1. Precipitate DNA dissolved in NaC1 solution.
2. Use cold alcohol to extract DNA with less impurities.
3. DNA is dyed blue by diphenylamine in the boiling water bath.
1. Extract cell nucleus material: stir clockwise, a little faster and a little heavier. 5 min
2. Dissolve DNA:
3. Precipitate a viscous substance containing DNA: 300mL of distilled water, stirring counterclockwise, slowly
4. Filtration: take the viscous material
5. Redissolve: Stir clockwise, slower. 3 min
6. Filtration: Take the filtrate.
7. To extract DNA with less impurities, stir counterclockwise, slightly slower. 5 min
8. DNA identification: boiling water bath for 5 minutes
DNA extraction is divided into three basic steps, and the specific method of each step can be different according to the type of sample, the substance that affects the extraction, and the subsequent steps.
1.The cells are broken by grinding or ultrasound, and the membrane lipid is removed by adding detergent.
Add protease, acetate precipitation, or phenol/chloroform extraction to remove intracellular proteins, such as histones bound to DNA.
Precipitating the DNA in cold ethanol or isopropanol, because the DNA is insoluble in alcohol and sticks together, this step can also remove the salt.
In addition, there are also commercial kits for extracting DNA by column adsorption.
2. The fragmentation of cells
Bacteria have a hard cell wall, so the meridians must be broken first. There are three methods; ① mechanical method: ultrasonic treatment, grinding method, homogenization method; ② chemical reagent method: treat cells with SDS; ③ enzymatic method: add lysozyme or snail enzyme to break the cell wall.
3. Several methods of DNA extraction
(1). Thick salt method
A. Using the different solubility of RNA and DNA in the electrolytic solution to separate the two, the common method is to extract with 1M sodium chloride, and the obtained DNP mucus is shaken with chloroform containing a small amount of octanol to emulsify, and then Centrifuge to remove the 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.
B. 0.15 M NaCL solution can also be used to repeatedly wash the cell crushing solution to remove RNP, and then 1 M NaCL to extract deoxyribonucleoprotein, and then remove the protein by the chloroform-iso-alcohol method.
Comparing the two methods, the latter method may reduce nucleic acid degradation.
C. 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, 15MNaCL, 0.015M sodium citrate, and called SSC solution, are used to extract DNA (2). Anionic detergent method; Detergents such as SDS or sodium xylate are used to denature protein, and DNA can be extracted directly from biological materials. Because the DNA and protein in the cell often use electrostatic attraction or coordination Bond bonding, because anionic detergents can destroy this valence bond, anionic detergents are often used to extract DNA
(3) Phenol extraction method: Phenol is used as a protein denaturant, and at the same time inhibits the degradation of DNase. When the homogenate is treated with phenol, because the protein and DNA bond has been broken, the surface of the protein molecule contains many polar groups and Phenol is similarly compatible. Protein molecules dissolve in the phenol phase, while DNA dissolves 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 being diffused into a ball with glass. The characteristic of this method is to keep the extracted DNA in its natural state
(4) Water extraction method: using the nature of nucleic acid to dissolve in water, after the tissue cells are broken, RNA is removed with a low-salt solution, then the precipitate is dissolved in water to fully dissolve the DNA in water, and the supernatant is collected 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 out with stirring. Then wash with 66%, 80% and 95% ethanol and copper acetone respectively, and finally in the air Medium-drying, existing DNA samples. The protein content of the DNA extracted by this method is high, so it is generally not used. In order to remove protein, this method can be improved, and SDS is added during the extraction process.
The above methods are some methods of DNA extraction. On the road of continuous exploration and experimentation, we have developed more efficient equipment, such as DNA extractor, RNA extractor, DNA extraction kit, RNA extraction kit, 8 Strip PCR Tube & 96 Well PCR Plate, etc. And so on, can provide you with multiple types of research and development equipment.
