SS Phagemid gradients, KI or CsCl?

Warren Wakarchuk num208ww at MBDS.NRC.CA
Wed Jul 22 22:10:26 EST 1992


In article <1992Jul21.154049.25431 at usenet.ins.cwru.edu>, djt2 at po.CWRU.Edu (Dennis J. Templeton) writes:
>
>We're trying to purify a mg or so of phagemid DNA, and need it to be free
>from contaminating double stranded DNA, such as might come from broken
>cells.  The protocol (sketchy) we're following suggests using EtBr/KI
>> stuff deleted
>dennis

Dennis, you might have better luck if you band the phage particles
themselves in CsCl, then simply extract DNA from the isolated phage
particles.  I used this technique about 10 years ago when I worked
with T7 phage particles.  I don't see why it wouldn't work with
phagemid.  Since the pariticles are lighter than DS DNA you should get
a good clean up.  I can't remember the exact reference, but I suspect
some of the earlier Methods in Enzymology (vol 65? 68?) may contain
references to banding phage in CsCl.  There is a series of books
published by the Cold Spring Habour Press on Lambda, T phage and SS
DNA phage, I am certain there will be references to how to do it. 
Good luck!
Dr. Warren Wakarchuk   Prefered email: wakarchu at biologysx.lan.nrc.ca
                       Alternate     : NUM208WW at MBDS.NRC.CA
IBS-Protein Structure and Design
National Research Council Canada
Ottawa, Ont.,
Canada K1A 0R6   ***THESE ARE MY OPINIONS NOT THOSE OF NRC***



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