does not require of a lot of input DNA and allows for the cloning of fragments for which

the whole sequence may not be known. Begin by amplifying the DNA fragment of interest

with a polymerase using primers that are complementary to the DNA sequence of interest. With each

cycle, the resulting DNA is amplified exponentially. The resulting amplified DNA will have either

a single-A overhang, if Taq DNA polymerase was used, or a blunt end, if a high-fidelity

polymerase, such as Q5 High-Fidelity DNA Polymerase, was used.

Next, join the amplified DNA fragments to the appropriate vector. This can be done by

the use of a DNA ligase, or by use of activated vectors that are covalently-bound to an effector

enzyme, which facilitates vector:insert joining. Depending on the vector chosen, the fragment

of interest may be inserted non-directionally at the ligation junction, such as in TA cloning.

Some supplied vectors can contain a toxic gene. The use of toxic gene fusions ensures

that the only competent cells that survive are those that have taken up the vectors with

successful insertion of the target gene sequence, as the insertion disrupts the expression of

the toxic gene. These vectors are known as “suicide vectors”.

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 gene

or fragment of interest. In recent years, the introduction of fast

and robust high-fidelity polymerases has made this approach easy and reliable.

The NEB PCR Cloning Kit allows quick and simple cloning of all your PCR amplicons, regardless

of the polymerase used. This kit utilizes a novel mechanism for background colony suppression,

and allows for direct cloning from your reaction, with no purification step.

Visit CLONEWITHNEB.com for a full list of products available for this application.