Buffering Phenol

szcooley at chip.ucdavis.edu szcooley at chip.ucdavis.edu
Tue Jun 21 12:13:41 EST 1994


Jim Owens (jow at helix.nih.gov) wrote:
: 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

Phenol will recrystalize provided its not completely buffered. At least 
that is my theory. I recently buffered phenol from USB in 1M Tris and 
then 0.1M Tris and the ph was close to 7 and yet crystalized at 4 oC. 
Buffering then again with 0.1M Tris did not allow crystallization.

Mike Cooley



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