Unwanted DNA degradation by addition of restriction enzyme buffer

[Michael Sullivan]

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.

Mike


On Jul 24, 2005, at 3:16 PM, Magnus Sundstrom wrote:

> Hi
>
> 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
>
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---
Michael L. Sullivan
Plant Research Molecular Geneticist
US Dairy Forage Research Center
ARS-USDA
1925 Linden Drive West
Madison, WI 53706
(608) 890-0046 (Phone)
(608) 890-0076 (FAX)