Buffering Phenol

at umich.edu
Thu Jun 9 14:04:53 EST 1994

In article <199406071653.JAA27400 at net.bio.net>, MUNGO at UVVM.UVIC.CA wrote:

>  We have on occaison found that it can be very difficult to buffer phenol to
> pH 8.  For reasons nobody here can explain certain lots of high quality phenol
> are impossible to get to pH 8 even after overnight extraction with Tris base
> 0.5 M unbuffered.  The phenol remains at pH 6.8.  However someone in the lab
> added a few drops of 10 M NaOH and the two phases then mixed and wouldn't
> seperate.  Upon addition of 50 mM Tris pH 8 the pahses seperated and the phenol
> was at pH 8 (sort of...).  Repeated extraction with 50 mM then 10 mM Tris pH 8
> does not change the pH of the phenol.  While I don't wish to address this
> rather novel approach to buffering phenol can anyone tell me what is going on?
> Why does adding NaOH cause the phases to mix ?  Is it the salt ?  Why do we
> have trouble buffering the phenol in the first place if it easily buffers once
> the phases mix ?  If the salt is causing the phases to mix why does the phenol
> remain buffered after repeated extractions with low salt buffer ?  And
> is this "solution" usable ?
> Thanks
> Mungo

First of All how are you measuring the pH?  You cannot use a pH meter; you
must use pH paper. Second of all what kind of water is the Tris made from?
If it has been DEPC treated  it will not separate properly. Next never add
NaOH to phenol because it will never help you pH the phenol. Next you must
add several changes of pH 8 Tris (I use 2M) to equilibrate and it takes
usually a week to equilibrate. It will never overnight unless some
chloroform is added and I don't think it pHs right because it becomes to
water insoluble. 50mM or 10mM is way to weak to ever change the pH of the
solution. Use 2M Tris.  For RNA extractions the phenol need not be
equilibrated to pH 8. Only when you are using it for DNA. At low Phs DNA
migrates to the Phenol phase will RNA stays in the aqueous. Any questions
Email me.

Steve Coon
sdcoon at umich.edu
University of Michigan School of Dentistry

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