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Construction of a restriction‐endonuclease‐Eam1105I‐generated T‐vector for high‐throughput cloning and expression
Author(s) -
Wang Baoli,
Liang Hui,
Liu Rui,
Li Xiaoxia,
Sun Bei,
Zhang Rui,
Guo Shanyi,
Guo Gang,
Zhang Jingyu,
Dai Chenlin
Publication year - 2007
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20070033
Subject(s) - cloning (programming) , restriction enzyme , vector (molecular biology) , cloning vector , biology , genetics , endonuclease , multiple cloning site , molecular cloning , expression vector , throughput , computational biology , microbiology and biotechnology , dna , gene , gene expression , recombinant dna , computer science , programming language , telecommunications , wireless
A novel T‐vector was constructed that could be used for direct cloning and expression of PCR‐amplified cDNA. The technique was based on the insertion into the parent vector of two endonuclease‐Eam1105I restriction sequences spaced by an expression cassette of the full‐length β‐galactosidase, which helped to improve cloning efficiency and to minimize the non‐recombinant background of the T‐vector when used to clone PCR products. Moreover, this method took advantage of the reconstitution of the rarest restriction sequence of MssI to enable directional cloning. These advantages make the T‐vector suitable for high‐throughput expression and analysis.