question about making phenol/TE and

[Zhiqun Li]

Hey I have a question regarding making the phenol/TE saturated buffers. 
I've heard from others that they only need to repeat the mixing of 
phenol and TE for 2~4 times before the pH of the aqueous phase reaches 
7.5 or higher (depends on the pH of the TE that's used). However, I've 
tried to mix the TE (pH 7.7~8) with phenol for more than 10 times 
(phenol around 150 ml, and TE around 100~150 ml each mix), the pH of the 
aqueous phase still remains lower than 6, but more than 5. I wonder why 
this happened, could it be because of the phenol is too old and 
therefore hard to be equilibrated (e.g., too acidic? i checked the pH of 
the bottom layer and it's between 3~4, after I found the aqueous phase 
is always lower than 6).

I also tried to make phenol/water saturated buffer in the same time, the 
aqueous phase is always staying below or close to 5, but not 5. I wonder 
if it's okay for extracting RNA, as acidicity is needed for separating 
DNA from RNA in the samples. In the mean time, the first time I added 
DEPC-dH2O into phenol, they were separated as clear as phenol and 
Tris-Cl do; however, after adding the second time of DEPC-dH2O with 
shaking vigorously afterwards, the bottom layer started getting 
foam-like looking and took forever to separate. I am very confused and 
not sure if these are normal and couldn't find anywhere for helpful 
suggestion.

If you can give out some tips and suggestions, that is highly 
appreciated, thanks a lot!

li