Re- Cloning PCR Products

Andrew J. Doherty doherty at bsa.bristol.ac.uk
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 msmtp.idde.saci.org
> 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                               
   Bristol                                            
   UK
   BS8 1TD

   e-mail  Doherty at bsa.bristol.ac.uk

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