now you see it now you don't

Dr Rob Harling ESA009 at ED.SAC.AC.UK
Fri Nov 10 10:03:04 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
Scotland, UK
tel: +44 (0)131 535 4000
fax: +44 (0)131 667 2601
e mail: esa009 at ed.sac.ac.uk




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