Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Protocols and Methods Forum
Register Search Today's Posts Mark Forums Read

Protocols and Methods Forum Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


Double band in phage CsCl gradient prep

Double band in phage CsCl gradient prep - Protocols and Methods Forum

Double band in phage CsCl gradient prep - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 01-19-2005, 11:18 AM
C Coward
Guest
 
Posts: n/a
Default 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

Reply With Quote
  #2  
Old 01-20-2005, 09:10 AM
Duncan Clark
Guest
 
Posts: n/a
Default Double band in phage CsCl gradient prep

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.
Reply With Quote
  #3  
Old 01-26-2005, 03:45 PM
Dr Engelbert Buxbaum
Guest
 
Posts: n/a
Default Double band in phage CsCl gradient prep

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.
Reply With Quote
  #4  
Old 01-28-2005, 11:20 AM
C Coward
Guest
 
Posts: n/a
Default Double band in phage CsCl gradient prep

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.
Reply With Quote
  #5  
Old 01-31-2005, 12:50 PM
peter willingmann
Guest
 
Posts: n/a
Default Double band in phage CsCl gradient prep

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
Reply With Quote
  #6  
Old 02-01-2005, 08:55 AM
Dr Engelbert Buxbaum
Guest
 
Posts: n/a
Default Double band in phage CsCl gradient prep

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.

Reply With Quote
Reply

Tags
band , cscl , double , gradient , phage , prep


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
Question about CsCl gradient purify chloroplast DNA!!!! Mr.Q Protocols and Methods Forum 2 10-22-2003 02:25 AM


All times are GMT. The time now is 01:49 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.13862 seconds with 16 queries