Introduction
You may wish to subclone your PCR product into a plasmid cloning vector. When PCR was in its infancy, researchers found that subcloning PCR products by simple blunt-ended ligation into blunt-ended plasmid cloning vectors was not easy. Thermostable DNA polymerases, like Taq DNA polymerase, add a single nucleotide base extension to the 3′ end of blunt DNA in a template-independent fashion (1,2). These polymerases usually add an adenine, leaving an “A overhang.” Historically, researchers have used several approaches to overcome the cloning difficulties presented by the presence of A overhangs on PCR products. One method involves treating the product with the Klenow fragment of E. coli DNA Polymerase I to create a blunt-ended fragment for subcloning. However this technique is not particularly efficient.