blue-white colour selection

blue-white colour selection

A routine technique used by molecular biologists to simplify differentiation between bacterial colonies/plaques that contain a cloning vector (plasmid) without an insert (blue) and those containing a vector with an insert of interest (white).

Cloning vectors may have polycloning sites within a part of the LacZ gene that encodes galactosidase, which provides complementation in an appropriate mutant E coli strain; a re-ligated (empty vector) produces blue colonies when grown on plates containing IPTG and X-gal; colonies with a large insert in the plasmid’s polycloning site cannot produce functional galactosidase and thus produce white colonies.

The technique is based on vectors such as the pUC and the M13mp series that carry a fragment of the -galactosidase gene encoding an -fragment of -galactosidase. The exploitation of these vectors requires the use of a bacteria strain carrying the complementing gene fragment to allow the assembly of an active complex. The transformation of the -galactosidase gene fragment containing vectors to the appropriate cells, in the presence of the -galactosidase chromogenic substrate X-gal5 and the inducer IPTG,6, results in the formation of blue colonies/plaques. Disruption of the -galactosidase gene by insertion of a DNA fragment into the vector’s multiple cloning site results in the loss of functional -galactosidase activity. Therefore, the colonies/plaques bearing a vector containing an insert will remain white and may be easily distinguished from those bearing an intact cloning vector.