Cloning in Plasmid Vectors: Blunt-End Cloning

Abstract

This protocol describes procedures for cloning blunt-ended DNA fragments into linearized plasmid vectors. To obtain the maximum number of “correct” ligation products when cloning blunt-ended target fragments, the two components of DNA in the ligation reaction must be present at an appropriate ratio. If the molar ratio of plasmid vector to target DNA is too high, then the ligation reaction may generate an undesirable number of circular empty plasmids, both monomeric and polymeric; if too low, the ligation reaction may generate an excess of linear and circular homopolymers and heteropolymers of varying sizes, orientations, and compositions. For this reason, the orientation of the foreign DNA and the number of inserts in each recombinant clone must always be validated by restriction endonuclease mapping or some other means.