On 17 Mar 1999,
Andrew G. wrote:
> I am seeking advice on storing low concentrations of DNA fragments for
> competitive PCR.
> What buffer + additives?
> What temperature for storage?
In its PCR-MIMIC kit Clontech uses 10 mM tris, pH 7.5, 0.1 mM EDTA, 10
ug/ml glycogen as dilution buffer for DNA competitors (store -20oC). They
do not recommend multiple freeze-thaw cycles, so after the third use an
aliquot should be discarded. Indeed, diluted competitors (PCR fragments
150-600 bp long, 1 attomol/ul and less) were not stable and I saw a
profound decrease in their effective concentration after each use. In my
hands, addition of 1 ng/ml sonicated E.coli genomic DNA (XL-Blue MRF',
home-made extraction) keeps their effective concentration much more
stable, so I could use aliquots as many times as I needed, say, 10 times,
storing them at -20oC (but I did not make comparative experiments to
measure precisely the effect of my storage/use conditions on diluted
competitors). Moreover, as to my experience, addition of heterologous DNA
increases PCR efficiency on diluted targets, and in any case it does not
decrease it, at least in reasonably small amounts.
Hope this helps. Good luck.
University of Kuopio