Question

Explain the method that would help in selection of recombinant colonies from non recombinant ones

Answer 1

1. Blue-white screening

Blue-white screening is a widely used technique to examine successful cloning. In this method the insert is cloned into a vector containing a lacΖα sequence encoding the α-peptide, a functional subunit of the β-galactosidase enzyme. The multiple cloning site lies within the lacΖα sequence. The plasmid must be transformed into a special strain of E. coli with thelacΖΔΜ15 mutation. An empty vector will produce blue colonies since the activity of the α-peptide (β-galactosidase) remains intact. The colorless X-gal (lactose analog) provided in the screening plates is hydrolyzed by β-galactosidase to form a blue pigment (5,5'-dibromo-4,4'-dichloro-indigo). If the vector contains the DNA insert disrupting the lacΖαsequence, then the α-peptide will not be expressed and X-gal will not be hydrolyzed. Thus, the colonies will be white if the DNA insert is present. It is possible to obtain false positives (white colonies with no insert), so further confirmation of the insert in white colonies is recommended.