CsCl-gradient pure whole phage M13, how?

Duncan Clark Duncan at genesys.demon.co.uk
Wed Oct 18 08:08:36 EST 1995


In article: <4606p7$7v8 at biovax.biobase.dk>  wind at biobase.dk (Troels Wind) 
writes:
> 
> Hi all,
> 
> Could anybody direct me to a protocol for purification of whole 
filamentous
> phage particles (M13) by CsCl-gradient?
> 

I did this a long long time ago. Basically PEG ppt phage as normal for ss 
DNA prep. I resuspended in standard SM lambda phage buffer (see Maniatis).
Add solid CsCl and ultracentrifuge as per plasmid CsCl. You will see a 
white phage band against a dark background. The only problem I have is that 
I cannot find my notes on how much CsCl I used. For lambda I use 0.7g/ml 
but I know I had to play for the M13. I'm not sure if I added more or less. 
If you can find the density of the M13 phage then you can adjust the CsCl 
accordingly. Failing that try it and see. A 1 litre prep gives a lot of 
phage. Also two PEG/NaCl pptn's help and centrifugation of the phage prep 
after ressuspension of the PEG pellet in SM cleans it up a lot. This is 
also applicable to lambda preps. PEG/NaCl ppts a lot of junk as well as 
phage. This extra centrifugation just gives a cleaner CsCl prep.

Duncan  
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