Molecular Biology. A Practical Guide - Velikov V.A. 2013

Isolation of Total DNA from Cells
DNA Concentration by Alcohol Precipitation

The most frequently used method for DNA concentration is ethanol precipitation. By dissolving the resulting pellet in a smaller volume of buffer, a more concentrated DNA solution is obtained. Reprecipitating DNA with alcohol also provides an additional purification step.

Ethanol acts as a dehydrating agent, reducing the solubility of Nucleic Acids (their salts ^a) in Water. DNA (RNA) aggregates in 70% ethanol in the presence of a salt that neutralizes phosphate groups. Nucleic acid aggregation proceeds more efficiently at lower temperatures and requires some time. The resulting aggregates are then pelleted by centrifugation.

To precipitate DNA from solution, 2.5 volumes of ethanol or 1 volume of isopropanol are used. For large-scale Procedures, to economize, it is permissible to use a minimum of 0.6 volumes of isopropanol. It should be kept in mind that 70% is the optimal ethanol concentration for nucleic acid precipitation. If the ethanol concentration is too low, the DNA will not transition into a crystalline state (cholesteric liquid crystals); if it is too high, residual Proteins and other impurities will co-precipitate with it. The optimal Ionic strength of the solution for DNA precipitation is 0.2M NaCl; lowering this strength will impair DNA precipitation.

After precipitation, if storage is intended, the DNA should be resuspended in a solution with a neutral or slightly alkaline pH.

In addition to alcohols, nucleic acids can be precipitated using polyethylene glycol (PEG), cetyltrimethylammonium bromide (CTAB), and other chaotropic agents. However, the standard Procedure remains ethanol precipitation.

Materials and Equipment

Low-concentration DNA sample of any origin, microcentrifuge, Glycogen.

Solutions

- 5M NaCl. Dissolve 292.5 g in water and bring the volume to 1 L.

- 3M sodium acetate, pH 5.2 (Topic 1).

- Glycogen solution in water, 10 mg/mL.

- 96% and 70% ethanol.

Procedure

1. Adjust the salt concentration in the sample to 0.2M NaCl (or 0.3M sodium acetate) using concentrated stock solutions of these salts. Most commonly, 1/25 volume of 5M NaCl or 1/10 volume of 3M sodium acetate, pH 5.2, is added to the DNA solution, as these Reagents are readily available to researchers working with DNA and proteins ^b.

2. If the DNA concentration is low, a coprecipitant such as glycogen should be added to a final concentration of 50 µg/mL ^c.

3. Add 2.5 volumes of ice-cold 96% ethanol ^d to the DNA solution, accounting for the volume of the salt solution already added.

4. Incubate at room Temperature for 5 minutes or overnight ^e at -20^оС, depending on the DNA concentration.

5. Centrifuge at maximum microcentrifuge speed for 10 minutes. (Low-concentration DNA preparations are pelleted for 1–2 hours in a high-speed refrigerated centrifuge at 27,000 rpm and a constant temperature of +4^оС).

6. Wash the DNA pellet with ice-cold 70% ethanol. To do this, mix the contents by inverting the tube several times, discard the alcohol, and place the tube inverted on filter paper to drain any residual alcohol. Air-dry the pellet.

7. Resuspend the DNA in TE buffer or H₂O.

Notes

^a Any DNA or RNA in a (colloidal) solution is not an acid, but a nucleic acid salt. Its cation is the same as that of the salt present during precipitation.

^b Acetates, citrates, and sulfates are kosmotropic salts, whereas cyanates are chaotropic. NaCl is most frequently used to precipitate DNA from SDS-containing solutions, as other salts precipitate SDS significantly more effectively.

^c Working concentrations of other coprecipitants: tRNA — 5–10 µg/mL, linear polyacrylamide — 10–20 µg/mL.

^d Isopropanol can be used instead of ethanol. In this case, 1 volume is added rather than 2.5 volumes. Isopropanol precipitation has one advantage over ethanol: the tubes can be smaller in volume. The main drawback is that isopropanol is less volatile, making a subsequent wash with 70% ethanol mandatory. Alcohol precipitation of DNA, particularly low-molecular-weight or low-concentration DNA, is enhanced by adding MgCl₂ to a final concentration of 10 mM.

^d It has been shown that increasing the incubation time, as well as lowering the temperature, does not significantly affect the DNA precipitation efficiency. This holds true for DNA solutions at concentrations above 20 ng/mL, although this approach is still frequently followed out of habit. When DNA is present in very small amounts, the most noticeable improvement comes from extending the centrifugation time to 1 hour at +4^oC, rather than prolonging the time the DNA spends under ethanol or lowering the incubation temperature down to -70^oC.

^e The microtube should be filled with 70% ethanol no more than 2/3 full. It is advisable to centrifuge the sample for 2–3 minutes at maximum centrifuge speed before discarding the 70% ethanol: although the pellets are fairly compact, they can be accidentally poured out along with the diluted ethanol. This occasionally happens, particularly when the DNA is poorly purified from proteins, without which it never exists in vivo. Within The Cell, DNA always exists as a DNP complex, and outside the cell, it is likewise coated with protein molecules (as in Viruses or Bacteriophages). In working with DNA, quality metrics take precedence over quantitative ones.