酚氯仿法提取DNA原理及方法.doc

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.1. Experimental Principles1) 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.2) 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. 3) Extract DNA from bufferOnce 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. 4) DNA precipitation The ethanol can precipitate DNA from water phase 2. Materials and SolutionsAll reagents are precooled or kept at 4C before use.1) Proteinase K 2) Phenol saturated with TE (pH 8.0)3) Chloroform 4) Isoamyl alcohol5) RNase stock (30 mg/ml, Catalog No.R4642-10MG, Sigma ) 6) 10% SDS 7) 0.5 mol/L EDTA, PH=8.08) 1 mol/L Tris-HCl, PH=8.09) 1 mol/L NaCl10) Extraction solution (ES) (100 mM EDTA, 200 mM NaCI, 50 mM Tris-HCI (pH 8.0), 0.5% SDS, 50 g/ml RNase)1 L50 ml0.5 mol/L EDTA, PH=8.0200 ml10 ml1 mol/L Tris-HCl, PH=8.050 ml 2.5 ml1 mol/L NaCl200 ml 10 ml 10% SDS50 ml 2.5 mlRNase stock (30 mg/ml )1.666 ml83.3 lDD Water498.3 ml25 ml3. Experimental protocol 1) Harvest cells and wash cells with PBS(106 cells)2) Suspend cells into 500 l ES3) Slowly add10 l proteases K (5mg/ml, Final concentration of 100 g/ml) to the above cell suspension while gently mixing. Incubate this solution at 55C for a minimum of 2-3 h with occasional manual or mechanical gentle mixing. 4) An equal volume of phenol (500 l) is added to the cell lysate. Centrifuge at 12,000 rpm for 5 min to separate the two phases. The aqueous (top) phase is transferred to a new tube using a wide bore transfer pipette. Note: cut a tip using scissors or blade to get a wide bore pipette.5) Add an equal volume of Phenol/chloroform/isoamyl alcohol (500 l) into the aqueous phase, and centrifuge at 12,000 rpm for 5 min to separate the two phases. The aqueous (top) phase is transferred to a new tube.6) Add an equal volume of chloroform (500 l) into the aqueous phase, and centrifuge at 12,000 rpm for 5 min to separate the two phases. The aqueous (top) phase is transferred to a new tube.7) Add 2 volume of absolute ethanol (900 l) to the aqueous phase and mix gently. Keep at -20C for 30 min, and centrifuge at 12,000g for 5 min. DNA pellet should be washed with 70% ethanol to decrease residual salt and briefly dried under vacuum or air-dried at 37C to evaporate the ethanol. 8) The ethanol-free DNA is dissolved in 50 l TE (pH=8.0) or DD water.Note: To get higher concentration of DNA solution, add smaller volume TE or water.9) Measure the absorbance of DNA solution at 260 and 280 nm. The purity can be estimated from the ratio of A260/A280. A ratio of 1.8-2.0 suggests minimal protein contamination. 10) The DNA solution is best stored at 4C or -20C.Quick Guide for Traditional DNA Extraction technologyQuick Guide for DNA Extraction KitAppendix 1: Protocol for removal of paraffin 1) Place a small section (not more than 25 mg) of paraffin-embedded tissue in a 2 ml microcentrifuge tube (not provided).2) Add 1200 l xylene. Vortex vigorously.3) Centrifuge at full speed for 5 min at room temperature.4) Remove supernatant by pipetting. Do not remove any of the pellet.5) Add 1200 l ethanol (96100%) to the pellet to remove residual xylene and mix gently by vortexing.6) Centrifuge at full speed for 5 min at room temperature (1525C).7) Carefully remove the ethanol by pipetting. Do not remove any of the pellet.8) Repeat steps step 57 once.9) Incubate the open microcentrifuge tube at 37C for 1015 min until the ethanol has evaporated.10) Resuspend the tissue pellet in lysis buffer.Appendix 2: Protocol for tissue on glass slides1) Add a drop of absolute ethanol on slide2) Scratch the tissue and transfer to a 2ml microtube3) Evaporate the ethanol in the air at room temperature4) Add