Phenol extraction of phage DNA

Duncan Clark blackhole at abuse.plus.com
Thu May 19 06:59:35 EST 2005


Historians believe that in newspost <d6cs28$92o$1 at gemini.csx.cam.ac.uk> 
on Tue, 17 May 2005, C Coward <cc122 at mole.bio.cam.ac.uk> penned the 
following literary masterpiece:
>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?

You have SDS in the aqueous phase and that is possibly the problem. A 
small amount of phenol will dissolve into the top layer. chloroform will 
remove sufficient phenol from the top layer to clear it.

I see this all the time when doing phage extractions so wouldn't worry. 
Pool your top layers and complete the extractions finishing with just 
chloroform. Dialyse 2 or 3x against TE using sterile buffer with 10mM 
EDTA (you want no possibility of any DNase still present being able to 
work) and well boiled dialyse tubing. EtOH or isopropanol ppt. as 
normal.

Duncan
-- 
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.



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