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 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.

2. Positive Selection Vector

An effective method to simplify screening is to use a positive selection vector. Positive selection vectors conditionally express a lethal gene, such as a restriction enzyme that digests the genomic DNA of the bacterial host. The expression of the lethal gene is disrupted by ligation of a DNA ｉnsert into the cloning site. As a result, only cells with recombinant plasmids are able to grow. This approach can save time ａnd costs since it typically yields >99% recombinant clones. Thermo Scientific CloneJET PCR Cloning Kit uses the positive colony selection method.

3. Diagnostic Restriction Digest

Restriction enzyme digests can also be performed to determine the correct insert. First, isolate plasmid DNA from an overnight bacterial culture using a plasmid miniprep kit such as Thermo Scientific GeneJET Plasmid Miniprep Kit. Our REsearch restriction site mapping tool can be used to identify restriction sites in a given sequence. Choose restriction enzymes that allow you to easily determine if the plasmid contains an insert. Then, digest the purified plasmid DNA from recombinant clones using restriction enzymes. For rapid results, we recommend Thermo Scientific FastDigest restriction enzymes. Run the digested plasmid on an agarose gel to verify that the vector backbone ａnd ｉnsert are of the expected sizes.

4. Colony PCR

The presence of the DNA ｉnsert can also be determined by screening bacterial colonies using PCR. The primers may be insert-specific, vector-specific, or both to detect the insert. To determine the orientation of the insert, a combination of vector-specific ａnd insert-specific primers is recommended. Colony screening by PCR is suitable for inserts shorter than 3 kb. Individual colonies can be subjected directly to the PCR ｍaster mix using Thermo Scientific PCR reagents. The remaining portion of the colony may be used to inoculate a culture plate or liquid LB media with appropriate antibiotic for downstream applications.

5. Sequencing

The most accurate way to verify the recombinant colonies is by sequencing. Plasmid DNA is first isolated from an overnight bacterial culture. The ｉnsert can be identified by Sanger sequencing using sequencing primers appropriate for the vector. Sequencing across the entire ｉnsert is required to verify the exact sequence of insert.

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