DNA precipitation is a laboratory technique used to separate DNA from a solution. This process isolates DNA from contaminants or concentrates it from a dilute solution. By removing impurities and increasing its concentration, DNA becomes suitable for molecular biology applications.
The Science Behind DNA Precipitation
DNA molecules possess a negative charge due to phosphate groups in their backbone. In an aqueous solution, these charges cause DNA strands to repel, keeping them dissolved. Precipitation involves introducing positively charged ions, typically from a salt. These ions bind to the negatively charged phosphate groups on the DNA.
This binding neutralizes repulsive forces, allowing DNA molecules to come closer. Once neutralized, alcohol, like ethanol or isopropanol, is added. Alcohol reduces the solution’s dielectric constant, which reduces DNA solubility. Water molecules, which hydrate and keep DNA dissolved, are displaced by the alcohol.
As DNA molecules lose their hydration shell and charges are neutralized, they begin to aggregate. This aggregation leads to the formation of a visible pellet of DNA that can be separated from the solution.
Materials and Reagents
Successfully precipitating DNA requires key components. The DNA sample is typically dissolved in water or a buffer solution.
A salt solution, often 3M sodium acetate, is added to neutralize the negative charges on the DNA molecules. Alternatively, ammonium acetate can be used, which is sometimes preferred because it precipitates fewer proteins and nucleotides. Following the salt, cold alcohol, such as 100% ethanol or isopropanol, is introduced. Ethanol is commonly used at 2 to 2.5 times the volume of the aqueous DNA solution, while isopropanol is used at a 0.7 to 1 times volume ratio.
After precipitation, a wash buffer, usually 70% ethanol, is necessary. This diluted alcohol helps wash away residual salts and contaminants without redissolving the DNA pellet.
Step-by-Step Procedure
First, add a salt solution to the DNA sample. Typically, one-tenth volume of a 3M sodium acetate solution is added. This mixture should be thoroughly inverted several times to ensure even distribution of the salt.
Following the salt addition, cold alcohol is introduced to the solution. Usually, two to two-and-a-half volumes of 100% ethanol are added, or about 0.7 to 1 volume of isopropanol. The tube should then be gently inverted multiple times to mix the contents, ensuring the DNA has maximum exposure to the alcohol.
Incubate the mixture at a low temperature, such as -20°C or -80°C. An incubation period of at least 20 minutes is common, though overnight incubation can sometimes increase recovery, especially for dilute samples. This cold incubation promotes the aggregation of DNA molecules into a stable precipitate.
After incubation, the aggregated DNA is collected by centrifugation. The sample is spun at high speed, typically around 12,000 x g, for 10 to 15 minutes in a refrigerated centrifuge, usually at 4°C. This high-speed spin forces the DNA pellet to the bottom of the tube. The supernatant, which contains impurities and excess reagents, is then carefully poured off or aspirated, leaving the DNA pellet behind.
Handling and Storage of Precipitated DNA
After centrifugation, the DNA pellet often contains residual salts and contaminants. To remove these impurities, the pellet is washed with a cold 70% ethanol solution. This wash helps dissolve and rinse away the remaining salts without affecting the precipitated DNA.
The tube is then centrifuged again to re-pellet the DNA. Carefully remove the 70% ethanol wash solution, ensuring the DNA pellet remains undisturbed. Any remaining ethanol is allowed to evaporate, usually by air-drying the pellet.
Over-drying the DNA pellet can make it difficult to re-dissolve. Once the pellet is dry, it is re-dissolved in a suitable buffer, such as TE buffer or nuclease-free water. The choice of buffer depends on the downstream application, with TE buffer providing better long-term stability for the DNA. The re-dissolved DNA can be stored at 4°C for short periods or -20°C for long-term preservation.