Alcohol precipitation is a common technique in molecular biology. It purifies and concentrates nucleic acids (like DNA and RNA) and some polysaccharides by separating them from other components in an aqueous solution.
How Alcohol Precipitation Works
The effectiveness of alcohol precipitation relies on altering the solubility of nucleic acids in a solution. When salt is introduced, its positively charged ions associate with the negatively charged phosphate backbone of nucleic acids. This interaction effectively neutralizes the nucleic acid molecules’ overall negative charge, reducing their natural repulsion. Without these repulsive forces, nucleic acids are more inclined to aggregate.
Following salt addition, an alcohol like ethanol or isopropanol is introduced to the mixture. Water possesses a high dielectric constant, which allows it to efficiently shield the charges of dissolved ions and molecules, keeping them separated. In contrast, alcohols have a significantly lower dielectric constant, which diminishes water’s ability to shield these charges. This reduction in shielding allows the neutralized nucleic acids to interact more strongly with each other and with the positive salt ions, forming stable aggregates.
As nucleic acids aggregate, their solubility drastically decreases within the alcohol-water mixture. This change in solubility causes them to come out of solution in a solid form, a process known as precipitation. The precipitated nucleic acids can then be easily separated from the remaining liquid. This change in the solvent environment drives the nucleic acids to form pellets.
The Step-by-Step Process
Alcohol precipitation begins by ensuring the correct concentration of positive ions in the nucleic acid solution, often by adding a salt like sodium acetate or ammonium acetate. Next, alcohol (typically two to three volumes of 95% ethanol or one volume of isopropanol) is introduced and thoroughly mixed. The choice of alcohol and its volume depends on the specific protocol and desired outcome.
After alcohol addition, the mixture is incubated, sometimes at low temperatures (e.g., -20°C for at least 30 minutes), though room temperature incubation can also be effective. This allows time for nucleic acids to aggregate and precipitate. After incubation, the solution undergoes high-speed centrifugation (usually around 12,000 x g for 10-20 minutes) to force the precipitated nucleic acids into a compact pellet.
The supernatant (liquid above the pellet) is then carefully decanted, leaving the nucleic acid pellet. To remove residual salts and impurities, the pellet is washed with 70% ethanol. This dissolves unwanted contaminants while keeping nucleic acids insoluble. After washing, the ethanol is removed, and the pellet is air-dried briefly to evaporate any remaining alcohol. It is then resuspended in a desired buffer, such as Tris-EDTA (TE) buffer or nuclease-free water.
Factors Affecting Precipitation
Several variables influence alcohol precipitation efficiency. Nucleic acid length and concentration play a role; shorter fragments or very low concentrations may require extended incubation or higher salt concentrations for efficient precipitation. The specific type and concentration of salt are also important, as different salts offer varying precipitation efficiencies for particular nucleic acid types. For example, sodium acetate is common, but lithium chloride or ammonium acetate can be preferred for RNA precipitation.
Incubation time is another factor; longer durations can yield better recovery for dilute samples, though overly long incubations are not necessary for concentrated samples. Centrifugation speed and duration are also crucial; insufficient force or time prevents complete pelleting of precipitated nucleic acids. Speeds ranging from 10,000 to 16,000 relative centrifugal force (RCF) for 10 to 30 minutes ensure a compact pellet.
Ethanol is widely preferred due to its ability to selectively precipitate nucleic acids and its ease of evaporation; however, isopropanol offers an alternative. Isopropanol requires a smaller volume (one volume compared to ethanol’s two to three volumes), making it useful for larger sample volumes. However, isopropanol is less volatile, evaporates more slowly, and tends to co-precipitate more salts and impurities, which can affect final nucleic acid sample purity.