Several methods of DNA extraction
(1), concentrated salt method
The RNP and DNP are separated in the electrolytic solution by different solubility. The common method is to extract with 1M sodium chloride extract, and the obtained DNP mucilage is shaken with chloroform containing a small amount of octanol to emulsifie and then centrifuge. Protein, at this time the protein gel stays in the middle of the aqueous phase and the chloroform phase, and the DNA is in the upper aqueous phase. The DNA sodium salt can be precipitated with 2 volumes of 95% ethanol. Shanghai Chuangsai provides 2,4-dinitro Benzoquinone (DNP), AR, 119-26-6, product number: D16-1011186-25g, price 40 yuan
The cell disruption solution can also be repeatedly washed with 0.15 MNaCL solution to remove RNP, then deoxyriboprotein is extracted with 1MNaCL, and then the protein is removed by chloroform---isool method. The latter methods may make the nucleic acid degradation less.
When DNA is extracted with dilute hydrochloric acid solution, the addition of an appropriate amount of detergent, such as SDS, can help separate the protein from the DNA. In the extraction process, in order to inhibit the degradation of DNA by DNase in the tissue, sodium citrate is added as a metal ion in the sodium chloride solution. Usually, the DNA is extracted with .15MNaCL, 0.015M sodium citrate, and called SSC solution. Shanghai Creations offers sodium citrate, AR, 99%, 6132-04-3, item number: D23-RT1109-500g, price 48 yuan
(2), anionic detergent method
Using a detergent such as SDS or sodium diformate to denature proteins, DNA can be extracted directly from biological materials. Because DNA and protein in cells often combine with electrostatic attraction or coordination bonds, because anionic detergents can destroy this. Kind of valence bond, so commonly used anionic detergent to extract DNA.
(3), phenol extraction method
Phenol acts as a protein denaturant and inhibits the degradation of DNase. When the homogenate is treated with phenol, the protein and DNA linkage bonds are broken, and the surface of the protein molecule contains many polar groups similar to phenol. The protein molecule is soluble in the phenol phase and the DNA is soluble in the aqueous phase. After centrifugation and stratification, the aqueous layer was taken out, and the operation was repeated several times, and then the aqueous phase containing DNA was combined, and the DNA was precipitated with ethanol by using the insoluble alcohol property of the nucleic acid. At this time, the DNA is a very viscous substance, which can be wrapped in a long glass and taken out. This method is characterized by keeping the extracted DNA in a natural state.
(4), water extraction method
Using the nature of the nucleic acid dissolved in water, after disrupting the tissue cells, the RNA is removed with a low salt solution, and then the precipitate is dissolved in water to fully dissolve the DNA in water, and the supernatant is collected after centrifugation. Solid chlorination is added to the supernatant. Sodium was adjusted to 2.6 M. Two times the volume of 95% ethanol was added and immediately stirred with stirring. Then washed with 66% ? 80% and 95% ethanol and copper, respectively, and finally dried in air to obtain a DNA sample. The extracted DNA has a high protein content, so it is generally not used. This method can be modified to remove the protein, and SDS is added during the extraction process.
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