DNA degradation

Greg gavis at uoknor.edu
Mon Oct 9 05:37:30 EST 1995


> Hi Netters,
> l routinely use the miniprep method by Applied Biosystems Inc., since this 
> method is supposed to give really clean DNA that can be used for
sequencing. l 
> have never had any problem with DNA degradation, however, recently,
every time 
> l use the DNA obtained using this method in restriction digests, the DNA 
> degrades (l see a smear on the gel). l know that the degradation is not
due to 
> the plasmid construct, because l have used DNA obtained using a different 
> method in digests and the DNA does not degrade. Has anyone had a similar 
> problem, and if so, how did you solve it? Thanks in advance!
> 
> Jackie

Jackie,

I just spent a week solving the same problem and my solution was this: 
use a strain that is negative for endonuclease (endA-).  Endonuclease
always makes it through my mini-prep procedure and degrades my DNA during
digests.  A good endA- strain is E. coli DH5-aplha (available from
Clontech).  When I use this strain I get NO DNA smears.  It was simply
wonderful.

Good Luck,
Greg Davis
gdavis at uoknor.edu
University of Oklahoma
School of Chemical Engineering and Materials Science
Biotechnology Research



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