Not able to digest plasmid with NotI

Marc Rosoff mrosoff at u.washington.edu
Thu Nov 6 18:38:47 EST 1997


Not-1 doesn't cut supercoiled plasmids very well.  The NEB catalogue
says 5 fold more enzyme is required.  I would try  a longer incubation
with more enzyme.  If that fails sequence it.

				good luck,
				Marc











On Wed, 5 Nov 1997, Hadi wrote:

> In article <345E2DD8.7611 at lacdr.leidenuniv.nl>, 
> santbrin at CHEM.LEIDENUNIV.NL says...
> > Hello fellow netters,
> > 
> > Recently I ligated a synthetic linker in the XhoI/SpeI site of a
> > plasmid.
> > The linker contains (from 5' to 3') a XhoI-, a BamHI-, a EcoRV-,a NotI-
> > and a SpeI-site. I checked the presence of the linker by digestion of
> > the plasmid (isolated by a standard alkaline lysis procedure) with the
> > restriction enzymes mentioned above. They all linearize the plasmid except
> > Not I: 
> 
> ....truncated....
> 
> > Peter van Santbrink
> > University of Leiden
> > Leiden, The Netherlands
> > E-mail: santbrin at lacdr.leidenuniv.com
> > 
> >  
> Hmmm....sounds weird. Personally, I had never these kind of problems with 
> NotI or ligation of NotI containing linkers. But before you start 
> fiddeling around, I would sequence the plasmid to verify your NotI site. 
> 
> Hadi Al-Hasani
> hadi at nojunkmailsplease.helix.nih.gov
> 
> 




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