The principle of various experimental reagents in DNA extraction
DNA extractor: the role and principle of different experimental reagents
1. STE is a mixture of Tris-Hcl , EDTA and Nacl. Its overall role is to provide a protective environment for DNA, in which DNA can be stable and minimally damaged. Provide a buffer system in which DNA is stable. EDTA: It is an inhibitor of DNase, which can prevent DNase from degrading DNA after the cell is broken. NaCl is conducive to the dissolution of DNA.
2. The function of SDS: Use high-concentration anionic detergent SDS (Sodium dodecyl sulfate) to separate DNA from protein, lyse cells under high temperature (55～65℃), and separate chromosomes , The protein is denatured and nucleic acid is released, then the protein and polysaccharide impurities are precipitated by increasing the salt concentration and lowering the temperature, and the precipitate is removed after centrifugation.
3. The role of phenol: it denatures the protein while inhibiting the degradation of DNase. When the homogenate is treated with phenol, because the bond between the protein and DNA is broken, the surface of the protein molecule contains many polar groups that are compatible with phenol. Protein molecules dissolve in the phenol phase, while DNA dissolves in the water phase.
The advantages of using phenol: 1. effectively denature protein; 2. inhibit the degradation of DNase.
Disadvantages: 1. can dissolve 10-15% of water, thereby dissolving part of poly(A)RNA. 2. Cannot completely inhibit the activity of RNase. The color of Trisphenol is generally yellow. If it turns pink, it means it is oxidized and can no longer be used.
4. The role of chloroform overcomes the shortcomings of phenol; accelerates the separation of organic phase and liquid phase. Finally, extract with chloroform: to remove traces of phenol in the nucleic acid solution. (Phenol is easily soluble in chloroform)
5. Isoamyl alcohol: Reduce bubbles generated during protein denaturation operations. Isoamyl alcohol can reduce surface tension, thereby reducing bubble generation. In addition, isoamyl alcohol helps to separate the phases and stabilizes the upper DNA-containing aqueous phase, the middle denatured protein phase, and the lower organic solvent phase after centrifugation.
6. When using ethanol to precipitate DNA, why add monovalent cations when ethanol is used to precipitate DNA, usually add monovalent cations in the solution, such as NaCl or NaAc, Na+ neutralizes the negative charge on the DNA molecules and reduces the difference between the DNA molecules Charges of the same sex repulsive force, and it is easy to aggregate and precipitate. 7. Why in the process of preservation or extraction of DNA, TE buffer is generally used in genetic manipulation experiments, the main principle of selecting buffer is to consider the stability of DNA and the composition of the buffer does not cause interference. Phosphate buffer system (pKa=) and boric acid system (pKa=) are also in line with the physiological range (pH) of the intracellular environment
7. Different reagents have different effects on DNA extraction.
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