Problem with mtDNA-Degradation
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.
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