Using a modified TA cloning method to create entry clones.
We describe a noncommercial alternative method to create entry clones compatible with all kinds of destination vectors based on an improved TA cloning approach. To generate Gateway T vectors, we first constructed gentamicin- and chloramphenicol-resistant entry vectors designated pGWG and pGWC, respectively. Each entry vector contains an AhdI cassette flanked by attL sites, with each AhdI cassette containing two AhdI restriction enzyme sites spaced by the ccdB killer gene, which is lethal to most Escherichia coli strains. Gateway T vectors can be prepared by simple digestion of these entry vectors with the AhdI enzyme or its isoschizomers. The use of the ccdB gene as a negative selection marker is an important improvement over conventional TA cloning in that it eliminates the necessity of blue/white color screening based on alpha-complementation. Another important improvement that we have implemented is to retail the T vectors using Taq polymerase and dTTP so as to improve the cloning efficiency. Together, these improvements allow TA cloning to realize its full potential. Using Gateway T vectors prepared by this improved method, entry clones for PCR products or restriction enzyme fragments can be created simply, efficiently, and inexpensively while at the same time introducing greater compatibility.
Chen
Qi-Jun
QJ
State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100094, China.
Zhou
Hai-Meng
HM
Chen
Jia
J
Wang
Xue-Chen
XC
D002701:Chloramphenicol / Q000494:pharmacology; D003001:Cloning, Molecular / Q000379:methods*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D005822:Genetic Vectors / Q000138:chemical synthesis*; D005839:Gentamicins / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods*
D005839:Gentamicins; D002701:Chloramphenicol; D004262:DNA Restriction Enzymes