now you see it now you don't
Michael G. Kurilla
mgk2r at UVA.PCMAIL.VIRGINIA.EDU
Fri Nov 10 10:30:59 EST 1995
On Nov 10, 9:30am, "Dr Rob Harling" wrote:
> Subject: Re: now you see it now you don't
> > 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.
The rationale for TE as a storage buffer is straightforward. The EDTA
chelates Mg that all nucleases require. That doesn't seem to be your problem
(although it could develop over time if your go in and out of that tube
repeatedly. The Tris (pH8.0) is where DNA is most stable. Basic solution
promote strand breakage (again not your problem). Acid pH can be bad because
of protonation of some of the bases. This can lead to ring cleavage that
would certainly cause PCR headaches.
Check the pH of your water. Even ultrapure H2O tends to be a little on the
acidic side. Why not prepare a weak storag buffer, like tris-HCl 1.0mM at
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