核酸的浓缩精制

(adapted from Bruce A. Roe, Department of Chemistry and Biochemistry, The University of Oklahoma, Norman, Oklahoma 73019 broe@ou.edu)

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 -20℃ 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 -20℃ overnight at this stage.

2. Centrifuge at 12,000 rpm in a microcentrifuge (Fisher) for 15 min at 4℃, 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 min and centrifuge again for 5 min, 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 min, 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 ℃. The DNA or RNA then may be pelleted by centrifugation at 10 - 13,000 x g for 15 min at 4℃. 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 -70℃ for at least 30 min, or at -20℃ 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 -20℃.

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

3. Place at at -70℃ for at least 30 min or -20℃ 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.

The key component in DNA precipitation is the centrifugation step. Be sure that centrifugation occurs at least 12,000 x g for 15 min or more.

B. Oligonucleotides

Add one-tenth volume of 3M NaOAc, pH 6.5, and three volumes of cold 95% ethanol. Place at -70℃ 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 -20℃ overnight. Small volume samples may be precipitated by placing in powdered dry ice or dry ice-ethanol bath for 5 to 10 min.

D. Isobutanol concentration of DNA

DNA samples may be concentrated by extraction with isobutanol. Add slightly more than one volume of isobutanol, vortex vigorously and centrifuge to separate the phases. Discard the isobutanol (upper) phase, and extract once with water-saturated diethyl ether to remove residual isobutanol. The nucleic acid then may be ethanol precipitated as described above.