Colony plating test for successful DNA recombination

    The medium in the petri dish contains an antibiotic, as well as the sugar X-galactose (Xgal), which when digested by beta-galactosidase produces a blue by-product. Beta-galactosidase is the proeduct of the LacZ gene. The antibiotic & Xgal markers provide a selection scheme for recombinant bacterial colonies, as follows. First, only bacterial colonies that contain a plasmid-borne antibiotic-resistance gene can grow at all. Second, of those that grow, only those whose plasmids carry a functional LacZ in the polylinker can metabolize Xgal.  Thus, those with an intact polylinker (and therefore no insert) have a functional LacZ gene: they produce blue colonies. Those with a polylinker disrupted by an insert have a non-functional LacZ gene, and are unable to metabolize Xgal: they therefore produce white colonies. Thus, the white colonies are the ones of interest.

    The double-selection scheme cuts down on the work required to evaluate the experiment.  Of the millions of bacteria on the plate, and the hundreds of growing colonies that have taken up a plasmid, only the few tens of white colonies stem from single bacteria that contain recombinant plasmids and need to be examined further. Other tests will show if the recombinant inserts contain the gene of interest.

All text material © 2008 by Steven M. Carr