Unwanted DNA degradation by addition of restriction enzyme
mlsulliv at wisc.edu
Mon Jul 25 09:32:34 EST 2005
There is probably DNAse contamination of your prep or some reagent
you are using for your restriction digest. The prep itself probably
won't degrade on it's own, because there is not Mg++ present, but
once you add restriction buffer with Mg++, DNase can be active and
degrade your prep.
How are you doing your plasmid preps? Some kits require an extra step
for some E. coli strains that are endA+ (these include JM series
strains, HB101, and others) to get rid of endogenous nucleases. E.g.,
Qiagen preps suggest an optional wash step with buffer PB, which
contains Guanadine HCl. I used to use Promega Wizard preps and I
think they suggested something similar for EndA+ strains.
Hope this helps.
On Jul 24, 2005, at 3:16 PM, Magnus Sundstrom wrote:
> Anyone experienced degradation of plasmid DNA after addition of the
> restriction enzyme buffer. Why is it happening and how can it be
> avoided?? I get a smear on the agarose gel and it is enough to add the
> buffer to get this. Very frustrating...
> Thanks for your help / magnus
> Methods mailing list
> Methods at net.bio.net
Michael L. Sullivan
Plant Research Molecular Geneticist
US Dairy Forage Research Center
1925 Linden Drive West
Madison, WI 53706
(608) 890-0046 (Phone)
(608) 890-0076 (FAX)
More information about the Methods