Trouble finding DNA concentration on Spec..
William B. Melchior, Jr.
wmelchior at fdant.nctr.fda.gov
Tue Feb 13 16:23:26 EST 1996
Charles A Miller wrote:
> I am am having trouble determining DNA concentrations on a
> spectrophotometer. When I measure the UV absorbance, I consistantly get
> 0 or even a negative number when it is obvious through gel
> elctrophoresis of the supercoiled plasmid that there is plently of DNA.
Assuming you have enough DNA (which you can estimate from a gel, by
comparison with your standards), it sounds like your blank may be giving
you problems. The most common trouble I have is with solutions
containing EDTA; even at 0.1 mM, these solutions may have a high OD.
EDTA absorbance is strongly influenced by pH, and I have seen it change
with time in a cuvet. When using a single-beam spectrophotometer, I try
to zero the machine with a blank solution, and then dilute the unknown
into the same solution, hopefully adding not more than 5% by volume of
Cuvets can be cleaned with varying degrees of vigor, but if their
absorbance (filled with clean water) is constant with repeated filling
and measuring, you're probably ok; consistency is the key; you don't want
things dissolving off the walls during a series of measurements. If
you're using a dual beam instrument, swap cuvets a couple of times
between reference and sample sides to make sure they match. (Another
hint, especially if the cuvets aren't completely clean: Mark one side of
each cuvet, or make a note of where the label is, and then always put it
into the machine in the same orientation -- for example, label always to
the front.) Of course, avoid fingerprints. When I start to use a cuvet,
I wipe the outside GENTLY and slowly with a damp--not wet--tissue, one
slow swipe at a time. I then repeat with a tissue barely damped with a
couple of drops of methanol; even if I can detect a slight film on the
INSIDE, it usually doesn't matter because it isn't water soluble. Be
very careful when loading the cuvets so that you don't spill liquid on
the outside of the windows, forcing you to reclean them.
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