Traditional cloning evolved from the discoveries of site-specific nucleases (restriction enzymes) and DNA ligases in the 1970s, to enable the era of molecular cloning. At the beginning, cloning a gene into a plasmid allowed the host organism to replicate the cloned sequence and this procedure fueled the early gene sequencing, protein expression, and gene function studies.
The first step is to identify unique restriction sites in your source DNA that can be used to isolate the fragment of interest. We recommend using NEBCutter an online tool available at NEBCutter.neb.com.
In many cases, PCR can be used to add the necessary restriction sites to the gene-of-interest to facilitate directional cloning. Digest your vector and DNA fragment-of-interest with a single restriction enzyme for non-directional cloning, or a pair of restriction enzymes possessing different cleavage sites for directional cloning. Use of two different enzymes that produce blunt ends creates a non-directional cloning strategy. If you are using a single restriction enzyme or two enzymes that produce blunt ends, additional screening may be required.
To avoid the possibility of vector self-ligation in the ligation step, it is common practice to dephosphorylate the vector, to remove the 5' phosphate group that the DNA ligase needs for phosphodiester bond formation. Depending on how you generate your vector and insert, other end treatments may be required. These include blunting, A-tailing, and phosphorylation.
Select a ligase to covalently join the vector and insert. Transform the recombinant plasmid into competent E. coli. Spread onto agar plates that contain the appropriate antibiotic for selection Screen the resulting colonies for the DNA fragment of interest.
Although the basic workflow in traditional cloning has not changed much since the 1970s, the introduction of faster and much more robust enzyme and buffer combinations, as well as wider specificities in the last decade has made this approach easier and faster than ever before. Visit CLONEWITHNEB.com for the full list of products available for this application.
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