Double band in phage CsCl gradient prep

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
linear.

-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?

TIA

Chris

Historians believe that in newspost <cslfm8$j8n$[Only registered users see links. ].cam.ac.uk>
on Wed, 19 Jan 2005, C Coward <[Only registered users see links. ].cam.ac.uk> penned the
following literary masterpiece:

I've seen lambda preps (100's of mgs DNA scale) where one gets two bands
but vastly different intensities. I've always thought one band is
failed/ empty or mispackaged phages. Long time since I've done this
though.

As to whether the DNA's are identical. Not too difficult to quickly
test. Do an RE digest on them with a variety of RE's and see what you
get on an agarose gel.

Duncan
--
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.

C Coward wrote:


Why don't you look at the sample in the electron microscope or place
them onto bacteria, to see which ones are active? Call me old-fashioned,
but one should not forget basic biology over all that nucleic acid
fiddeling.

Dr Engelbert Buxbaum <[Only registered users see links. ]> writes:


Nor am I - I've done a phage assay on both of the bands, and both appear
to contain phage. Once I manage to purify some phage DNA (see a new post
below) I'll try & RE map them to determine whether they are the same. In
the meantime, I'm still wondering whether it's possible for there to be
something structurally different in a single phage that would account for
a difference in density on a CsCl gradient.

C Coward wrote:


Hi,
one reason could be that there are DI particles (defective interfering
particles). Make a plaque assay, you should see different size of plaques.
best wishes
peter

C Coward wrote:


Ok, thats half your rent. Now I would try and repurify the phages from
both samples, to see whether you get homogeneous bands or whether there
will be two bands again in each of the samples.