酚氯仿法提取DNA原理及方法.docVIP

Chapter 1 DNA extraction 说明：本原理及方法是个人整理，用来给研究生教学用的，用本方法可以提取到理想的DNA。各实验室提供的细胞裂解液浓度各有不同，只要经过实验证实的，都可以用来提取到理想的DNA. Experimental Principles Cell lysis (lysis buffer, containing SDS, EDTA, Tris-HCl, and RNase) SDS, a detergent is added to the buffer to break open the cell membranes; it also helps remove proteins and lipids in the cell. Ethylenediaminetetraacetic acid (EDTA), a chelator to remove metal ions in solution to preven DNase from cutting up the DNA. RNase is also present in the buffer at this step, to break up the RNA present in the cells. Remove protein Proteinase K, it remains active at elevated temperatures, so the solution can be heated to about 55 °C to aid protein inactivation and removal by the detergent. Extract DNA from buffer Once the cells are broken open and the RNA, proteins, and lipids have been dissolved in the buffer, the DNA must be separated from these materials. Phenol: remove the proteins, leaving DNA and other water-soluble materials behind by centrifugation. The DNA is then extracted from the water phase using chloroform and precipitated from the chloroform using ethyl alcohol mixed with sodium acetate salt. DNA precipitation The ethanol can precipitate DNA from water phase Materials and Solutions All reagents are precooled or kept at 4°C before use. Proteinase K Phenol saturated with TE (pH 8.0) Chloroform Isoamyl alcohol RNase stock (30 mg/ml, Catalog No.R4642-10MG, Sigma ) 10% SDS 0.5 mol/L EDTA, PH=8.0 1 mol/L Tris-HCl, PH=8.0 1 mol/L NaCl Extraction solution (ES) (100 mM EDTA, 200 mM NaCI, 50 mM Tris-HCI (pH 8.0), 0.5% SDS, 50 μg/ml RNase) 　 1 L 50 ml 0.5 mol/L EDTA, PH=8.0 200 ml 10 ml 1 mol/L Tris-HCl, PH=8.0 50 ml 2.5 ml 1 mol/L NaCl 200 ml 10 ml 10% SDS 50 ml 2.5 ml RNase stock (30 mg/ml ) 1.666 ml 83.3 μl DD Water 498.3 ml 25 ml Experimental Harvest cells and wash cells with PBS(~106 cells) Suspend cells into 500 μl ES Slowly add10 μl proteases K (5mg/ml, Final concentration of 100 μg/ml) to the above ce