Blunt-end Cloning of PCR Products

Abstract

INTRODUCTION Incubation of a blunt-end ligation reaction in the presence of an excess amount of an appropriate restriction enzyme can dramatically increase the yield of recombinant plasmids. The role of the restriction enzyme is to cleave circular and linear concatemers at restriction sites that are re-formed when linear, blunt-ended plasmid molecules ligate to themselves. In almost all cases, ligation of the PCR product to the plasmid destroys the restriction site. The constant reclamation of vector molecules drives the equilibrium of the ligation reaction strongly in favor of the recombinants between vector and blunt-ended PCR product.