now you see it now you don't
Dr Rob Harling
ESA009 at ED.SAC.AC.UK
Fri Nov 10 09:25:42 EST 1995
> Subject: Re: now you see it now you don't
> To: mx%"diagnost at net.bio.net"
> ESA009 at ED.SAC.AC.UK ("Dr Rob Harling") wrote:
> >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?
On Thu, 09 Nov 95 17:47:16
<cubano67.ix.netcom.com at net.bio.net> asked:
"What is your storage buffer (if any) and why do you store at 4C?"
I use water for storage (because TE might mess up my carefully
calibrated PCR reactions) and I store at 4C because I continually
take out samples for conducting PCR and do not wish to freeze/thaw.
Thanks for your response.
Dr Rob Harling
SAC (Scottish Agricultural College)/
University of Edinburgh
West Mains Road
Edinburgh EH9 3JG
tel: +44 (0)131 535 4000
fax: +44 (0)131 667 2601
e mail: esa009 at ed.sac.ac.uk
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