DNA提取原理和方法讲义.pptVIP

细胞内的核酸包括DNA与RNA两种分子，均与蛋白质结合成核蛋白。 真核生物的DNA又有染色体DNA与细胞器DNA之分。前者为双链线性分子；后者为双链环状分子。 除此之外，在原核生物中还有双链环状的质粒DNA；在非细胞型病毒颗粒内，DNA的存在形式多种多样。;极性化合物，溶于水，不溶于乙醇、氯仿等有机溶剂，钠盐比游离核酸易溶于水。 在酸性溶液中，DNA、RNA易水解，在中性或弱碱性溶液中较稳定。 天然状态的DNA 是以脱氧核糖核蛋白(DNP)形式存在于细胞核中。 ;提取DNA总的原则 :;DNA提取的几种方法;　基因组DNA的提取;　吸附材料结合法：;　浓盐法： 　有机溶剂抽提法： 　密度梯度离心法： 　　　　　　　; SDS法流程图 （以动物组织为例） ;Approximately ~1 cm of tail is cut and put into an microfuge tube; Add 0.5 ml tail solution and 25 ml Proteinase K, mix well and incubate at 55oC overnight or until tissue is dissolved; More Proteinase K may be added if digestion is not complete after 24 hr; Add 0.5 ml Phenol/Chloroform and vortex at top speed for 15 min at RT; Spin at 13,000 rpm for 30 min at RT; Transfer the top aqueous phase to a fresh microfuge tube; Add 0.5 ml Chloroform and vortex at top speed for 5 min at RT; Spin at 13,000 rpm for 5 min at RT; Transfer the top aqueous phase to a fresh microfuge tube, add 50 ml 3 M NaOAc (pH=6.0) and 0.5 ml 100% ethanol, invert several times; A NaOAc solution with pH lower than 6.0 will cause the EDTA to precipitate; Spin at 13,000 rpm for 5 min at RT; If there is no pellet, place samples in -80oC for 10 min and spin at 13,000 rpm for 25 min at 4oC; Wash pellet once with 70% ethanol, air dry; Resuspend DNA in ddH2O. Use ~100-200 ml ddH2O to get final concentration as 100ng/ml for PCR. ;1.SDS ：十二烷基硫酸钠;3. More Proteinase K may be added if digestion is not complete after 24 hr; ;4. Add 0.5 ml Phenol/Chloroform and vortex at top speed for 15 min at RT; 5. Spin at 13,000 rpm for 30 min at RT; ; ;10.Transfer the top aqueous phase to a fresh microfuge tube, add 50 ml 3M NaOAc (pH=6.0) and 0.5 ml 100% ethanol, invert several times; 11.Spin at 13,000 rpm for 5 min at RT;;12. If there is no pellet, place samples in -80oC for 10 min and spin at 13,000 rpm for 25 min at 4oC; 13. Wash pellet once with 70% ethanol, air dry; 14. Resuspend DNA