notice of cloning problem

Bernard P. Murray, PhD bpmurray*STUFFER* at socrates.ucsf.edu
Wed Dec 9 20:49:36 EST 1998


In article <Pine.HPP.3.96.981209151215.2288A-100000 at holmes.ipfw.edu>,
smitam01 at HOLMES.IPFW.EDU (Alan Smith) wrote:

> I have had this same cloning problem in the past and after sequencing the
> PCR generated fragement I found that the primer was made incorrectly and
> an A had never been added in the restriction site by the manufacturer.
> I remeber reading in this methods group that some restriction enzymes have
> trouble cutting at the ends of linear DNA, because of where they land on
> the DNA.  If this is the case it would be nioce for manufacturer to
> include this information in their catalogs about certain restriction which
> exhibit these properties. 
> Alan Smith

But they do (at least for some enzymes).  Check out the NEB catalogue
(eg. pages 258 and 259 in the '98/'99 catalogue) for data for many
of the common enzymes.  There are quite a few that don't cut well
with a 4 bp extension but can be pursuaded to perform a little letter
with an extended incubation.
  
> On Wed, 9 Dec 1998, Long-Cheng li wrote: 
> > Hi, there:
> > A couple of weeks ago I posted  my question regarding transformation.
> > After pcr, I could not cut the products. Usually 4bp outside cut site should
> > be enough. However I failed to cut it several times. Later I switched to TA
> > cloning and succeed in cutting the segment from the plasmid. It seems that
> > 4bp is not enough for cutting. 
> > Long

     I hope that this helps (in the future at least),
          Bernard

[No affiliation with NEB but I am frequently grateful for the mine
of information the back of their catalogue - though maybe they could
expand it at some point]
-- 
Bernard P. Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA



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