PCR RE lab questions regarding blue and white bacterial ins

PCR & RE lab questions regarding blue and white bacterial insert colonies

In what case could a “wrong” color colony have an insert

All of your colonies are white, what is the most likely problem?

Solution

The blue-white screening tests for disruption of coding sequence by introduction of another piece of DNA based upon disruption of beta-gal expression leads to removal of enzymatic activity required to cleave X-gal and produce the blue color. Vectors such as E.Coli strain can often self-ligate without incorporating an insert, thus produces non-recombinant plasmid that gives rise to the blue colonies. In rare cases, when the insert is very small and does not disrupt the reading frame leads to wrong color colony.

Colonies containing the plasmid will turn blue, a result of the -galactosidase activity. Those colonies consisting of plasmids with an insert can be differentiated from those without an insert by the color of the colony. If the insert used disrupts the -galactosidase gene, those colonies will appear as white.