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A. Phenol extraction of DNA samples

Phenol extraction is a common technique used to purify a DNA sample (1). Typically, an equal volume of TE-saturated phenol is added to an aqueous DNA sample in a microcentrifuge tube. The mixture is vigorously vortexed, and then centrifuged to enact phase separation. The upper, aqueous layer carefully is removed to a new tube, avoiding the phenol interface and then is subjected to two ether extractions to remove residual phenol. An equal volume of water-saturated ether is added to the tube, the mixture is vortexed, and the tube is centrifuged to allow phase separation. The upper, ether layer is removed and discarded, including phenol droplets at the interface. After this extraction is repeated, the DNA is concentrated by ethanol precipitation.

Protocol

1. Add an equal volume of TE-saturated phenol to the DNA sample contained in a 1.5 ml microcentrifuge tube and vortex for 15-30 seconds.

2. Centrifuge the sample for 5 minutes at room temperature to separate the phases.

3. Remove about 90% of the upper, aqueous layer to a clean tube, carefully avoiding proteins at the aqueous:phenol interface. At this stage the aqueous phase can be extracted a second time with an equal volume of 1:1 TE-saturated phenol:chloroform, centrifuged and removed to a clean tube as above but this additional extraction usually is not necessary if care is taken during the first phenol extraction.

4. Add an equal volume of water-saturated ether, vortex briefly, and centrifuge for 3 minutes at room temperature. Remove and discard the upper, ether layer, taking care to remove phenol droplets at the ether:aqueous interface. Repeat the ether extraction.

5. Ethanol precipitate the DNA by adding 2.5-3 volumes of ethanol-acetate, as discussed below.

Typically, 2.5 - 3 volumes of an ethanol/acetate solution is added to the DNA sample in a microcentrifuge tube, which is placed in an ice-water bath for at least 10 minutes. Frequently, this precipitation is performed by incubation at -20C overnight (1). To recover the precipitated DNA, the tube is centrifuged, the supernatant discarded, and the DNA pellet is rinsed with a more dilute ethanol solution. After a second centrifugation, the supernatant again is discarded, and the DNA pellet is dried in a Speedy-Vac.

Protocol

1. Add 2.5-3 volumes of 95% ethanol/0.12 M sodium acetate to the DNA sample contained in a 1.5 ml microcentrifuge tube, invert to mix, and incubate in an ice-water bath for at least 10 minutes. It is possible to place the sample at -20degC overnight at this stage.

2. Centrifuge at 12,000 rpm in a microcentrifuge (Fisher) for 15 minutes at 4 degC, decant the supernatant, and drain inverted on a paper towel.

3. Add 80% ethanol (corresponding to about two volume of the original sample), incubate at room temperature for 5-10 minutes and centrifuge again for 5 minutes, and decant and drain the tube, as above.

4. Place the tube in a Savant Speed-Vac and dry the DNA pellet for about 5-10 minutes, or until dry.

5. Always dissolve dried DNA in 10 mM Tris-HCl, pH 7.6-8.0, 0.1 mM EDTA (termed 10:0.1 TE buffer).

6. It is advisable to aliquot the DNA purified in large scale isolations (i.e. 100 ug or more) into several small (0.5 ml) microcentrifuge tubes for frozen storage because repeated freezing and thawing is not advisable.

Notes on precipitation of nucleic acids

A. General rules

Most nucleic acids may be precipitated by addition of monovalent cations and two to three volumes of cold 95% ethanol, followed by incubation at 0 to -70 degC. The DNA or RNA then may be pelleted by centrifugation at 10 to 13,000 x g. for 15 minutes at 4degC. A subsequent wash with 70% ethanol, followed by brief centrifugation, removes residual salt and moisture.

The general procedure for precipitating DNA and RNA is:

1. Add one-tenth volume of 3M NaOAc, pH 5.2* to the nucleic acid solution to be precipitated,

2. Add two volumes of cold 95% ethanol,

3. Place at -70degC for at least 30 minutes, or at -20degC overnight.

or alternatively

1. Combine 95 ml of 100% ethanol with 4 ml of 3 M NaOAc (pH 4.8) and 1ml of sterile water. Mix by inversion and store at -20degC.

2. Add 2.5 volumes of cold ethanol/acetate solution to the nucleic acid solution to be precipitated.

3. Place at at -70degC for at least 30 minutes or -20degC for two hours to overnight.

* 5M NH4OAc, pH 7.4, NaCl and LiCl may be used as alternatives to NaOAc. DNA also may be precipitated by addition of 0.6 volumes of isopropanol.

B. Oligonucleotides

Add one-tenth volume of 3M NaOAc, pH 6.5, and three volumes of cold 95% ethanol.

Place at -70degC for at least one hour.

C. RNA

Add one-tenth volume of 1M NaOAc, pH 4.5, and 2.5 volumes of cold 95% ethanol.

Precipitate large volumes at -20degC overnight.

Small volume samples may be precipitated by placing in powdered dry ice or dry ice-ethanol bath for five to 10 minutes.

D. Isobutanol concentration of DNA