Need advice on resolving bacteriophage mix

Michael Benedik benedik at uh.edu
Tue Mar 9 10:07:04 EST 1999


In article <bAzuRAAFnA52EwKa at genesys.demon.co.uk>
"Dr. Duncan Clark" <Duncan at nospam.demon.co.uk> writes:

> In article <36E0451B.A7435F48 at sph.unc.edu>, John Scott Meschke
> <jmeschke at sph.unc.edu> writes
> >I am currently having a problem resolving a mixture of male-specific RNA
> >coliphages from male-specific DNA coliphages.  I am interested in
> >purifying the RNA phage.  I have tried to plaque purify (3 times in
> >sequence), as well as plaque purifying using a composite antisera
> >against prototype F+ DNA coliphages (again three times in sequence) with
> >no luck. It seems to me that these techniques should have worked ( I
> >would have bet that I would have better odds of winning the lottery than
> >failing with both techniques).  Please give me any insight you may have
> >regarding the persistence of this mix or anyway to resolve it.  Thank
> >you very much in advance.
> >
> >Scott Meschke
> >
> >
> 
> Maybe passage through a host containing a type II restriction enzyme
> system would eliminate the DNA phages. That is assuming that they have
> one or more sites for the RE. 
> 
> Duncan
> -- 
> The problem with being on the cutting edge is that you occasionally get 
> sliced from time to time....
> 
> Duncan Clark
> DNAmp Ltd.
> Tel: +44(0)1252376288
> FAX: +44(0)8701640382
> http://www.dnamp.com
> http://www.genesys.demon.co.uk

Duncan's suggestion might work if you have such plasmids. There are
also host mutants which fail to support the growth of the DNA phages
which you could get. However I would strongly suspect the problem is in
methodology. These phages diffuse rapidly, spread by aerosols, and are
quite stable. You likely are just getting repeat contamination.

1) when you plaque purify are you picking plaques which are well
resolved and at least 1 cm away from any other plaque? There is much
diffusion in the plate.

2) be sure all your dilution buffers, tubes etc are free of phage. If
you are doing serial dilutions with a pipetperson, be sure that the
barrel is phage free and / or use aerosol tips. Best would be to use
regular pipets. 

3) After doing a purification and titer do a negative control titer.
Set up a dilution series with tubes, dilution buffer, pipets, cells etc
but don't add phage. This will show if you are getting phages from your
environment.

4) Are you sure that you have both RNA and DNA phages mixed together?
How do you know? Maybe one of your assumptions or criteria in
distinguishing them is not valid.

Hope some of these suggestions help.

__________________________________________________________
Michael Benedik                                         
Department of Biology and Biochemistry                      
University of Houston                   Tel: 713-743-8377
3201 Cullen Boulevard, HSC 402          Fax: 713-743-8351
Houston, TX 77204-5513              email: benedik at uh.edu
__________________________________________________________



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