The steps and principles of blue and white spot screening, and the reasons for the failure of the bl

Blue and white spot screening is a method of recombinant screening, which is the second screening based on antibiotic screening and screening recombinants based on the genetic characteristics of the vector. In this method, fragments are cloned into multiple cloning sites (MCS), with the MCS region located in the gene lac Ζα Internally, that is to say, the vector of successful cloning, its LAC Ζα Genes are disrupted. This article introduces the steps and principles of blue and white spot screening, and analyzes the reasons for experimental failure.

Principle of Blue and White Spot Screening Experiment

Blue and white spot screening is a fast and effective recombinant bacterial identification technique that relies on β- The activity of galactosidase. β- Galactosidase is an enzyme present in Escherichia coli that cleaves lactose into glucose and galactose.

To screen clones containing recombinant DNA, X-gal chromogenic substrates were added to agar plates. If there is a β- Galactosidase hydrolyzes X-gal to form 5-bromo-4-chloroindolyoxy, which spontaneously dimerizes to produce an insoluble blue pigment called 5,5 '- dibromo-4,4' - dichloro indigo. Colonies formed by non recombinant cells therefore appear blue, while colonies formed by recombinant cells appear white. Thus, it is easy to select and cultivate the required recombinant colonies.

Isopropyl group β- D-1-Thiopyranogalactoside (IPTG) is used in combination with X-gal for blue and white spot screening. IPTG is an unmetabolible galactose analogue that induces the expression of the lacZ gene. It should be noted that IPTG is not β- The substrate of galactosidase, rather than just an inducer. For visual screening purposes, a chromogenic substrate like X-gal is required.