Equilibriation of Phenol

Caution: Phenol and chlorofom are very toxic. Wear gloves, protective eye wear and lab coat. Use hood.

Phenol dissolves and denatures proteins. It dissolves protein that is tightly associated to DNA and DNA will not enter the organic phase. However, phenol is quite toxic (taken orally, an average fatal human dose is 15 g), and is a suspected carcinogen, It is rapidly absorbed through skin. Small qunantities are metabolized by the body: so, it is not accumulated poison. If phenol is spilt on body or clothes, immediatly remove the contaminated clothing and wash small spills under running water for at least 5 min (a spill is small if the skin area involved can fit under the stream of a faucet). Spills on legs can be rinsed in the sink. Do not hesitate to use the lab shower in the next room on large spill. For help in cleaning a spill in the hood or on the floor, notify Dr. Kohel or other colleges. Do not rinse contaminated skin with alcohol to dissolve the phenol: it increase the surface area and can cause accelerated absorption through the skin. A laboratory safety manual reported an instance where a relatively minor spill was fatal because of the use of ethanol.

Add 1 g of 8-hydroxyquinoline to 1 L phenol (melt at 65℃, if necessary), stir it in the bottle until dissolved, pour into a 2 L separatory funnel, add 5 ml saturated NaCl, 900 ml 1 M Tris-Base (121 g Tris-Base/1L H₂O, reserve 100 ml), mix and allow to separate. Keep lower (yellow) phenol phase and discard H₂O, add 1 L 0.1 M Tris-Base (made from the reserved 1 M), mix, allow to separate. Keep lower (yellow) phenol phase and discard H₂O phase, add 1 L CHCl₃:isoamyl alcohol (96:4, v/v), mix, and store in brown bottle at 4℃.

Phenol/CHCl₃ Extraction of DNA sample

Caution: Phenol and chlorofom are very toxic. Wear gloves, protective eye wear and lab coat. Use hood.

Make sure the DNA sample has a little (25 mM to 1 M or so) NaCl in it (but not too much: no salt phase do not separate; too much there is not a sufficient difference in density and they still do not separate), add an equal volume of phenol/CHCl₃, shake briefly, spin to 3 K rpm (12 K in MFT, less if using the 2 ml MFTs) to separate, keep H₂O phase, repeat 1 time with phenol/CHCl₃ again and 2 times with CHCl₃:isoamyl alcohol (96:4, v/v), and then give it another high speed (9 K rpm, 5 min) spin to be able to get rid of the rest of the CHCl₃:isoamyl alcohol. Finally give the sample either an NH₄Ac:EtOH precipitation spin or CsCl density gradient spin. Dump waste phenol and CHCl₃ in the solvent waste can.