I am concerned that a phage prep I've done contains more than one phage.
This should be unlikely and I'm wondering whether the symptoms are due to
a different state of the phage DNA, but would this affect the mobility of
the phage through CsCl? Anyway, here's the situation:
-Phage suspension (harvested from overlays) precipitated with PEG,
resuspended in SM buffer.
-0.75g/ml CsCl added and spun 160000g x24hr (Beckman SW50.1 rotor) to
purify through equilibrium gradient.
-3 bands seen - one right at the top of the tube (assuming debris), one a
couple of mm below that (assuming phage) - designated "U", and a third
around 5mm below that - designated "L".
-Obtained bands U & L, did a quick DNA prep to visualise on agarose gel
(1ul sample + 9ul dH2O + 4ul 2.5X SDS-EDTA sample buffer; heat 65Cx5min &
run on agarose). I also ran 1ul samples in normal gel loading buffer (i.e.
I would expect to see no band as any phage should not have been popped
open to release their DNA)
-no DNA seen in the unpopped samples (as expected)
-3 bands seen in each of the popped samples. Bands the same apparent size
for both U & L but the intensity varied (in the U sample the larger size
band was more intense, in the L sample the lower). Are these bands
indicative of different forms of phage DNA? The phage are dsDNA myoviridae
but I don't have any information regarding whether the chromosome is
-phage assays on both L & U indicate that phage is present
So, what's the reason for the 2 bands in the CsCl gradient? Could there be
differences in the physical structure of the phage chromosomal DNA? Could
one band be phage that have lost their tail fibres?