What does X-gal do in blue white screening?

What does X-gal do in blue white screening?

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.

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

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.

Is X-gal expensive?

The main advantage of this system is that is less expensive than other systems since media do not contain chromogenic markers such as X-gal, which is both expensive and cumbersome to prepare and use, or inductors such as IPTG.

How do you read a blue white screen?

The blue–white screen is a screening technique that allows for the rapid and convenient detection of recombinant bacteria in vector-based molecular cloning experiments. This method of screening is usually performed using a suitable bacterial strain, but other organisms such as yeast may also be used.

Can you do blue white screening without Iptg?

In some blue/white screening systems, an additional reagent must be used: IPTG (isopropylthiogalactoside). IPTG is an inducer that de-represses lacZ expression (it turns the gene on). 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 blue white screening in biotechnology?

How many base pairs are in pBR322?

4,361
coli (1). The molecule is a double-stranded circle 4,361* base pairs in length (2). pBR322 contains the genes for resistance to ampicillin and tetracycline, and can be amplified with chloramphenicol.

How do I choose a transformant?

Method I: Selection of Transformants in Liquid Media

1. i. Pipette the medium up and down to remove cells from the plate. Save the original plate until the clones are stably growing as a backup in case of contamination.
2. ii. Dilute the cells to yield one cell per well.
3. iii. Distribute into microtiter well plates.

Are there any drawbacks to X-gal blue screening?

There are some drawbacks with such X-gal blue screening. Besides instability to temperature and light, the screening process may lead to false positive (unless you pick up strictly white colonies).

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.

How are non recombinant colonies picked for blue-white screening?

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. Isopropyl β-D-1-thiogalactopyranoside (IPTG) is used along with X-gal for blue-white screening.

How is IPTG used in blue / white screening?

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