question about phenol/water saturated solution

David F. Spencer via methods%40net.bio.net (by DSpencer from NO!!SPAM.Dal.CA)
Sun Feb 10 20:05:37 EST 2008


Z.L. K wrote:
> Hi all,
> I have a question with regard to the phenol/water saturated solution. I
> didn't add any acids into it when i made it, when i test the pH of the
> aqueous phase it said below 5 with the pH paper. the phenol was a fresh one
> from some commercial company back then. currently the pH of the aqueous
> phase is still the same after half a year sitting in the cold room at 4 C.
> Today my labmate brought up a question for me and asked me what acid I added
> into to to bring down the phenol solution pH. I told him I didn't add
> anything. based on my understanding from reading online literatures before,
> phenol is an organic acid and its pH stays low unless you use some buffer to
> bring it up to the point you want it to be (such as pH 7 for extracting
> DNA). I wonder if my understanding is correct or not, as i couldn't find any
> detail online information now, which is weird.
> 
> My question here then should include two parts: 1, i have had RNA extracted
> with this phenol/water saturated buffer (well then it's from the in vitro
> transcription reaction, quite pure an RNA yield anyway), does it mean that
> the water-saturated phenol really has the right pH to extract RNA (just to
> answer my labmate's question); 2, if not, what acid should be used in
> adjusting the pH?
> 
> Thanks!
> 
> z.

The classic name for phenol is carbolic acid and it is indeed a very weak acid, 
pka of a little under 10, and pH of a water solution of about 6 or less.
I wouldn't trust pH paper with phenol but instead would use a pH meter.
For protocols where RNA is desired but not DNA, the usual strategy is to have 
the solution contain an acetic acid/sodium acetate buffer (say 0.3M) at about pH 
4.5, and use phenol equilibrated against a similar buffer.
Your approach is essentially the same, although as with any method, using 
unbuffered solutions will occasionally lead to unpredictable results.

David Spencer


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