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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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Title An alternative approach in Gateway ® cloning when the bacterial antibiotic selection cassettes of the entry clone and destination vector are the same
 
Creator Kumar, Kamal
Yadav, Saurabh
Purayannur, Savithri
Verma, Praveen K.
 
Subject Gateway cloning
Kanamycin selection
Entry vector
Destination vector
LR recombination
 
Description Accepted date: 04 May 2012
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.
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 Onder (Paracelsus Private Medical University Salzburg,
Austria) and Tsuyoshi Nakagawa (Shimane University, Japan) for
pBD-gate1 and pGWB441 destination vectors, respectively.
 
Date 2015-11-05T09:51:09Z
2015-11-05T09:51:09Z
2013
 
Type Article
 
Identifier Mol. Biotechnol., 54(2): 133-140
1559-0305
http://172.16.0.77:8080/jspui/handle/123456789/331
http://link.springer.com/article/10.1007%2Fs12033-012-9549-0
10.1007/s12033-012-9549-0
 
Language en_US
 
Publisher Springer