Construction of a Zero-background T-vector for Cloning PCR Products

This experiment aimed to improve the shortcomings of low ligation efficiency and weak positive selection in cloning PCR products with T-vector.The approach called for an addition of T-overhang to a blunt-end plasmid. Firstly, a 3'-T overhang was attached to the blunt-end pBluescript SK (+) plasmid utilizing a terminal transferase, Taq DNA polymerase. Secondly, thepBS vector was ligated by T4 DNA ligase to form thesupercoiled blunt-end pBS plasmid without a 3'-T overhang, whereas the plasmidwith a 3'-T overhang remained linear in appearance. Finally, the DNA fragment of 3 000 bp, which was pBST-vector for cloning PCR products, could be purified byagarose gel electrophoresis followed by usinga gel extraction kit. The 500 bp and 1 000 bp DNA fragments were cloned into pBS T-vector with a100% cloning efficiency. It appeared that the newly developed procedures provided a valid zero-background T-vector for cloning PCR products.