Why isopropanol is used in dna extraction

This causes the DNA to become less hydrophilic and precipitate out of solution. Ice to chill the sample. Lower temperatures promote the flocculation of the nucleic acids so they form larger complexes that pellet under the centrifugal forces of a microcentrifuge. A nucleic acid concentration high enough to force the DNA out of solution if the concentration is not high enough, you can add a carrier nucleic acid or glycogen to enhance the recovery.

DNA is less soluble in isopropanol so it precipitates faster even at low concentrations. With ethanol, the DNA needs to be at a higher concentration to flocculate but the salt tends to stay soluble, even at colder temperatures.

So for the typical precipitation protocol, isopropanol is added from between 0. If you are precipitating small volumes of DNA, and you can fit the required amount of solvent into the sample tube, then ice-cold ethanol is the preferred choice. Alcohol is used to precipitate the DNA out of the extraction solution, so we can wash all those salts and chemicals away and then dissolve it in our final solvent—usually water or some variant of Tris-EDTA solution.

DNA remains dissolved in aqueous solution because DNA has phosphodiester backbone, which is hydrophilic in nature. Water molecule forms hydration shell around DNA by forming hydrogen bonds. Isopropanol is a good choice for precipitation of DNA. The amount of isopropanol requirement is less 0. Isopropanol also dissolves nonpolar solvents such as chloroform, thus the impurities form previous step can also be removed. Using ice-cold isopropanol is generally practiced, but many researchers say that it should be used at room temperature, otherwise it will precipitate polysaccharides also [ 21 ].

Though the yield of DNA will be increased at low temperature, it may increase impurities [ 22 ]. The role of the salt in the extraction protocol is to neutralize the charges on the sugar phosphate backbone of the DNA.

Sodium acetate with pH 5. This is useful in aggregation and formation of tangled mass. It is also called as salting out. Nevertheless, it is not seen when salt alone is used.

It requires the solution with low dielectric constant, which allows this interaction. Role of salt in DNA precipitation [ 25 ]. A DNA molecules in aqueous solution have the negative charge and repel each other; B sodium acetate dissociates into the water into sodium and acetate ion; and C sodium ion shields the negative charge on the DNA molecules by neutralizing it and helps in aggregation and precipitation.

Precipitate is air-dried or vacuum-dried. Over drying should be avoided as DNA converts B form to D form, which is difficult to dissolve later [ 25 ]. Nowadays, for long-term storage, it is prudent to store DNA in a buffer that maintains its pH and keeps it from getting degraded. For DNA isolation, the pH is usually set to 7. Tris amino constituent of TE buffer has the ability to protect DNA strands from radiation damage, in both solid state and fluid solution. As radiation produces free radicals, it may break DNA strands.

Thus, in the fluid solution at ambient temperature Tris acts by scavenging hydroxyl radicals [ 26 ]. Sterile water can be utilized for short-duration storage of DNA. In fact, in a large number of cases, they do not. Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3. Help us write another book on this subject and reach those readers. Login to your personal dashboard for more detailed statistics on your publications. Edited by Oana-Maria Boldura.

By Niu J. Tan, Leona D. Daim, Amilia A. Alcohol precipitation is commonly used for concentrating, desalting, and recovering nucleic acids. Since less alcohol is required for isopropanol precipitation, this is the preferred method for precipitation of DNA from large volumes. In addition, isopropanol precipitation can be performed at room temperature, which minimizes co-precipitation of salt that interferes with downstream applications.

Topics: Molecular Biology. This assay is suitable for the simple and rapid estimation of protein concentration.

This assay is based on a single Coomassie dye based reagent. The binding of protein to the dye results in a change of color from brown to blue. The change in color density is proportional to protein concentration. Protein estimation can be performed using as little as 0. Question: What is the work of salt, isopropanol and ethanol in DNA extraction? The Protein Man Says: To identify bacteria and viruses in an environmental sample, diagnose disease pathologies, or examine a biological sample for forensic purposes, the DNA can be removed from the nucleus of a cell and its proteins can be separated by electrophoresis.