Re- Cloning PCR Products

Brett Beitzel brett_beitzel at MSMTP.FS.SACI.ORG
Mon Aug 19 09:06:45 EST 1996


Subject:Re: Cloning PCR Products                   Date: 8/19/96

If you are doing TA cloning, and your vector has been phosphatased, I would
think that you would have to kinase your PCR product.  Oligos are usually
5'OH (unless you specifically order 5'-phosphate), so your PCR product will
be 5' and 3' OH.
I've never done TA cloning, but I wonder if it is really necessary to
phosphatase the vector.  Since both 3' overhangs (on your vector) are T, you
shouldn't need to worry about vector ligating to itself.
I've always cloned my PCR products by digesting them with an enzyme that cuts
in the primer region (pretty straight-forward,) or by UDG cloning, in which
there is no need for a ligation.

Hope this helps,
Brett  Beitzel
brett_beitzel at msmtp.idde.saci.org
Cancer Therapy and Research Center
San Antonio, TX




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