Abstract

To increase a recombinant ratio of cloning blunt DNA fragments, for example, polymerase chain reaction (PCR) products, a vector DNA is usually digested by a blunt-end producing restriction endonuclease and then dephosphorylated in order to avoid self-ligation of the linearized vector. In this paper, we described a simple method to directly ligate a blunt DNA fragment to a non-dephosphorylated vector with a high recombinant ratio. Firstly, a vector DNA was digested by a blunt-end producing restriction enzyme, such as SmaI in our experiment. The linearized vector was then directly ligated to a blunt DNA fragment in a standard ligation buffer in the presence of SmaI. The results showed that the restriction enzyme SmaI in the ligation reaction can efficiently minimize the self-ligated vector and keep a very high recombinant ratio.

Key words: Blunt-end ligation, non-dephosphorylation, high efficiency.