Construction of TA cloning vector

Dr. C. Dolphin cdolphin at
Sat Jan 29 11:24:11 EST 1994

Rasmussen at (Randy P. Rasmussen) wrote:

>I have heard that you can add a single 3' overhanging T to a blunt end by
>incubating your blunted DNA with Taq polymerase and dTTP.  This would be a
>dandy way to make your own PCR cloning vectors.  Can anyone confirm or deny
>this?  Does anyone have a reference?

you could also incubate blunt-cut vector with dideoxyTTP (ddTTP) and terminal
transferase which will add single ddTTP to each 3'end. Addition of extra dATP
in the final elongation stage of PCR may also increase proportion of products
with A overhangs (question - does Taq add just a single dATP or does it also
have terminal transferase-like activity?). We have used this for succssefully
for sub-cloning of many PCR products. Also include a touch of the enzyme used
to blunt cut vector in the ligation reaction to reduce background.


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