Why are some colonies blue and some white?

Any colony containing the plasmid (and therefore the functioning β-galactosidase gene) will turn blue, a result of the β-galactosidase activity. The insert disrupted the β-galactosidase gene, and therefore these colonies remain white.

What is blue white screening and what is alpha complementation?

Blue-white screening in the lab Providing DNA encoding this section of amino acids (called the α-peptide) to a lacZΔM15-mutant bacterial cell in trans complements the mutation allowing for a functional enzyme. This process is called α-complementation.

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 blue-white screening used for?

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.

What is insertional activation?

Definition. Insertional activation/inactivation refers to either activation of an endogenous gene which is located near an integrated transgene, or to disruption of a gene or other functional sequence by insertion of a transposable element.

Why is Iptg used in blue white screening?

How Does Blue White Screening Work? Isopropyl β-D-1-thiogalactopyranoside (IPTG) is used along with X-gal for blue-white screening. IPTG is a non-metabolizable analog of galactose that induces the expression of lacZ gene. It should be noted that IPTG is not a substrate for β-galactosidase but only an inducer.

How blue white screening is carried out Slideshare?

I) BLUE-WHITE SCREENING The use of chromogenic substrate to detect a particular enzymatic activity is the basis to screen the desired clone. The colourless compound X-gal or 5-bromo-4-chloro-3-indolyl-β-D-galactoside used in this screening method is a substrate for β-galactosidase.

What is recombinant selection?

RECOMBINANTS. A genetic screen or mutagenesis screen is an experimental technique used to identify and select for individuals who possess a phenotype of interest in a mutagenised population.

Is the blue white technique a selection technique?

The blue-white technique is only a screening procedure; it is not a selection technique. The lacZ gene in the vector may sometimes be non-functional and may not produce β-galactosidase. The resulting colony will not be recombinant but will appear white.

How is blue white used in colony selection?

Blue-white color selection of recombinant bacteria using X-gal. The blue-white technique is only a screening procedure; it is not a selection technique. The lacZ gene in the vector may sometimes be non-functional and may not produce β-galactosidase. The resulting colony will not be recombinant but will appear white.

How are positive selection vectors used in blue white screening?

Positive selection vectors encode a gene which, when expressed, is lethal to the cell. Cloning fragments are inserted into an MCS in the center of this gene, disrupting the lethality. This is similar to α-peptide DNA disruption in the blue-white screen.

What are the steps in blue white screening?

Protocols for Blue-White Screening. The complete protocol of blue-white screening includes 3 important steps: Ligation: ligation of foreign DNA into MCS of the plasmid vector; Transformation: introduction of plasmid vector with foreign DNA insert into competent E. coli; Screening: blue-white screening to identify recombinant bacterial colonies