Re- Cloning PCR Products
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
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
More information about the Methods