To further break down cell components and then draw off the DNA associated proteins, researchers typically add ammonium, sodium acetate or similar salts during this stage of the procedure. Since DNA is insoluble in ethanol and isopropanol, the addition of alcohol, followed by centrifugation, will cause the DNA proteins to come out of the solution.
When DNA concentration in the sample is heavy, the addition of ethanol will cause a white precipitate to form immediately. If the DNA concentration in the sample is low, isopropanol may work better than ethanol to precipitate the available proteins. In addition, isopropanol is often used for precipitating DNA from large volumes as less alcohol is used see protocols below.
The ethanol and isopropanol can also wash away the remaining salt residue. After being washed in alcohol and subjected to a centrifuge, the precipitated DNA protein will form a pellet, which can be washed in alcohol again, dried, and re-suspended in a Tris or TE buffer.
Be careful not to overdry the sample, since this can denature the DNA; just leave the washed pellet on the lab table for a few minutes. If isopropanol has been used during the extraction instead of ethanol, the sample may not adhere as tightly to the tube and may require a longer drying time. Incubate on ice for 15 minutes. In case of small DNA fragments or high dilutions overnight incubation gives best results.
Because of these charges, polar molecules like DNA or RNA can interact electrostatically with the water molecules, allowing them to easily dissolve in water. Nucleic acids are hydrophilic due to the negatively charged phosphate PO 3 — groups along the sugar-phosphate backbone.
OK, so back to the protocol. The role of salt in the protocol is to neutralize the charges on the sugar-phosphate backbone. A commonly used salt is sodium acetate. The positively charged sodium ions neutralize the negative charge on the PO 3 — groups on the nucleic acids, making the molecule far less hydrophilic and, therefore, much less soluble in water.
This shields its charge and makes the nucleic acid less hydrophilic, thus causing it to drop out of the solution. However, according to Maniatis et al. This explanation should bring you up to speed on how ethanol precipitation works. If you want to learn more about the ins and outs of ethanol precipitation and other DNA clean-up approaches, you might want to check these out…. Two typical protocols are alkaline lysis for extraction of bacterial plasmid DNA and phenol-chloroform extraction.
In both methods, ethanol or isopropanol precipitation of nucleic acids is one of the final steps. Both ethanol and isopropanol mix well are miscible with water, but they have lower dielectric constants than water, meaning that their ability to shield positive and negative charges in the solution and keep them segregated is much poorer. The dielectric constant for water, for example, is DNA is negatively charged, so it's attracted to positive ions in the solution like potassium or sodium.
Ethanol has a poorer ability than water to keep the positively charged ions and the DNA apart. Ethanol also makes the DNA less soluble for another reason. Since the ethanol molecules can form interactions called hydrogen bonds with water molecules, they decrease the number of water molecules available to hydrate the DNA.
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