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Absorption Spectroscopy and Quantitation of Filamentous Phage
Below is a typical UV absorption spectrum of purified filamentous phage dissolved in
TBS, which also serves as the blanking solution in the reference cuvette. Unlike spherical phage
like T4 and , which have roughly equal weight ratios of protein to DNA, filamentous phage
have about 6 times more protein than DNA; the protein therefore contributes substantially to the
absorption spectrum, accounting for the broad plateau at 260–280 nm, with a shallow maximum
at 269 nm. Based on the measurements of Day and Wiseman [Day, L.A. and Wiseman, R.L.: A
comparison of DNA packaging in the virions of fd, Xf, and Pf1. In: Denhardt, D.T., Dressler, D.
and Ray, D.S. (Eds.), The Single-Stranded DNA Phages. Cold Spring Harbor Laboratory, Cold
Spring Harbor, NY, 1978, pp. 605–625], we calculate the concentration of phage in virions/ml
from the difference between A269 and A320 as follows:
( A269  A320 )  6  1016
virions/ml 
number of bases/viri on
Subtracting A320, a wavelength where there is little light absorption from phage chromophores, is
meant to correct crudely for light scattering from phage particles and non-phage particulate
contaminants. Applying this formula to the spectrum below, in which the phage clone has 9312
bases/virion, we get a physical particle concentrations of
(0.511  0.067)  6 1016
 2.86 1012 virions/m l
9312