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
