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An Alternative Approach in Gateway® Cloning when the Bacterial Antibiotic Selection Cassettes of the Entry Clone and Destination Vector are the Same

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Abstract

The Gateway® recombination technology has revolutionized the method of gene cloning for functional analyses and high-throughput ORFeome projects. In general, Gateway cloning is highly efficient because after LR recombination and bacterial transformation, only cells containing the recombinant destination clone are selected on an antibiotic selection plate. However, when the antibiotic resistance gene for bacterial selection is the same in the entry and destination vectors, the direct selection of recombinant destination clones on an antibiotic plate is difficult. Here, we demonstrate an efficient and comprehensive approach to obtain positive destination clones directly on an antibiotic selection plate in this situation. The strategy involves polymerase chain reaction (PCR)-mediated amplification of the entry clone using entry vector-specific primers that bind outside the attL sequences and the subsequent use of this purified PCR product for LR recombination with the destination vector. Our results suggest that cloning of linear DNA fragments into circular destination vectors through LR recombination is an efficient method for inserts up to 7 kb in size. Using this approach, the yield of colony PCR positive destination clones was 100 % for genes of various sizes tested in our experiments.

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Acknowledgments

This work is supported partially by research grant provided by Department of Biotechnology, Government of India for Next Generation Challenge Programe on Chickpea Genomics project (Sanction No. BT/PR12919/AGR/02/676/2009), and National Institute of Plant Genome Research (N.I.P.G.R.), New Delhi. K.K. acknowledges NIPGR for Postdoctoral fellowship. We thank Kamil Önder (Paracelsus Private Medical University Salzburg, Austria) and Tsuyoshi Nakagawa (Shimane University, Japan) for pBD-gate1 and pGWB441 destination vectors, respectively.

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Correspondence to Praveen Kumar Verma.

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Kumar, K., Yadav, S., Purayannur, S. et al. An Alternative Approach in Gateway® Cloning when the Bacterial Antibiotic Selection Cassettes of the Entry Clone and Destination Vector are the Same. Mol Biotechnol 54, 133–140 (2013). https://doi.org/10.1007/s12033-012-9549-0

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  • DOI: https://doi.org/10.1007/s12033-012-9549-0

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