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 fuelled 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.

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.