D.J.Glover at bham.ac.uk
Thu Mar 2 15:14:15 EST 1995
I am using a Molecular Dynamics Phosphorimager to
obtain quantitative data from northern blots. I have a couple of questions
that other users around me have been unable to answer to my satisfaction.
One of the advantages of a phosphorimager over
autoradiography and densitometry is that the response of a phosphor screen is
linear over a much greater range than an autorad. film. Nevertheless, there
is a limit to the linearity of a phosphor screen.Therefore, should we always
calibrate our screen to check our signal is within the linear range of the
screen. I don't know anyone here who does. My 32P signals are often quite weak
so I am not worried about saturation but rather the lineararity of the screen
at low range signals.
My second question regards the production of a
publication quality hardcopy of the image. I vaguely remember some discussion
on this newsgroup about this a long time ago.If I remember rightly the best
solution anybody offered was to expose the blot to an autorad. after exposure
to the phosphor screen and photograph it. This has also been suggested to me
by others here. I find this very disappointing for such an expensive piece of
equipment. I do a lot of these so if I had to expose to an autorad for several
days after quantitation on the phosphorimager I'd never get to finish my PhD!
At the moment I expose to the phosphor screen for 3-4 days so that I get a
good contrast between the signal and background and then print it out on an HP
laserjet at 600 dpi. I'm not totally satisfied with this but I guess I'll have
to make do unless anyone else has any suggestions.
BTW, thanks to all those who take part in the
phosphorimager discussions. I find them most informative.
Thanks in advance,
School of Biochemistry,
University of Birmingham, UK.
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