质粒提取常见问题（Common problems of Plasmid Extraction）.docVIP

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质粒提取常见问题（Common problems of Plasmid Extraction）.doc

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质粒提取常见问题（Common problems of Plasmid Extraction）

质粒提取常见问题（Common problems of Plasmid Extraction） Common problems of Plasmid Extraction 2010-11-30 17:00:03 source: Life Science Review at Zaozhuang University: sub 1. I lysate solution: Lysozyme: it is the main chemical composition of glycoside hydrolases, can hydrolyze the peptidoglycan of cell wall in the beta -1,4 glycosidic bond, which has the function of. When pH is less than 8 in solution, the action of lysozyme is inhibited. Glucose: increase the viscosity of the solution, maintain the osmotic pressure, and prevent the DNA from degrading by mechanical shearing force. EDTA (1): Mg2 +, Ca2 + chelating metal ions, inhibit the degradation of DNA (DNase to certain metal ions as a cofactor of DNase); (2) the existence of EDTA, is conducive to the effect of lysozyme, because lysozyme response requires low ionic strength of the environment. 2. IINaOHSDS liquid solution: NaOH: nucleic acids are stable in solutions of pH greater than 5 and less than 9. But when pH 12 or pH 3, it will lead to the dissociation of hydrogen bonds between double chains. When the concentration of NaOH in solution II is 0.2mo1 / L, the pH of the system is up to 12.6 as the extraction solution is added, thus promoting the denaturation of chromosome DNA and plasmid DNA. SDS:SDS is an ionic surfactant. Its main functions are as follows: (1) to dissolve lipid and protein on the cell membrane, and thus to dissolve the membrane protein and destroy the cell membrane. (2) nucleoprotein in depolymerization cells. (3) SDS binds to proteins and becomes R-O-SO3-... R + - protein complex that causes protein denaturation and precipitation. But SDS can inhibit the action of RNase, so in the future extraction process, it must be removed clean, to prevent interference in the next operation (using RNase to remove RNA). 3. solution III--3mol / L NaAc (pH4.8) solution: The aqueous solution of NaAc is alkaline. In order to regulate pH to 4.8, a large amount of glacial acetic acid must be added. So the solution i