Production of recombineering substrates with standard-size PCR primers.

ABSTRACT: Recombineering is a powerful method for DNA manipulation. It has advantages over restriction endonuclease-based methods and is usually rapid. Typically, recombineering uses long PCR primers (c. 65 bases), each of which contains a small region of target homology (c. 45 bases). We have developed a simple, albeit somewhat less rapid, strategy to create recombineering substrates that can use primers of ≤ 35 bases for all steps. The regions of homology can be several hundred base pairs in length to (1) increase the chance of obtaining the desired clone and/or (2) allow coliphage-based recombineering in some non-Escherichia coli bacteria. The method uses cloning techniques to construct a template for the generation of the recombineering substrate. Because the template is made from cloned DNA seg

SUBMITTER: Xu K 

PROVIDER: S-EPMC3510332 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature