3 Purification of Nucleic Acids From Eukaryotic Cells
53
1c) Stir the mixture on a magnetic stirrer for 15 min and then turn off the
stirrer. Allow phases to separate and decant as much as possible of the
top ( aqueous) phase into waste receptacle, removing remainder with a
pipet and suction bulb.
1d) Add an equal volume of0.1 M Tris-HCl (pH 8.0) to the phenol. Proceed
as described in 1c). Repeat the extraction, until phenol phase reaches
pH 7.8 (as checked with pH paper).
1e) After the phenol is equilibrated and the final aqueous phase has been
removed, add 0.1 vol of0.1M Tris-HCl (pH 8.0) containing 0.2% ß-mercaptoethanol. The phenol solution may be stored up to 2 months at 4°C
in light-tight bottles. Phenol for high-molecular-weight RNA extractions should be made freshly melted and the excess should be discarded.
1f) Mix 50 ml phenol, 48 ml chloroform and 2 ml isoamyl alcohol. This
mixture may be storedunder 100 mM Tris-HCl (pH 8.0) in a light-tight
bottle at 4 oc for periods of up to 1 month.
Note: If RNA is to be extracted, treat all water used directly or for buffer
preparation with diethyl pyrocarbonate (DEPC) to inactivate RNAses (see
Subprotocol 3, Precautions)
2. Add an equal volume of phenol:chloroform:isoamyl alcohol to the nu- Phenol extraction
cleic acid sample (prefentially 200 J..Ll) in a polypropylene microcentrifuge
tube (only these are resistant to the organic solvents).
3. Mix the contents of the tube until an emulsion forms. When isolating
small DNA molecules (<10 kb), this can be done by vortexing, or by
gentle shaking for DNA of moderate size ( 10-30 kb ). When isolating large
DNA molecules, the two phases should be mixed by rotating the tube
slowly (20 rpm) on a wheel.
4. Spin at 12000 rpm, room temperature (RT), until the two phases are separated (15 sec-2 min).
5. Transfer top (aqueous) phase to a fresh tube. Avoid carry-over of the
interphase.
Note: Normally, the aqueous phase forms the upper phase. However, ifthe
aqueous solution is dense because of salt (>0.5 M) or sucrose (> 10% ), it will
form the lower phase. The organic phase is easily identifiable because of its
yellow colour.
53
1c) Stir the mixture on a magnetic stirrer for 15 min and then turn off the
stirrer. Allow phases to separate and decant as much as possible of the
top ( aqueous) phase into waste receptacle, removing remainder with a
pipet and suction bulb.
1d) Add an equal volume of0.1 M Tris-HCl (pH 8.0) to the phenol. Proceed
as described in 1c). Repeat the extraction, until phenol phase reaches
pH 7.8 (as checked with pH paper).
1e) After the phenol is equilibrated and the final aqueous phase has been
removed, add 0.1 vol of0.1M Tris-HCl (pH 8.0) containing 0.2% ß-mercaptoethanol. The phenol solution may be stored up to 2 months at 4°C
in light-tight bottles. Phenol for high-molecular-weight RNA extractions should be made freshly melted and the excess should be discarded.
1f) Mix 50 ml phenol, 48 ml chloroform and 2 ml isoamyl alcohol. This
mixture may be storedunder 100 mM Tris-HCl (pH 8.0) in a light-tight
bottle at 4 oc for periods of up to 1 month.
Note: If RNA is to be extracted, treat all water used directly or for buffer
preparation with diethyl pyrocarbonate (DEPC) to inactivate RNAses (see
Subprotocol 3, Precautions)
2. Add an equal volume of phenol:chloroform:isoamyl alcohol to the nu- Phenol extraction
cleic acid sample (prefentially 200 J..Ll) in a polypropylene microcentrifuge
tube (only these are resistant to the organic solvents).
3. Mix the contents of the tube until an emulsion forms. When isolating
small DNA molecules (<10 kb), this can be done by vortexing, or by
gentle shaking for DNA of moderate size ( 10-30 kb ). When isolating large
DNA molecules, the two phases should be mixed by rotating the tube
slowly (20 rpm) on a wheel.
4. Spin at 12000 rpm, room temperature (RT), until the two phases are separated (15 sec-2 min).
5. Transfer top (aqueous) phase to a fresh tube. Avoid carry-over of the
interphase.
Note: Normally, the aqueous phase forms the upper phase. However, ifthe
aqueous solution is dense because of salt (>0.5 M) or sucrose (> 10% ), it will
form the lower phase. The organic phase is easily identifiable because of its
yellow colour.