PSDTV vector: a modification of the pBluescript SK+ plasmid in order to perform PCR-fragments TA-cloning using Eam1105I restriction endonuclease

Abstract

Some of the approaches for cloning PCR products obtained with conventional Taq-polymerases which do not involve modifications of the ends of the vector or the insert are based on the use of restriction enzymes which can generate 3' thymine single nucleotide overhangs, such as Eam1105I (AhdI). Due to the presence of Eam1105I restriction site within the β-lactamase gene, this is not achievable with a number of the most widely used cloning vectors descending from the pUC family, for which the selection is based on the ampicillin resistance. In this report we describe the construction of a vector for TA-cloning, based on the abolishment of the Eam1105I recognition site within the β-lactamase gene by site-directed mutagenesis, and the introduction of a stuffer flanked by Eam1105I target sites within the polylinker of the pBluescript SK+ plasmid.