Phenol extraction of phage DNA

C Coward cc122 at mole.bio.cam.ac.uk
Tue May 17 08:37:12 EST 2005


I'm wondering whether I've done something unwise. My intention was to 
extract phage DNA (from a plate wash propagation of the phage) by 
Proteinase K/SDS treatment.

The phage was in SM buffer. I adapted a protocol from Sambrook:

-incubated with DNase & RNase to remove any contaminating nucleic acids.
-added EDTA, proteinase K, SDS & incubated at 56C for 1 hr to break the 
phage particles
-extracted with phenol (with the intention os subsequently extracting with 
phenol:chlorofom & then chlorofom and then precipitating with ethanol)

However at this stage the aqueous phase was cloudy following 
centrifugation (a large pellet at the interface was also seen, as 
expected).
A 2nd extraction with phenol had the same effect.
I tried then a phenol:chloroform extraction, here the aqueous phase was 
clear.

I then tried another phenol (no chloroform) extraction, and once again the 
aqueous phase was cloudy.

It has occurred to me that the SM buffer contains gelatin, may this be 
accounting for the observations?
What is it that is causing the cloudiness with phenol, but not 
phenol:chloroform?

More importantly, am I going to be able to extract my DNA from the mixes 
that I have?



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