《III提取DNA》PPT课件

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1、Chapter3DNA的提取与纯化基因工程需要三种不同的DNA:总DNA质粒DNA噬菌体DNAFigure3.1ThebasicstepsinpreparationoftotalcellDNAfromacultureofbacteriaBasicSteps1总DNA提取1.1培养、收集细菌细胞两种类型的细菌培养基的成分：M9培养基：Na2HPO4(6.0)KH2PO4(3.0)NaCl(0.5)NH4Cl(1.0)MgSO4(0.5)Glucose(2.0)CaCl2(0.015)LB培养基：typtone(10)Yeaste

2、xtract(5)NaCl(10)1.1培养、收集细菌细胞37°C，150-250rpm达到最大浓度2-3×109cell/ml。600nm下的OD值，1OD相当于0.8×109cell/mlFigure3.2EstimationofbacterialcellnumberbymeasurementofopticaldensityFigure3.3HarvestingbacteriabycentrifugationFigure3.4Preparationofacellextract.Preparationofacellextra

3、ct1.2裂解细胞溶菌酶、EDTAFigure3.5Removalofproteincontaminantsbyphenolextraction.3PurificationofDNAfromacellextract1.4DNA的浓缩与浓度测定最常用的方法是乙醇（同时含有Na离子）、异丙醇沉淀法。260nm下测定吸光度，1OD相当于50μg双链DNA/ml。A260/A280=1.8，<1.8表示含有酚或蛋白质,>1.8说明有RNA。Figure3.6CollectingDNAbyethanolprecipitation.◆Co

4、ncentrationofDNAsamplesFigure3.7TheCTABmethodforpurificationofplantDNA◆PlantDNA(CTAB法)十六烷基三甲基溴化铵Figure3.8Theuseofananion-exchangechromatographyresininDNApurification.阴离子交换层析法2质粒DNA提取质粒DNA的大小（8%）。DNA的构造◇Alkalinedenaturation◇Ethidiumbromide-caesiumchloridedensitygradi

5、entcentrifugationsphaeroplasts裂解细胞可以破坏部分细胞壁，而保持细胞膜的完整。问题：裂解液中包含染色体DNA质粒分子非常大时，将与染色体DNA共沉淀Figure3.9Preparationofaclearedlysate.Sphaeroplast残壁细胞碱裂解法基本原理：在pH12.0~12.5范围内，非螺旋化的DNA会变性，而闭合环状的质粒的DNA则不会被变性或只有少量的氢键断裂；当恢复中性pH值时便会迅速地复性。但变性的线性染色体DNA分子复性时不准确，也不迅速，因此彼此聚集形成网状结构，通过

6、离心分离便与变性的蛋白质及RNA一起沉淀下来，而仍滞留在上清液中的质粒DNA则可用酒精等沉淀收集。Figure3.11Plasmidpurificationbythealkalinedenaturationmethod.AlkalinedenaturationFigure3.10Twoconformationofcirculardouble-strandedDNA.Twoconformationofcirculardouble-strandedDNACircularformsofDNAmigrateinagarosedisti

7、nctlydifferentlyfromlinearDNAsofthesamemass.Typicallyuncutplasmidswillappeartomigratemorerapidlythanthesameplasmidwhenlinearized.Additionally,mostpreparationsofuncutplasmidcontainatleasttwotopologically-differentformsofDNA,correspondingtosupercoiledformsandnickedcir

8、cles.Theimagetotherightshowsanethidium-stainedgelwithuncutplasmidintheleftlaneandthesameplasmidlinearizedatasinglesiteintherightlane.Figur