Preparation of phenol/phenol:chloroform https://www.mrc-lmb.cam.ac.uk/ms/methods/phenol.

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Preparation of phenol/phenol:chloroform
DANGER!
Phenol is one of the nastiest things you can find in the lab. Small amounts can KILL. It causes burns, is
absorbed through the skin, and takes out your kidneys. A guy I know had a lucky escape after spilling ~500
ml phenol down his legs. Despite immediately standing in the sink with lots of running water he spent 3
months in Stoke Mandeville having skin grafts and dialysis.

When working with more than say 10 ml phenol, always ensure there is a bottle of glycerol or PEG300 close
by. If you are silly enough to spill some on you, absorb it with the glycerol or PEG before washing with lots
and lots of water. Water alone just spreads the stuff around. Always wear gloves, always wear glasses or work
behind a screen.

In other words, be extremely cautious when making up these stocks. Better yet, get someone who knows what
they're doing to do it.

Ordering
Buy phenol cast in the bottle, 100g maximum, Sigma catalogue #P1037. Once saturated, store under Tris at
-20°C.

Phenol preparation
1. Take 100g phenol bottle to fume hood, open it, and pour in ~ 100 ml 50 mM TrisCl pH 8. Close lid
tightly and shake gently. Leave to stand for an hour or two until the phenol liquifies and the phases are
separated.
2. Remove the supernatant with a pipette (dispose into the 'chlorinated solvent waste' container). Add a
further 100 ml 50 mM TrisCl pH8 and shake gently. Let stand for a few minutes to separate.
3. Repeat step #2 four or five times until the pH of the supernatant is between 7 and 8. Use pH strips to
check.
4. Make 10 - 20 ml aliquots in Falcon tubes. Add 10 ml 50 mM TrisCl pH 8 to each.
5. Store upright in polystyrene trays in the freezer at -20°C.

25:24:1 Phenol:chloroform:IAA preparation

1. Take 100g phenol bottle to fume hood, open it, and pour in ~ 100 ml 50 mM TrisCl pH 8. Close lid
tightly and shake gently. Leave to stand for an hour or two until the phenol liquifies and the phases are
separated.
2. Remove the supernatant with a pipette (dispose into the 'chlorinated solvent waste' container). Add a
further 100 ml 50 mM TrisCl pH8 and shake gently. Let stand for a few minutes to separate.
3. Repeat step #2 four or five times until the pH of the supernatant is between 7 and 8.
4. Combine 96 ml chloroform with 4 ml isoamylalcohol (3-methyl-1-butanol) in a glass measuring
cylinder to make 24:1 CHCl3:IAA.

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Preparation of phenol/phenol:chloroform https://www.mrc-lmb.cam.ac.uk/ms/methods/phenol.html

5. Make 10 ml aliquots (accurately) in Falcon tubes. To each, add 10 ml 24:1 CHCl3:IAA and mix gently.
Overlay with 10 ml 50 mM TrisCl pH 8.
6. Store upright in polystyrene trays in the freezer at -20°C.

Important Note

It is possible to buy phenol with added stabilizer. This sounds like a good idea until you realize that the
stabilizer is yellow. Pure phenol is colourless and only turns yellow (and then red) when oxidized. Therefore
if you buy it with the yellow stabilizer added you won't be able to tell when it's gone off. Storing phenol in
small aliquots under Tris at -20°C minimizes the risk of oxidation, so it shouldn't be a problem if you follow
these instructions.

Contents

RPG 12/16/2005 08:42:15

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