Julius Kühn-Institut
Question
Asked 10th Oct, 2012
How to prepare phenol for a phenol/chloroform extraction?
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?
Most recent answer
The link below will help to prepare your phenol
2 Recommendations
Popular answers (1)
Haven't heard of anyone doing this for a long time - it's too easy to purchase it already prepared.
1. Extract liquid phenol using a saturating amount (s.t. 2 liquid phases form) of 1M Tris pH 8.0. This will saturate the phenol with water as well as raise the pH to the desired 8.0.
2. Extract the phenol layer using standard TE buffer to lower the Tris concentration and add EDTA to the system.
3. Remove the phenol layer to a clean glass bottle (do not use plastic) and add a little TE buffer to keep it saturated. Aliquot and freeze if necessary. Always store the neutralized/saturated phenol cold in the dark.
Regarding phenol 'burns': For contact of phenol with skin, safety people always recommend extensive flushing with water. This has never been good in my experience - 'burns' still occur. I found an immediate rinse with EtOH, to pull the phenol out of the skin, followed with a water flush minimizes phenol burns.
9 Recommendations
All Answers (8)
Haven't heard of anyone doing this for a long time - it's too easy to purchase it already prepared.
1. Extract liquid phenol using a saturating amount (s.t. 2 liquid phases form) of 1M Tris pH 8.0. This will saturate the phenol with water as well as raise the pH to the desired 8.0.
2. Extract the phenol layer using standard TE buffer to lower the Tris concentration and add EDTA to the system.
3. Remove the phenol layer to a clean glass bottle (do not use plastic) and add a little TE buffer to keep it saturated. Aliquot and freeze if necessary. Always store the neutralized/saturated phenol cold in the dark.
Regarding phenol 'burns': For contact of phenol with skin, safety people always recommend extensive flushing with water. This has never been good in my experience - 'burns' still occur. I found an immediate rinse with EtOH, to pull the phenol out of the skin, followed with a water flush minimizes phenol burns.
9 Recommendations
Chaudhary Charan Singh University
You have to repeat the treatment of phenol with TE buffer several times until you get the desired pH
Université du Québec à Montréal
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
Tabriz University of Medical Sciences
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
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