Open AccessProduction of recombineering substrates with standard‐size PCR primers
Author(s) -
Xu Ke,
Hua Jianyuan,
Roberts Kelsey J.,
Figurski David H.
Publication year - 2012
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/1574-6968.12016
Subject(s) - recombineering , biology , plasmid , restriction enzyme , genetics , homology (biology) , cloning (programming) , cloning vector , escherichia coli , restriction site , computational biology , multiple cloning site , dna , recombinant dna , gene , vector (molecular biology) , computer science , programming language
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‐ E scherichia 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 segments, the segments (including those for the homology regions) can be readily changed. During construction of the template plasmid, potential background transformants arising from the vector without insert are significantly reduced by cloning each segment with two restriction endonucleases that produce noncompatible ends. We have used this method to change the bla gene of p ACYC 177 to aadA , to add the MCS ‐ lacZα region from p BBR 1 MCS to I nc Q plasmid vectors, and to make an oriT IncP ‐ aacC1 cassette and add it to a plasmid.