Vectors for ligation-independent construction of lacZ gene fusions and cloning of PCR products using a nicking endonuclease.

ABSTRACT: Several ligation-independent cloning methods have been developed that offer advantages for construction of recombinant plasmids at high efficiency while minimizing cloning artifacts. Here we report new plasmid vectors that use the nicking endonuclease Nt.BspQI to generate extended single stranded tails for direct cloning of PCR products. The vectors include pLacCOs1, a ColE1-derivative plasmid imparting resistance to ampicillin, which allows facile construction of lacZ translational fusions and pKanCOs1, a pSC101-derivative cloning vector that imparts resistance to kanamycin, for cloning of PCR amplicons from genomic DNA as well as from ampicillin-based plasmids. We have successfully used these plasmids to directionally clone and characterize bacterial promoters that exhibit temperature re

SUBMITTER: Oster CJ 

PROVIDER: S-EPMC3220417 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature