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.


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.

Once you have identified the correct recombinant,
you have successfully reached the end of your cloning workflow!