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J Microbiol Methods. 2014 May;100:46-51. doi: 10.1016/j.mimet.2013.11.013. Epub 2014 Jan 10.

A universal cloning method based on yeast homologous recombination that is simple, efficient, and versatile.

Author information

1
Department of Animal Sciences, Rutgers, The State University of New Jersey, School of Environmental and Biological Sciences, 59 Dudley Road, New Brunswick, NJ, USA. Electronic address: joska@aesop.rutgers.edu.
2
Department of Biochemistry and Microbiology, Rutgers, The State University of New Jersey, School of Environmental and Biological Sciences, 76 Lipman Dr., New Brunswick, NJ, USA.
3
Department of Biochemistry and Microbiology, Rutgers, The State University of New Jersey, School of Environmental and Biological Sciences, 76 Lipman Dr., New Brunswick, NJ, USA. Electronic address: boyd@aesop.rutgers.edu.
4
Department of Animal Sciences, Rutgers, The State University of New Jersey, School of Environmental and Biological Sciences, 59 Dudley Road, New Brunswick, NJ, USA. Electronic address: belden@aesop.rutgers.edu.

Erratum in

  • J Microbiol Methods. 2014 Jul;102:65.

Abstract

Cloning by homologous recombination (HR) in Saccharomyces cerevisiae is an extremely efficient and cost-effective alternative to other methods of recombinant DNA technologies. Unfortunately, it is incompatible with all the various specialized plasmids currently used in microbiology and biomedical research laboratories, and is therefore, not widely adopted. In an effort to dramatically improve the versatility of yeast gap-repair cloning and make it compatible with any DNA plasmid, we demonstrate that by simply including a yeast-cloning cassette (YCC) that contains the 2-micron origin of replication (2μm ori) and the ura3 gene for selection, multiple DNA fragments can be assembled into any DNA vector. We show this has almost unlimited potential by building a variety of plasmid for different uses including: recombinant protein production, epitope tagging, site-directed mutagenesis, and expression of fluorescent fusion proteins. We demonstrate the use in a variety of plasmids for use in microbial systems and even demonstrate it can be used in a vertebrate model. This method is remarkably simple and extremely efficient, plus it provides a significant cost saving over commercially available kits.

KEYWORDS:

Heterologous protein expression; Ligation-independent DNA cloning; Staphylococcus aureus; Yeast homologous recombination; Zebrafish

PMID:
24418681
PMCID:
PMC4521215
DOI:
10.1016/j.mimet.2013.11.013
[Indexed for MEDLINE]
Free PMC Article

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