Problem with mtDNA-Degradation

Frederik Intelmann intelman at uni-hohenheim.de
Fri Aug 16 04:12:31 EST 1996


I'm currently trying to extract the mitochondrial DNA of a zoosporic fungus 
via differential centrifugation, lysis of the mitochondria via 2% SDS 
(including proteinase K addition) and preciptiation of the DNA (after removing 
SDS and protein).
What baffles me is the fact that even though I use solutions from the same 
batch (aliquoted), do everything on ice / in cooled centrifuges, and add 20 mM 
EDTA to the Lysismix to inhibit nucleases, I sometimes get one clear band of 
undegraded mtDNA, and at other times (unfortunately the majority of times) 
just degraded mtDNA on an agarose gel. I'm wearing gloves to protect the 
samples and sterilize solutions and reaction cups.
So, does anybody have a suggestion as to what I could be doing wrong ?
Any help would be greatly appreciated.


Frederik Intelmann
Universität Hohenheim
Germany



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