Re- Cloning PCR Products

S. Wang sw59 at cornell.edu
Mon Aug 19 16:15:48 EST 1996


I have done TA cloning many times, it worked every time without kinase
treatment.

Shaojiu Wang

In article <n1371676715.52021 at msmtp.idde.saci.org>,
brett_beitzel at MSMTP.FS.SACI.ORG ("Brett Beitzel") wrote:

> 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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