Cutting with MboII

Phillip San Miguel pmiguel at purdue.edu
Tue Nov 30 12:16:09 EST 1999


    Another possibility: the enzyme is cutting to completion, but your PCR band
contains more than one restriction-site variant. If these variant(s) were unequally
amplified, then you will get bands that appear to be non-stoichiometric (that is, a
faint band above a more intense band).
    I once attempted to clone a PCR product and found I got two different variants
of clones by restriction digest and sequencing. One was the product I wanted. The
other was something different--totally unrelated. Note that these came from a PCR
reaction that produced a clean, single band.
Phillip San Miguel

Johns053 at MC.DUKE.EDU wrote:

> Are you sure it is incomplete digestion.  See the November issue of
> BioTechniques.  Adding excess enzyme causes aberrant migration of the DNA bands.
> Apparently, you can avoid this problem by heat inactivating before loading the
> samples.  I have seen the same phenomenon with FokI.  It may be universal for
> enzymes which cleave outside their recognition sites.
>
> Frank Bartel <frank.bartel at medizin.uni-halle.de> on 11/30/99 09:30:52 AM
>
> To:   methods at net.bio.net
> cc:
> Subject:  Cutting with MboII
>
> Does somebody have experiences with the restriction enzyme MboII. We try
> to cut PCR products, but the digestion seems to be incomplete, even when
> incubating overnight.
>
> Thank you for your help.





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