> We're using RAPD-PCR to study diversity in epiphytic Pseudomonas
> species. We prepare the template DNA using Flowgen's Puregene kit,
> which is basically an SDS cell lysis, ammonium acetate protein
> precipitation, RNAse, isopropanol DNA precipitation, then dissolve in
> water. The resulting DNA is of good spectrophotometric quality.
>> PCR works if we use freshly - prepared DNA, ie extracted from the
> bacteria then run on the same day. If we store the DNA at 4C for
> only one week, then PCR fails, we get zilch in the lanes at any
> concentration of template. The DNA still looks the same after one week
> when run on a gel, ie there doesn't seem to be nuclease breakdown
> of the DNA. Does anyone know what's happening to the DNA in storage?
>> Thanks
> Rob (still got my hair) Harling
>> Dr Rob Harling
> SAC (Scottish Agricultural College)/
> University of Edinburgh
> West Mains Road
> Edinburgh EH9 3JG
> Scotland, UK
> tel: +44 (0)131 535 4000
> fax: +44 (0)131 667 2601
> e mail: esa009 at ed.sac.ac.uk
This phenomenon has been observed repeatedly in a number of groups
(consult RAPD at net.bio.net archives). If I had to make predictions I
would guess your DNA was fairly dilute (1-25 ng/ul). Most people
find DNA to be much more stable at higher concentrations and that
dilute samples for RAPD should be stored frozen. We have observed
the same phenomenon with first strand cDNA preps for RT-PCR or
ddRT-PCR. But for these it is even worse. They don't last more than
a week even in the -20. We have tried adding a little DTT to inhibit
oxidation and that seems to help a little.
//========================================================\\
||Doug Rhoads || Dept. of Biological Sciences||
||drhoads at mercury.uark.edu || 601 Science Engineering ||
||drhoads at uafsysb.uark.edu || University of Arkansas ||
||501-575-3251 || Fayetteville, AR 72701 ||
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|| My Dogma Just Got Run Over by Someone's Karma ||
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