Universal CG cloning of polymerase chain reaction products |
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Authors: | Julian Stevenson Andrew J. Brown |
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Affiliation: | 1. School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia;2. Department of Nutrition and Toxicology, University of California, Berkeley, CA 94720, USA |
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Abstract: | Single-insert cloning of DNA fragments without restriction enzymes has traditionally been achieved using TA cloning, with annealing of a polymerase chain reaction (PCR) fragment containing a single overhanging 3′ A to a plasmid vector containing a 3′ T. In this article, we show that the analogous “CG cloning” is faster and far more efficient, using AhdI to generate a C-vector. For an afternoon ligation, CG cloning achieved double the cloning efficiency and more than 4-fold the number of transformants compared with TA cloning. However, blunt-end ligation was markedly more efficient than both. CG cloning could prove to be extremely useful for single-copy high-throughput cloning. |
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Keywords: | PCR Cloning Plasmid vectors |
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