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X-Gal Solution, ready-to-use

 
#R0941 10ml

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Certificate of Analysis: #R0941
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Description
X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galacto-pyranoside) is a non-toxic, ready-to-use, stable, 0.22 µm membrane filtered solution of a special formulation. It is designed for direct use in conjunction with IPTG. The X-Gal is an inert chromogenic substrate for beta-galactosidase, an enzyme that promotes lactose utilization. Beta-galactosidase hydrolyzes X-Gal into a colorless galactose and 4-chloro-3-brom-indigo, which forms an intense blue precipitate. Induction of the lacZ gene with IPTG leads to the hydrolysis of X-Gal and to the development of blue colonies (see the scheme below).

Application
Blue/white colony screening assay, distinguishing recombinant colonies (white) among non-recombinant ones (blue) (1), see protocol below.

Quality Control
Functionally tested in blue/white colony screening.

Concentration
20 mg/ml

Storage
Stable for at least 12 months at +4°C or at room temperature in the original dark vial.

Protocol for the Blue/White Colony Screening

For individual LB (Luria Broth) agar plates:

  1. Pour sterile warm LB agar (about 25 ml) into a Petri dish.
  2. Dry opened LB plates at room temperature under UV light for about 30 min.
  3. Add 40 µl of the X-Gal Solution (20 mg/ml), ready-to-use.
  4. Add 40 µl of 100 mM IPTG Solution, ready-to-use.
  5. Spread evenly on the plate with a sterile spatula.

For batch use, add the following directly per 1 ml of the liquid LB agar (kept at about 50°C):

  1. 1 µl of X-Gal Solution (20 mg/ml), ready-to-use.
  2. 1 µl of 100 mM IPTG Solution, ready-to-use.
  3. Mix well.
  4. Pour 25 ml of prepared LB agar into each Petri dish.
  5. Dry opened LB plates at room temperature under UV light for about 30 min.

Reference

  1. Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1.10, 1.123-1.125, 1.127, 1.150, 2.30, 16.13, 17.97, 17.99-1.125,A1.27, 2001.

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Updated vasario 27, 2008 12:36