1. Extraction of dna by concentrated salt method:
A. Using RNA and DNA in different solubility in the electrolytic solution, the two are separated. 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 emulsify. The protein was removed by centrifugation, at which time the protein gel stayed in the middle of the aqueous phase and the chloroform phase, and the DNA was in the upper aqueous phase. The DNA sodium salt was precipitated in 2 volumes of 95% ethanol.
B. It is also possible to repeatedly wash the cell disruption solution with 0.15 MNaCL solution to remove RNP, then extract the deoxyriboprotein with 1MNaCL, and then remove the protein by chloroform---isool method.
Comparison of the two methods, the latter method may make the nucleic acid degradation less.
C. When extracting DNA with dilute hydrochloric acid solution, adding an appropriate amount of detergent, such as SDS, can help separate the protein from 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. .
2. Anionic detergent removal method for DNA: Decolorization of proteins with SDS or sodium dibenzoate, etc., DNA can be extracted directly from biological materials. Because of the electrostatic attraction or coordination bond between DNA and protein in cells Binding, because anionic detergents can destroy this valence bond, so anionic detergents are often used to extract DNA.
3. Phenol extraction method for extracting dna: phenol as protein denaturant, and inhibiting 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. Phenol is similarly soluble. 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 the aqueous phase containing DNA was further 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 its natural state.
4. Extraction of DNA by water extraction: Using the nature of nucleic acid dissolved in water, after disrupting tissue cells, removing RNA with a low salt solution, then dissolving the precipitate in water, allowing the DNA to be fully dissolved in water, and collecting the supernatant after centrifugation. Add solid sodium chloride to the supernatant to adjust to 2.6M. Add 2 times the volume of 95% ethanol, immediately stir it with stirring. Then wash with 66%, 80% and 95% ethanol and copper, respectively, and finally in the air. Medium dry, obtained DNA sample. The DNA extracted by this method has higher protein content, so it is generally not used. This method can be modified to remove protein, and SDS is added during the extraction process.