3. Filter sterilize with a 0.22 μ syringe filter.
4. Store in 1mL aliquots at -20 °C.

What is the function of lacZ?

The lacZ gene encodes the portion of the mRNA that is responsible for the production of β-galactosidase (B) and translation of the lacY gene produces the section of mRNA that is ultimately responsible for the production of an enzyme permease (P).

What does blue mean in blue white screening?

Tips for blue-white screening All colonies on this plate should be blue, indicating that your IPTG and x-gal are working as they should be.

How much X gal to apply to agar plates?

A typical stock concentration of X-gal is 50mM (20mg/mL). To apply X-gal directly to the top of agar plates for blue/white screening, apply 40μL and wait to dry.

What’s the stock concentration of X-gal for IPTG?

Typically used in conjunction with IPTG for blue/white screening via the lac operon. A typical stock concentration of X-gal is 50mM ( 20mg/mL ). To apply X-gal directly to the top of agar plates for blue/white screening, apply 40μL and wait to dry.

What do you need to know about X-gal?

Jump to navigationJump to search. X-Gal is the common short name for 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside. It is a substrate for beta-galactosidase (lacZ) and turns blue upon being cleaved. It is commonly used for blue-white screening or detection of LacZ transgenes in cells and tissues.

How many mg of X-gal is in DMF?

A stock solution of X-gal is commonly made with 20 mg/ml X-gal in DMF (dimethylformamide). When pouring agar plates, a typical final concentration is 20 μg/ml. Note that DMF is toxic.

What is the specific function of X-gal in the LB agar? For screening the clones containing recombinant DNA, a chromogenic substrate known as X-gal is added to the agar plate. If β-galactosidase is produced, X-gal is hydrolyzed to form 5-bromo-4-chloro-indoxyl, which spontaneously dimerizes to produce an insoluble blue pigment called 5,5′-dibromo-4,4′-dichloro-indigo. What is X-gal solution?…