Site-directed, Ligase-Independent Mutagenesis (SLIM) for highly efficient mutagenesis of plasmids greater than 8kb

Joyce Chiu; Daniel Tillett; Ian W Dawes; Paul E March

doi:10.1016/j.mimet.2008.02.013

Site-directed, Ligase-Independent Mutagenesis (SLIM) for highly efficient mutagenesis of plasmids greater than 8kb

J Microbiol Methods. 2008 May;73(2):195-8. doi: 10.1016/j.mimet.2008.02.013. Epub 2008 Feb 26.

Authors

Joyce Chiu¹, Daniel Tillett, Ian W Dawes, Paul E March

Affiliation

¹ School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia. j.chiu@unsw.edu.au

PMID: 18387684
DOI: 10.1016/j.mimet.2008.02.013

Abstract

Modifying the Site-directed, Ligase-Independent Mutagenesis (SLIM) protocol from a single reaction mode to a two-reaction mode enables highly efficient mutagenesis of plasmid constructs that exceed 8kb. This modified approach reduces the complexity of the PCR step and is optimised for generation of heteroduplexes from long PCR products. The two-reaction mode SLIM has 92% efficiency.

MeSH terms

DNA, Bacterial / genetics*
Ligases / metabolism
Mutagenesis, Site-Directed / methods*
Plasmids*
Polymerase Chain Reaction / methods

Substances

DNA, Bacterial
Ligases