“Combinatorial Strategy”: A Highly Efficient Method for Cloning Different Vectors with Various Clone Sites

Gang Zhang^{1, 2,} and Anurag Tandon¹

¹Department of Medicine, Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada

²Division of Nephrology, Massachusetts General Hospital, Charlestown, USA

Cite this paper:
Gang Zhang and Anurag Tandon. “Combinatorial Strategy”: A Highly Efficient Method for Cloning Different Vectors with Various Clone Sites. American Journal of Biomedical Research. 2013; 1(4):112-119. doi: 10.12691/ajbr-1-4-7

Abstract

In this study, we generalized the “Combinatorial Strategy” for efficient cloning of different vectors with various clone sites. 1) Using originally existed clone sites from circular plasmids to prepare the inserts, if no appropriate sites available, performing SDM to create compatible sites, could achieve maximal correct digestion of the inserts. 2) Different vectors were digested with various restriction endonucleases, and then dephosphorylated after digestion. 3) Top10 competent cells were used for transformation to increase the transformant colonies. Our results showed that, when either blunt-sites or Xba I site was adopted for ligation, the percentages of positive clones were about 50%. Whereas, when different sites, including one blunt and another Pst I sites, Not I and Xho I sites, were used, the percentages of positive clones were nearly 100%. With this strategy, most vectors could be successfully cloned through “one ligation, one transformation, three to five minipreps”.

Keywords:
DNA recombination combinatorial strategy vector cloning site-directed mutagenesis Top 10 cells clone sites

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