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Dear RASMB:<br><br>
I should have realized that my comments might cause concern, especially
given the many successful prior publications. I'll try to be more
helpful. <br><br>
What absorbance range worked you, Peter? "Highly" turbid
solutions will cause nonlinearity in the signal-to-concentration
relationship. You can see this effect by shining a beam of visible
light through slightly turbid, and highly turbid, solutions. The
slight solution will clearly show the beam path. The highly turbid
solution will have a diffuse beam path, and show light outside of the
beam path due to more than one scattering event. Unless the
detector has special optics to see only the transmitted light path and
nothing else then there is a chance that the light signal will over-state
the concentration. My feeling is that if you keep ~0.1 OD or less
that the problem is negligible, while at 1 OD the problem is
significant. If the sample is highly scattering I would expect
problems in the diffusion / shape fitted parameters, but not necessarily
the size.<br><br>
My other concern is that the same scattering sample can produce different
absorbance signals on different instruments architectures, due to
different optical layouts and susceptibilities to scattered light.
Fortunately, that is not the goal of AUC experiments. Rather the
AUC measurements are relative to each other (much like fluorescence) with
the same sample and hardware. The analysis software is not as
sensitive to absolute amplitudes, but rather the relative shape(s) of the
radial scan(s). Therefore, for slightly turbid solutions I see no
problem for a single species. If there is a large range of particle
sizes, then I would expect the sizes to be correct, but the relative
concentrations to be off due to larger particles scattering light more
than small particles. <br><br>
My $0.02 for people who haven't already coped with these issues.<br><br>
Glen<br><br>
<br>
At 06:38 AM 3/9/2010, Peter Edward Prevelige Jr wrote:<br>
<blockquote type=cite class=cite cite="">We have determined (in control
experiments for our experiments using turbidity to monitor phage
assembly) that the turbidity increases linearly with the concentration of
phage capsids over the useful working range of a spectrophotometer
regardless of wavelength. As Borries mentioned you just need a standard
to get absolute number. We use phage with a particle/pfu ratio of very
nearly 1 and obtain absolute concentration by titer.<br>
<br>
Peter<br><br>
<br>
Peter E. Prevelige Jr.<br>
Professor<br>
Dept. of Microbiology, BBRB 416/6<br>
Univ. of Alabama @ Birmingham<br>
845 19th St. South<br>
Birmingham AL. 35294-2170<br>
Phone 205 975-5327<br>
FAX 205 975-5479<br>
prevelig@uab.edu<br>
<a href="http://www.microbio.uab.edu/faculty/prevelige/prevelige-p.htm" eudora="autourl">
http://www.microbio.uab.edu/faculty/prevelige/prevelige-p.htm</a><br><br>
-----Original Message-----<br>
From: rasmb-bounces@rasmb.bbri.org
[<a href="mailto:rasmb-bounces@rasmb.bbri.org" eudora="autourl">
mailto:rasmb-bounces@rasmb.bbri.org</a>] On Behalf Of Borries
Demeler<br>
Sent: Tuesday, March 09, 2010 5:17 AM<br>
To: Steve Harding<br>
Cc: rasmb@server1.bbri.org<br>
Subject: Re: [RASMB] determination of phage particle
concentration<br><br>
I wonder if one could mitgate the problems with light scattering by<br>
preparing a known, significant dilution, staining with sybrgreen<br>
and looking at fluorescence intensity in a velocity experiment.<br>
Sybrgreen bound to DNA is very sensitive and this can be done at
very<br>
low concentration.<br><br>
We have done this with good success using the Aviv fluorescence
machine<br>
to measure phage aggregation behavior. Dilutions provided linear
changes<br>
in fluorescence intensity. Sybrgreen seems to have no problem leaking
into<br>
the phages we investigated and lights them up like a christmas tree.
One<br>
issue though is that one would need some standard, and I am not sure
how<br>
to get this. At the very least one could use this to determine if
there<br>
is leaked DNA in the prep or other contaminating DNA, and sybrgreen
has<br>
a very low background.<br><br>
Serwer P, Hayes SJ, Thomas JA, Demeler B, Hardies SC. Isolation of
novel<br>
large and aggregating bacteriophages. Methods Mol Biol.
2009;501:55-66.<br><br>
Regards,<br>
-Borries<br>
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<x-sigsep><p></x-sigsep>
<br>
Glen Ramsay, Ph.D.<br>
Chief Scientist<br>
Aviv Biomedical, Inc.<br>
750 Vassar Avenue, Suite #2<br>
Lakewood, NJ 08701<br>
(732) 370-1300, Ext. 25<br>
(732) 370-1303, FAX<br>
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www.avivbiomedical.com<br>
</a> Skype: GlenAtAviv<br><br>
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