Blue–white screen

The vector is then inserted into a competent host cell viable for transformation, which are then grown in the presence of X-gal.

One of the early methods developed for the detection of insert is blue–white screening which allows for identification of successful products of cloning reactions through the colour of the bacterial colony.

This phenomenon of α-complementation was first demonstrated in work done by Agnes Ullmann in the laboratory of François Jacob and Jacques Monod, where the function of an inactive mutant β-galactosidase with deleted sequence was shown to be rescued by a fragment of β-galactosidase in which that same sequence, the α-donor peptide, is still intact.

[4] In this method, DNA ligated into the plasmid disrupts the α peptide and therefore the complementation process, and no functional β-galactosidase can form.

[5] β-galactosidase is a protein encoded by the lacZ gene of the lac operon, and it exists as a homotetramer in its active state.

However, when the two peptides are expressed together, as when a plasmid containing the lacZα sequence is transformed into a lacZΔM15 cells, they form a functional β-galactosidase enzyme.

[6] If the DNA is necessary to be sequenced, the plasmids from the colonies will need to be isolated at a point, whether to cut using restriction enzymes or performing other assays.

The correct type of vector and competent cells are important considerations when planning a blue–white screen.

Isopropyl β-D-1-thiogalactopyranoside (IPTG), which functions as the inducer of the lac operon, may be used in the media to enhance the expression of LacZ.

On occasion, the medium in which the colonies grow can influence the screen and introduce false-positive results.

The correct recombinant construct can sometimes give lighter blue colonies which may complicate its identification.