Buffering Phenol

David L. Haviland, Ph.D. HAVILAND at KIDS.WUSTL.EDU
Sat Jun 11 12:17:20 EST 1994


> Just make sure the aqueous, upper phase has a pH over 7.5 and less than
> 9.5 to make sure DNA and RNA remain in the aqueous phase of the phenol
> extraction and will not be degraded.

Jim:

I understand your note but now I'm confused.  I have read in a methods book 
long ago (Methods in Enzymology??) that when in an acidic state phenol has 
to potential to acid-depurinate DNA and RNA.  So it follows that the pH
needs to be readjusted above pH 6.5 to within in range you suggest.  We 
have usually Tris pH 7.5 adjusted our phenol.  Yet for low melt agarose 
excraction we've found that using Sodium Acetate equilibrated phenol (pH
5.5) is paramount to obtaining a good yeild.  Second, the Chomczynski and 
Sacchi Acid GTC Phenol isolation protocol for RNA simply uses water 
equilibrated phenol (which in our case is pH 6.5) but the system is 
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?

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?

David





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