[RASMB] determination of phage particle concentration

Tue Mar 9 03:38:07 PST 2010

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.

Peter

Peter E. Prevelige Jr.
Professor
Dept. of Microbiology, BBRB 416/6
Univ. of Alabama @ Birmingham
845 19th St. South
Birmingham AL. 35294-2170
Phone 205 975-5327
FAX 205 975-5479
prevelig at uab.edu
http://www.microbio.uab.edu/faculty/prevelige/prevelige-p.htm

-----Original Message-----
From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On Behalf Of Borries Demeler
Sent: Tuesday, March 09, 2010 5:17 AM
To: Steve Harding
Cc: rasmb at server1.bbri.org
Subject: Re: [RASMB] determination of phage particle concentration

I wonder if one could mitgate the problems with light scattering by
preparing a known, significant dilution, staining with sybrgreen
and looking at fluorescence intensity in a velocity experiment.
Sybrgreen bound to DNA is very sensitive and this can be done at very
low concentration.

We have done this with good success using the Aviv fluorescence machine
to measure phage aggregation behavior. Dilutions provided linear changes
in fluorescence intensity. Sybrgreen seems to have no problem leaking into
the phages we investigated and lights them up like a christmas tree. One
issue though is that one would need some standard, and I am not sure how
to get this. At the very least one could use this to determine if there
is leaked DNA in the prep or other contaminating DNA, and sybrgreen has
a very low background.

Serwer P, Hayes SJ, Thomas JA, Demeler B, Hardies SC. Isolation of novel
large and aggregating bacteriophages. Methods Mol Biol. 2009;501:55-66.

Regards,
-Borries
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