DNA degradation by incubation at 70 C

MICPRF at latvax8.lat.oz MICPRF at latvax8.lat.oz
Wed Jul 4 13:02:36 EST 1990


Greetings molbio gurus,
	Here is a problem that is puzzling us. We are working with
Dictyostelium nuclear DNA for various purposes and noticed unexpected
degradation of the DNA in our preps. occasionally. The problem has now
been traced to heat. The DNA is ok at temperatures up to 52 degrees C,
but at the next highest temperature we tested, 68 degrees C, a 30 minute
or 1 hour incubation leads to complete degradation of the DNA. This is
occurring in normal TE buffer, and since we do not work with thermophilic
organisms of any kind, it seems unlikely to me that it is enzymatic.
Everything we work with is, of course, sterilized appropriately. This has
no effect on our ability to probe genomic Dictyostelium DNA with bacterially
grown probes. Here the genomic DNA is already cut, denatured and bound to
the nitrocellulose before hybridization at 68-70 degrees C.
The DNA is from a nuclear prep. after gentle Triton lysis of the cells and
has been phenol extracted several times, then chloroform extracted
and ethanol precipitated. While it doesn't affect Southern blot analysis,
there are obviously other situations where it becomes a nuisance to say the
least. Does anyone out there in Netland know what the cause might be,
and how it can be avoided?
				Regards,
				Paul Fisher (micprf at latvax8.lat.oz.au)



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