DNA concentration measurement
elenag at imap1.asu.edu
Sat Jun 17 12:40:45 EST 2000
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>...
>> 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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