Home-Made TA cloning
horton at biosci.cbs.umn.edu
Tue Jul 11 12:28:03 EST 1995
nsaunders at molbiol.ox.ac.uk wrote:
: No, it must be dTTP. If it were ddTTP, you'ld have no free 3' OH group to form
: a phosphodiester bond to the A residue of the PCR insert and the ligation
: wouldn't work.
These are the classic references for construction of T-vectors:
Marchuk, D., Drumm, M., Saulino, A., and Collins, F.S. Construction of
T-vectors, a rapid and general system for direct cloning of unmodified
PCR products. Nucleic Acids Res. 19:1154, 1991.
(The blunt-ended vector is incubated with Taq and dTTP. One T is added.)
Holton, T.A., and Graham, M.W. A simple and efficient method for direct
cloning of PCR products using ddT-tailed vectors. Nucleic Acids Res.
(This does work, because the 3' end of the PCR-generated strands can be
ligated to the phosphate group of the vector. The 5' ends of PCR
generated strands don't normally have phosphates anyway.)
More information about the Methods