氯仿法或chelex-100法提取dna - 昆明医科大学.ppt

DNA提取和定量 The extraction and quantitative of DNA 许冰莹 教授 昆明医科大学法医学院;;毛发;the type of sample ;1. DNA的提取;The extraction of DNA specimens;; The theory of the extraction nDNA;?（1）最大程度的去除样品DNA溶液中的RNA、蛋白质(protein)、多糖(polysaccharide)和脂类分子(lipid molecules)等成分； （2）尽可能的保持提取的DNA分子的完整性； （3）减少或去除可能对酶(enzyme)有抑制作用(inhibition)的物质，如有机溶剂(organic solvent)、去污剂(decontaminant)、金属离子(metal ion)、染料(dyestuff)及其他抑制DNA分析所用酶活性的物质。 ;Commonly used extraction method;;;phenol - chloro extraction;;DNA沉淀干燥;ADVANTAGE：(1)A wide range of applications, suitable for all biological samples；(2)More effective against corruption, pollution, old samples and nails, bone and other materials；(3) DNA high purity, can be used for all DNA analysis。 ;DISADVANTAGE： The use of harmful chemical reagent, extraction process requires multiple centrifugation, may lead to more loss of DNA and may remain inhibited ingredients in the extract. ;Commonly used extraction method;;Chelex-100 extraction steps ;;注 意（attention） ;ADVANTAGE: simple and quick; Less amount of samples, suitable for extracting trace materials DNA; Extraction is always in the same tube, can reduce the loss of DNA； The degradation of DNA can be prevented by Chelex-100. ;DISADVANTAGE: (1)Inevitably some residual impurities in DNA product extraction; (2)Get after the treatment of Chelex-100 is single-stranded DNA, cannot be used for RFLP analysis.;Blood and blood stain samples;blood;Semen stain and mixed plaque samples;Other samples;2.Quantitative detection of DNA;Ethidium bromide fluorescence detection method;Analyzing DNA samples in a Classroom Lab;Ultraviolet spectrophotometric method and principle; 将提取的样本DNA溶解于0.1mol/LNaOH 液中，经分光光度计测定OD260 值，按照公式计算样本DNA的含量： 样本DNA浓度(μg/ml)＝OD260×40μg/ml×稀释倍数 蛋白质最大吸光波长是280nm，同时测定样本DNA液OD260值和OD280值，计算OD260/OD280 比值，如果比值为 1.8－2.1，样本DNA比较纯；如果比值小于1.6，样本液中含有过多的蛋白质杂质。