Buffering Phenol

Jim Owens jow at helix.nih.gov
Fri Jun 10 10:05:07 EST 1994


In article <-090694150453 at dent-226-97.dentistry.umich.edu> , @umich.edu
writes:
>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

1)  Neither pH meter nor pH paper will measure pH in an organic solvent. 
It will measure an electrical potential that probably has no relation to
the -(log H+).  These devices measure pH only in a dilute (> 0.01M ionic
strength) aqueous solutions, although they can approximate the pH up to
0.1M salt.

2)  Always measure the pH of the aqueous buffer that has been
equilibrated with the phenol.  This doesn't really give the pH of the
phenol either, but it does tell you the pH that the equilibrated phenol
will contribute to the aqueous, buffered TE (or TEN) in which the DNA (or
RNA) is dissolved when phenol and TE are shaken together.  

3)  The pH of the aqueous phase is the important thing.  DNA will remain
in the aqueous phase if the pH of the aqueous phase is over 7.5.

The way I do it is to shake the phenol with an equal volume of 0.1M
TrisHCl, pH8, and add 3 drops from a pasteur pipet of 5M NaOH per 10ml of
phenol + Tris solution.  This equilibrates the phenol in one pass to pH 8
in the aqueous phase.

This is not the only way to properly equilibrate phenol to extract
proteins from DNA or RNA.  But it works.  This method was developed by
Alan Williamson (a pioneer of molecular biology if not one of the
founders) at least 20 years ago.  He taught this method to his grad
students and postdocs, one of whom (Leona Fitzmaurice) taught me in 1978.
 She said he developed this method after a lot of experimentation.  It
also makes sense chemically because of the three points above.

Good luck,

Jim Owens



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