Suck off the organic phase (yellow). The organic phase (phenol solution) is usually
on the bottom however, high salt concentrations occasionally result in the inversion of
the layers. Either way, you want to get rid of the yellow one.
Add 1/10th volume of ammonium acetate (5M), finger vortex and add 2.5 vols of
ethanol or 0.6 vols isopropanol. Isopropanol is good for relativiely large pieces of DNA
(the size of a plasmid) as less RNA and free nucleotides are precipitated. Since
isopropanol is more deleterious to enzymes etc. always wash the DNA pellet with
70% ethanol. Because residual isopropanol is more of a problem than
residual ethanol I usually use ethanol for precipitating unless isopropanol is specified (ie plasmid
preps). Contrary to popular misconception there is no advantage to cooling (or
freezing) after adding ethanol or isopropanol. Room temp. Is fine and may even be better. Safety Notes
Spin 10 min in microfuge, suck off sup carefully, wash pellet by addition of 70%
ethanol (volume equal to the original total) and respin 5 minutes, suck dry carefully with
a pulled pipette, air dry at 37°C for a few minutes or with speed vac and dissolve in TE
(volume depends on the next step). After the wash the DNA pellet does not stick very
well to the side of the tube so be careful. If the DNA is small (less than 200 bases)
wash with 95% ethanol.
Phenol is nasty stuff. It denatures proteins so that is what it will do to the
proteins that make up the skin on your hands and arms, etc. Wear gloves, labcoat and safety glasses--NO EXCEPTIONS.
Ethanol
and isopropanol are flammable and should be stored in the flammable liquids cupboard
when not in use.
Read the MSDS for these compounds and don't be careless.
