The principle of nucleic acids extraction firstly guarantees the integrity of the primary structure of nucleic acid, and secondly eliminates the contamination of other molecules. When extracting, attention should be paid to shorten the extraction time, reduce the degradation of nucleic acid by chemical and physical factors, mechanical shearing force and high temperature, and prevent the biological degradation of nucleic acid. What are the methods of nucleic acid extraction?
1.Concentrated salt method
(1). Using the different solubility of RNP and DNP in the electrolytic solution to separate the two, the common method is to extract the DNP mucus obtained by extracting sodium chloride with 1M sodium chloride and shaking together with chloroform containing a small amount of octanol to make the emulsification and centrifugation to remove Protein. At this time, the protein gel stays between the water phase and the chloroform phase, while the DNA is in the upper water phase. The sodium salt of DNA can be precipitated with 2 times the volume of 95% ethanol.
(2). You can also use 0.15 M NaCL to repeatedly wash the cell crushing solution to remove RNP, then extract deoxyribonuclein with 1 M NaCL, and then remove the protein by chloroform—isoalcohol method. Comparing these two methods, the second method may reduce nucleic acid degradation.
(3). When extracting DNA with dilute hydrochloric acid solution, adding an appropriate amount of detergent, such as SDS, can help separate protein and DNA. In the extraction process, in order to inhibit the degradation of DNA by DNase in the tissue, sodium citrate was added to the sodium chloride solution as a melting agent for metal ions, usually 15M NaCL, 0.015M sodium citrate, and called SSC solution, to extract DNA.
2.Anionic detergent method
Detergents such as SDS or sodium xylate are used to denature proteins, and DNA can be extracted directly from biological materials. Because DNA and protein in cells are often combined by electrostatic attraction or coordination bonds, anionic detergents can destroy this. A kind of valence bond, so anionic detergents are often used to extract DNA.
3.Phenol extraction method
Phenol acts as a protein denaturant, and at the same time inhibits the degradation of DNase. When the homogenate is treated with phenol, the bond between the protein and DNA is broken, and the surface of the protein molecule contains many polar groups that are compatible with phenol, and the protein molecule is soluble in phenol. Phase, while DNA is soluble in the water phase. After centrifugation and layering, take out the water layer, repeat the operation several times, and then combine the DNA-containing water phase, and use the alcohol-insoluble property of nucleic acid to precipitate the DNA with ethanol. At this time, DNA is a very viscous substance, and it can be taken out by wrapping it around in a ball with glass. The characteristic of this method is to keep the extracted DNA in its natural state.
4. water extraction method
Utilizing the nature of nucleic acid to dissolve in water, after breaking tissue cells, remove RNA with low-salt solution and dissolve the precipitate in water to fully dissolve DNA in water. After centrifugation, collect the supernatant and add solid sodium chloride to the supernatant. To 2.6M, add 2 times the volume of 95% ethanol, stir out immediately, and then wash with 66%, 80%, and 95% ethanol and acetone respectively.
The above gives a brief introduction to what are the methods of nucleic acid extraction. Ascend provides professional and complete casting services for nucleic acid extraction products.