TA cloning vector

Ed Rybicki ED at micro.uct.ac.za
Wed Aug 4 02:49:33 EST 1993


> In <1993Aug3.205333.24726 at galileo.cc.rochester.edu> PHIP at BPHVAX.BIOPHYSICS.ROCHESTER.EDU writes:
> >
> > I'll be doing some cloning of PCR products in the near future and I was
> > planning on using TA cloning as described by Invitrogen.  I was hoping,
> > however, not to have to buy their kit.  Does anyone know of a vector
> > that is easily obtained and can be used for this purpose (i.e., can be
> > cut with an enzyme which leaves a single T overhang).  Thanks in advance,
> > I'll post a summary later.
>
> Check out the pT7Blue T-Vector Introductory Kit from Novagen (800-526-7319).
> For $75 you get vector, competent cells, SOC medium, positive control and

NONONONONONONONONONOoooooo....MAKE a kit....!  Marchuk et al, 1990, Nucl
Acids Res 19(5) 1156, and another 1-pager in same journal, both describe
simple and easy ways of T-tailing blunt-cut plasmid (pUC and pSK in our
hands, cut with SmaI or EcoRV) amd using same for reliable cloning of PCR
products.  Marchuk method works well in our hands, and is FAR, FAR CHEAPER
than any kit (a sop to Jim Graham).  If you don't want to use Taq to T-
tail, or to T-tail with terminal transferase and ddTTP, then you could do
what someone here has mooted, which is to cut a plasmid with any enzyme
that gives a T at the end of a 3'-overhang, then fill in with Klenow and
dC-, dG- and dTTPs (note: no dATP) to get (eg.):

                  vector              PCR amplimer        vector
        5'-XXXXXXXXXXXXXXXXXGACT XXXXXXXXXXXXXXXXXXXXXXA GTCXXXXX-3'
        3'-XXXXXXXXXXXXXXXXXCTG AXXXXXXXXXXXXXXXXXXXXXX TCAGXXXXX-5'

Good luck,

  ____________________________________________________________________
 | Ed Rybicki, PhD             |       "Lord, won't you buy me        |
 | (ed at micro.uct.ac.za)        |                                      |
 | Dept Microbiology           |         A Mer-ce-des Benz..."        |
 | University of Cape Town     |                                      |
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