Construction of a restriction-endonuclease-Eam1105I-generated T-vector for high-throughput cloning and expression.
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 beta-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.
Wang
Baoli
B
Key Research Laboratory of Hormone and Development Affiliated to the Ministry of Health, Institute of Endocrinology, Tianjin Medical University, Tianjin, People's Republic of China.
Liang
Hui
H
Liu
Rui
R
Li
Xiaoxia
X
Sun
Bei
B
Zhang
Rui
R
Guo
Shanyi
S
Guo
Gang
G
Zhang
Jingyu
J
Dai
Chenlin
C
D003001:Cloning, Molecular / Q000379:methods*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism*
D011994:Recombinant Proteins; C075914:endodeoxyribonuclease Eam1105I; D015252:Deoxyribonucleases, Type II Site-Specific