Principle:
1. DNA precipitation dissolved in NaC 1 solution.
2. Use cold alcohol to extract DNA with less impurities.
3.DNA was dyed blue by diphenylamine in boiling water bath.
Method steps:
1. Extraction of nuclear substances: stir clockwise, slightly faster and slightly heavier. 5 minutes
2. Dissolve DNA:
3. precipitate a sticky substance containing DNA: 300mL distilled water, and slowly stir counterclockwise.
Step 4: filter: take a sticky substance.
5. Re-dissolution: stir clockwise, which is slower. 3 minutes
6. Filtering: take the filtrate.
7. extract DNA with less impurities and stir it counterclockwise, slightly slower. 5 minutes
8.DNA identification: boiling water bath for 5min minutes.
university
DNA extraction is divided into three basic steps, and the specific method of each step can be different according to the sample type, substances affecting extraction and subsequent steps.
Crushing cells by grinding or ultrasonic wave, and removing membrane lipids by adding detergent.
Adding protease, acetate precipitation or phenol/chloroform extraction to remove protein in cells, such as histone binding to DNA.
Precipitate DNA in cold ethanol or isopropanol, because DNA is insoluble in ethanol and will stick together. This step can also remove salt.
In addition, there are commercial kits for extracting DNA by column adsorption.
2. Cell division
Bacteria have hard cell walls, and they must first break the warped cells. There are three methods; ① Mechanical methods: ultrasonic treatment, grinding and homogenization; ② Chemical reagent method: SDS was used to treat cells; ③ Enzymolysis: adding lysozyme or snail enzyme can break the wall.
3.3 Several methods. DNA extraction
(1). concentrated salt method
A.RNA and DNA are separated according to their different solubility in electrolyte. The common method is to extract with 1M sodium chloride. The obtained DNP mucus was shaken with chloroform containing a small amount of octanol, emulsified, and then protein was removed by centrifugation. At this time, protein gel stays between the water phase and chloroform phase, while DNA is in the upper water phase. The sodium salt of DNA can be precipitated with twice the volume of 95% ethanol.
B. RNP can also be removed by repeatedly washing the cell lysate with 0. 15 MNaCL solution, then extracting the deoxyribonucleoprotein with 1MNaCL, and then removing the protein with chloroform-isopropanol method.
Compared with these two methods, the latter method may degrade less nucleic acid.
C. When extracting DNA with dilute hydrochloric acid solution, adding appropriate detergent, such as SDS, is helpful to separate protein from DNA. In order to inhibit the degradation of DNA by DNase in tissues, sodium citrate was added to sodium chloride solution as a binder of metal ions. Generally, 0. 15 mnacl and 0.0 15 m sodium citrate, called SSC solution, are used to extract DNA. (2) anionic detergent method; Protein can be denatured by detergents such as SDS or sodium xylene, and DNA can be directly extracted from biological materials. Because DNA and protein in cells are often combined by electrostatic attraction or coordination bonds, anionic detergents are often used to extract DNA because they can destroy this valence bond.
(3) Phenol extraction: Phenol is used as protein denaturant to inhibit the degradation of deoxyribonuclease. When the homogenate is treated with phenol, because the bond between protein and DNA has been broken, the surface of protein molecule contains many polar groups similar to phenol. Protein molecules are soluble in the phenol phase, while DNA is soluble in the water phase. After centrifugation and stratification, take out the water layer and repeat the operation for many times. Then, the aqueous phases containing DNA were combined, and DNA was precipitated with ethanol by using the insolubility of nucleic acid in ethanol. At this time, DNA is a very viscous substance, which can be wrapped in glass and taken out. The characteristic of this method is to keep the extracted DNA in a natural state.
(4) Water extraction: breaking tissue cells by using the property that nucleic acid is soluble in water, then removing RNA with low-salt solution, then dissolving the precipitate in water to make DNA fully soluble in water, and collecting supernatant after centrifugation. Add solid sodium chloride to the supernatant to adjust it to 2.6M, add twice the volume of 95% ethanol, and immediately stir. Then, 66%, 80% and 95% ethanol were used respectively. A DNA sample has been obtained. The protein content of DNA extracted by this method is high, so it is generally not used. In order to remove protein, the method can be improved by adding SDS in the extraction process.