A Rapid and Simple, Recombination-Based Cloning Method in Escherichia Coli

Manoj Baliram Pohare^1* and Mitsuru Akita^{1, 2}  

¹The United Graduate School of Agricultural Sciences, Ehime University, 3-5-7  Tarumi, Matsuyama, 790-8566 Japan.

²Graduate School of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, 790-8566 Japan.

Corresponding Author E-mail: b742006b@mails.cc.ehime-u.ac.jp

DOI : http://dx.doi.org/10.13005/bbra/2415

ABSTRACT: Cloning is indispensable in molecular biology. Here we developed an in vivo homologous recombination-based cloning procedure and determined the optimal conditions. This procedure required two PCR products to be amplified from a gene of interest and desired plasmid vector. The 5’ ends of both primers that amplified one product had nucleotide sequences complementary to that used to amplify the other product. Once the mixture of those PCR products was introduced into Escherichia coli DH5α competent cells, transformants carried plasmids in which the gene of interest had been properly cloned. Optimizing the cloning conditions, at least 12-nucleotides overlaps between the terminal ends of two fragments were required to generate desired plasmids. This value was much shorter than the length of overlaps required for the same procedure employed in the yeast system. Therefore, this procedure is expected to be an attractive alternative for cloning in the E. coli system.

KEYWORDS: Cloning; PCR; plasmid vector; Escherichia coli; in vivo homologous recombination

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