About this item:

699 Views | 850 Downloads

Author Notes:

Address correspondence to Ichiro Matsumura, Department of Biochemistry, Center for Fundamental and Applied Molecular Evolution, Emory University, Atlanta, Georgia, 30322, USA. imatsum@emory.edu

We thank Natasha Degtereva and Joseph Kramer of Emory University for critical reviews of this manuscript.

The authors declare no competing interests.

Subject:

Research Funding:

This work was supported by the National Institutes of Health (NIH; grant nos. 1 R01 GM074264 and 1 R01 GM086824, to I.M.)

Keywords:

  • overlap extension PCR cloning
  • recombinant vector
  • Phusion
  • restriction enzyme ligation independent

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

Tools:

Journal Title:

BioTechniques

Volume:

Volume 48, Number 6

Publisher:

, Pages 463-465

Type of Work:

Article | Final Publisher PDF

Abstract:

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), β-D-glucuronidase (gusA), and β-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.

Copyright information:

© 2010 Author(s)

This is an Open Access work distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Creative Commons License

Export to EndNote