Send to

Choose Destination
Biotechniques. 2010 Jun;48(6):463-5. doi: 10.2144/000113418.

Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids.

Author information

Department of Biochemistry, Center for Fundamental and Applied Molecular Evolution, Emory University, Atlanta, Georgia 30322, USA.


Here we describe a straightforward, efficient, and reliable way to clone an insert of choice into a plasmid of choice without restriction endonucleases or T4 DNA ligase. Chimeric primers containing plasmid sequence at the 5' ends and insert sequence at the 3' ends were used to PCR-amplify insertion sequences of various sizes, namely the genes for GFP (gfp), beta-d-glucuronidase (gusA), and beta-galactosidase (lacZ), as well as the entire luxABCDE operon. These inserts were employed as mega-primers in a second PCR with a circular plasmid template. The original plasmid templates were then destroyed in restriction digests with DpnI, and the overlap extension PCR products were used to transform competent Escherichia coli cells. Phusion DNA polymerase was used for the amplification and fusion reactions, so both reactions were easy to monitor and optimize.

Comment in

[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Atypon Icon for PubMed Central
Loading ...
Support Center