what's the best way to clone PCR products?

Tracy Aquilla aquilla at salus.med.uvm.edu
Fri Sep 23 16:48:09 EST 1994

In Article <mikep.147.000F90D1 at uniwa.uwa.edu.au>, mikep at uniwa.uwa.edu.au
(Michael Poidinger) wrote:
>In article <aquilla.1130661858D at sadye.emba.uvm.edu> aquilla at salus.med.uvm.edu
(Tracy Aquilla) writes:
>>From: aquilla at salus.med.uvm.edu (Tracy Aquilla)
>>Subject: Re: what's the best way to clone PCR products?
>>Date: Thu, 22 Sep 1994 22:50:18 GMT
>>In Article <35si70$gt at mark.ucdavis.edu>, ez005528 at rocky.ucdavis.edu (Kevin
>>Morano) wrote:
>>>Regarding PCR cloning:
>>>1) Engineer restriction sites into the middle of your primers, leaving 
>>>5-8 bp flanking on each side.
>>>2) Geneclean/gel purify 5-10 ug of PCR band. 
>[stuff about home brew deleted]
...(more deleted)...
>Well I tried cloning 1 lousy PCR product with restriction sites (Eco RI and 
>Hind III) for over 4 months and never got close.  Some PCR products just will 
>not cut with enzymes, 8 base overhang or no.
>I routinely use Promega's PGEM-T  with electroporation now and never have any 
>ps And to resurrect an old flame war, when in doubt, use a kit :)
>Dr Mike Poidinger        
>Microbiology, UWA       ==> Animal Welfare NOT Animal Rights       
>Australia                        ==> Sonic the Hedgehog says "It's your cone"
>mikep at uniwa.uwa.edu.au 

I am curious as to why you say that some PCR products just can not be
cleaved by restriction endonucleases. If there is a site there, and the
conditions are right for the enzyme, it should work every time. If not, then
something is wrong with the reaction conditions or the DNA. Granted, for
many enzymes, an 8-base overhang may not be enough, especially if it isn't
completely double-stranded, but restriction digestion is not some magical
process. Did you test-cut your PCR product with an enzyme which cleaves it
somewhere in the middle? I have not observed this problem myself. Does
anyone have a rational explanation for why some PCR products don't cut
(other than 'it just won't cut')?
Tracy Aquilla, Ph.D.
Molecular Physiology and Biophysics
University of Vermont
aquilla at salus.med.uvm.edu

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