Re- Cloning PCR Products

Andrew J. Doherty doherty at
Tue Aug 20 04:24:19 EST 1996

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
> Cancer Therapy and Research Center
> San Antonio, TX
Ta very much - I'm actually doing a blunt-end ligation into an
expression vector, but I was very silly and did not put restriction
sites into the Primers. I'm using a proof-reading Taq, so I should have
a good population of blunt-ended products. But I didn;t realise that the
oligos would be -OH at both the 5' and 3' ends! So I'll kinase treat the
PCR product and try again.

Thanks very much

   Dr Andrew Doherty                          
   Department of Anatomy                   
   School of Medical Sciences             
   University Walk                               
   BS8 1TD

   e-mail  Doherty at


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