How do you prepare LB ampicillin IPTG X-gal plates?

How do you prepare LB ampicillin IPTG X-gal plates?

Blue/White Screening of Bacterial Colonies X-Gal/IPTG Plates

1. Add 10 µl X-Gal Solution (20 mg/ml) per 1 mL of Media.
2. Add 10 µl IPTG (100mM) per 1 mL of Media for a final concentration of 1mM.
3. Add screening antibiotic of choice (Ampicillin, Kanamycin, Carbenicillin, etc).

How does X-gal screening work?

For screening the clones containing recombinant DNA, a chromogenic substrate known as X-gal is added to the agar plate. The colonies formed by non-recombinant cells, therefore appear blue in color while the recombinant ones appear white. The desired recombinant colonies can be easily picked and cultured.

Can you do blue white screening without IPTG?

In some blue/white screening systems, an additional reagent must be used: IPTG (isopropylthiogalactoside). In some cases, without IPTG, not enough β-galactosidase is produced to turn the colony blue even if the lacZ gene is intact.

What is X-gal in blue white screening?

In gene cloning, X-gal is used as a visual indication of whether a cell expresses a functional β-galactosidase enzyme in a technique called blue/white screening. This method of screening is a convenient way of distinguishing a successful cloning product from other unsuccessful ones.

How to screen for X-gal in IPTG plates?

Blue/White Screening of Bacterial Colonies X-Gal/IPTG Plates  Prepare 20 mg/ml X-Gal solution in DMF (See X-Gal Stock Solution Procedure). For reduced DMF toxicity in media, you can alternatively make a 100 mg/ml X-Gal solution in DMF (this concentration is only stable at -20°C for ~1 week).

How to screen for bacteria on X-gal plates?

1. Cool autoclaved growth media agar to 50°C. 2. Add 10 µl X-Gal Solution**(20 mg/ml) per 1 mL of Media (or 2 µl X-Gal Solution (100 mg/ml) per 1 mL of Media). 3. Add 10 µl IPTG (100mM) per 1 mL of Media for a final concentration of 1mM. 4. Add screening antibiotic of choice (Ampicillin, Kanamycin, Carbenicillin, etc). 5.

What can X-gal be used for in cloning?

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.

How is X-gal used to screen for LacZΩ?

To perform blue-white screening after transformation, X-Gal is added along with Isopropyl β-D- 1-thiogalactopyranoside (IPTG), an inducer of lacZω gene expression. The blue colonies contain bacteria with functional β-galactosidase, indicating the plasmid taken up during transformation did not contain the DNA of interest.