A modified plasmid vector pCMV-3Tag-LIC for rapid, reliable, ligation-independent cloning of polymerase chain reaction products.
Here we present a modified vector pCMV-3Taq-LIC for a rapid, simple, and relatively cheap method to build expression constructs. After being digested by Nt.BspQI and EcoRV, a lineal vector with specific 11-base single overhangs is obtained. Polymerase chain reaction (PCR) products with complementary overhangs are created by building appropriate extensions into the primers and treating them with T4 DNA polymerase. The annealing of the insert and the vector is performed in the absence of ligase by simple mixing of the DNA fragments. This process is very specific because only the desired products can form. Using this vector, we successfully constructed hnRNP K full-length complementary DNA (cDNA) expression plasmid.
Du
Runlei
R
College of Life Sciences, Wuhan University, Wuhan 430072, China.
Li
Shangze
S
Zhang
Xiaodong
X
D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005822:Genetic Vectors / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D010957:Plasmids / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods; D014162:Transfection
D004247:DNA; D004259:DNA-Directed DNA Polymerase; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases