DNA concentration measurement
Frank O. Fackelmayer
Frank.Fackelmayer at uni-konstanz.de
Wed Jun 21 03:30:51 EST 2000
The way you prepare the DNA might be a problem. Commercial (as well as
homemade) calf thymus DNA or E.coli DNA have very high molecular masses
and are quite difficult to get into solution. It is quite common that
stocks made from the same jar vary considerably. What we do is the
following: When we receive a new jar of DNA, we take out ALL the stuff,
weigh it, and put into a suitable amount of TE (to yield a 5mg/ml
solution approximately). The DNA is allowed to dissolve at 65C
overnight, and then sheared by sonication until viscosity of the
solution is low. Sometimes, depending on the DNA lot, a second heating
is necessary. After sonication, spin down undissolved junk (you should
no see a significant pellet) for 15min at 12.000g and check a small
aliquot on a agarose gel. Fragments should be between 200 and 2000bp,
with most in the 500bp region. If the fragments are ok, check the
concentration by spectrometry (should be consistent with the amount of
DNA used!), and make small aliquots for storage at -20C. We have never
observed differences between such aliquots, and the reproducibility
between individual preparations is negligible.
> Thanks, Chris. Well, the difference between slopes of the standard curves is
> 2-5 folds. In fact, this is not just 2 stocks - practically every time we
> prepare a new stock, it gives a new curve. And they all are made from the
> same jar of DNA (calf thymus, Sigma, cat. # D-1501) in the same way using
> sterile water, autoclaved glassware, etc.
> The dye is Picogreen, which doesn't have any measurable background
> fluorescence and binds efficiently to dsDNA.
> I 've already run abs scans - they are identical. All tests at 325, 280, 260
> and 230 are fine.
> Frozen aliquots of a same stock give identical lines.
> Checking with std DNA is a good idea, I will do it. However, choosing "good"
> stock decoratively is not as I need absolute amounts to measure, not
> relative. And they have to be comparable throughout the years and labs.
> Thanks again. Elena
> Chris LaRosa wrote in message <394BAB7C.78F21930 at biocomp.unl.edu>...
> >elena wrote:
> >> What if two stocks of DNA (concentration 15-30 µg/ml determined by spec)
> >> produce absolutely different standard curves when used to prepare
> >> within 0 - 0.001 µg/ml and read on microplate fluorometer?
> >> Results are consistent and repeatable. Linearity in any case is great.
> >What kind of Fluorescent dye?? They are subject to different
> >interferences, and can bind differently to different types of dna.
> >How different? 2 fold , 10 fold?
> >Either one is off or both are off...
> >Also your fluorometer might be giving different gains at different
> >There are several possibilities....
> >You need to get a properly callibrated std DNA prep to start with.
> >These can be bought. You need to double check your homemade stocks.
> >Perhaps run absorbance scans.
> >You may have interfering compounds in the buffer of one of the stocks.
> >You can always arbitrarily declare one of your stocks the "good" stock
> >The measurments may be off in absolute terms, but relative differences
> >may be good enough for your work.
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