Buffering Phenol

Jim Owens jow at helix.nih.gov
Mon Jun 13 08:18:56 EST 1994


In article <2td0q6$9cq at server.st.usm.edu> Shiao Y. Wang,
sywang at whale.st.usm.edu writes:
>What about the 3 drops of 5 M NaOH per 10 ml phenol + Tris that you
>suggested in an earlier posting? Please clarify and be consistent.

That was a variation added when I got impatient with shaking the phenol
with several changes of 0.1M TrisHCl, pH8.  This way the pH of the
equilibrated water phase would be at pH 8 with the first shake.

In article <2tcrj0INNr5h at medicine.wustl.edu> David L. Haviland,
HAVILAND at KIDS.WUSTL.EDU writes:
>buffered again with sodium acetate pH 5.5...  I can't argue with the 
>success of the latter two methods but can't help but be concerned that 
>these latter two also "fly in the face" of acid-depurination of DNA and 
>RNA?  
>
>Any thoughts?

It is a question also of time and temperature as well as pH.  The lower
the pH the faster the depurination.   The DNA or RNA would be OK once it
is precipitated.  Depurination should not be very fast at room
temperature, but add the alcohol as soon as possible.  The DNA or RNA
should not be stored at pH 5 unless you want depurination.

I adjust the low-melt agarose slice to T.01E.001N.5 at pH 8, assuming
that the slice contributes about 1ml/g of mass.  After phenol, then
phenol:chloroform, then chloroform extraction, the DNA is precipitated
with 2 volumes of ethanol.

>Second:  is anyone having any problem of their phenol crystalizing in
the 
>fridge *after* equilibration.  We simply add 500ul BME, 5 mgs of 8 
>hydroxyquinaline, 100mM Tris pH 7.5 - shake madly, let the phases
separate, 
>and check the pH of the phenol 'til it's above pH 7.2-7.3.  Yet it 
>continues to crystalized in the fridge.
>
>Again , any thoughts?

We are able to store phenol equilibrated with water in the refrigerator
without it freezing so this has me stumped.  On the otherhand, the phenol
is safer (more stable) stored as a solid rather than a liquid.  What you
lose in convenience, you gain in shelf-life.  I think the phenol lasts
for us because the breakdown of phenol is an oxidation.  Storing it under
an aqueous phase greatly retards oxygen getting in contact with the
phenol.

Good luck,

Jim Owens



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