I am performing a phenol/chloroform extraction of DNA. Unfortunately, I have to equilibrate the pH of the phenol by myself, and mix it with chloroform afterwards.
The way I did the pH equilibrium is by pouring some TE buffer (pH=8.0) into the phenol and shaking it rigorously for 2 minutes, and letting it equilibrate and phase separate. After that, I measured the pH of the phenol. it is 6.8 instead of being 8.0, which is desired.
Is this correct?
You have to repeat the treatment of phenol with TE buffer several times until you get the desired pH
This link will help you for preparation of phenol.
http://ieg.ou.edu/protocol/Protocols/Phenol%20Saturation.pdf
I have the same problem and I added many times tris-cl (ph 8) but no resullt my ph stay 7.
Can some one help me please.
Thank you
you have to treat the phenol several times (3-4 times) and for long time (15-30 min while mixing them) to let more exposure of Tris to phenol. i think the problem of your procedure was the short time of treating.
and do this with equal volumes of tris. ( use tris ph8 ( or TE buffer ph8) in the same volume that you picked phenol.
and at the end, when phenol's ph was adjusted, left a thin layer of fresh TE buffer on phenol. and store at dark. in 4 centigrade for 3-6 months, and in -20 for longer time.
good luck
The link below will help to prepare your phenol
http://www.mrc-lmb.cam.ac.uk/ms/methods/phenol.html
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