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.
  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.

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