Home-Made TA cloning

Robert Horton 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. 
19:1156, 1991.
(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.)

-Bob





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