Methods to verify your gene of interest was successfully cloned.

You are approaching the end of your cloning workflow. The DNA fragment was ligated in the vector of choice and transformed into E.coli cells. The transformed cells were streaked across a culture plate resulting in a multitude of colonies overnight. How do you know that all of the steps have resulted in the correct recombinant clones? Before you can declare the cloning victorious, colonies must be screened for positive results. Listed below are 5 commonly used methods.

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 the lacΖΔΜ15 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 lacΖα sequence, 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.