Blue white spot screening is a method of recombinant screening, which is the second screening based on antibiotic screening, and is used to screen recombinant vectors according to their genetic characteristics. In this method, fragments are cloned into multiple cloning sites (MCS), and the MCS region is located inside the gene lac Ζ α, which means that the successfully cloned vector has its lac Ζ α gene disrupted. This article introduces the steps and principles of blue and white spot screening, and analyzes the reasons for experimental failure.
Principle of Blue White Spot Screening Experiment
Blue white spot screening is a rapid and effective recombinant bacterial identification technique that relies on the activity of β - galactosidase. β - galactosidase is an enzyme found in Escherichia coli that cleaves lactose into glucose and galactose.
To screen clones containing recombinant DNA, X-gal chromogenic substrate was added to agar plates. If β - galactosidase is produced, X-gal hydrolyzes to form 5-bromo-4-chloroindolyloxy, which spontaneously dimerizes to produce an insoluble blue pigment called 5,5 '- dibromo-4,4' - dichloroindigo. Colonies formed by non recombinant cells appear blue, while colonies formed by recombinant cells appear white. Thus, it is easy to select and cultivate the desired recombinant colonies.
Isopropyl β - D-1-thiopyranoside (IPTG) was used together with X-gal for screening blue white spots. IPTG is a non metabolizable galactose analogue that can induce the expression of the lacZ gene. It should be noted that IPTG is not a substrate for β - galactosidase, but only an inducer. For visual screening purposes, chromogenic substrates like X-gal are needed.