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Nucleic Acids Research Advance Access published online on April 19, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn167
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Rapid one-step recombinational cloning

Changlin Fu, Daniel R. Wehr, Janice Edwards and Brian Hauge*

Monsanto Company, 800 N. Lindbergh Blvd., St Louis, MO 63167, USA

*To whom correspondence should be addressed. Tel: +1 617 737 6810; Fax: +1 617 737 6757; Email: brian.hauge{at}monsanto.com

Received November 27, 2007. Revised March 24, 2008. Accepted March 24, 2008.

As an increasing number of genes and open reading frames of unknown function are discovered, expression of the encoded proteins is critical toward establishing function. Accordingly, there is an increased need for highly efficient, high-fidelity methods for directional cloning. Among the available methods, site-specific recombination-based cloning techniques, which eliminate the use of restriction endonucleases and ligase, have been widely used for high-throughput (HTP) procedures. We have developed a recombination cloning method, which uses truncated recombination sites to clone PCR products directly into destination/expression vectors, thereby bypassing the requirement for first producing an entry clone. Cloning efficiencies in excess of 80% are obtained providing a highly efficient method for directional HTP cloning.


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