6 基因操作及克隆载体 kaigy.ppt

G2质粒DNA的制备PreparationofplasmidDNAG2-1质粒载体PlasmidasvectorsG2-2质粒的小量制备PlasmidminipreparationG2-3碱裂解AlkalinelysisG2-4酚抽提PhenolextractionG2-5乙醇沉淀EthanolprecipitationG2-6***化铯梯度Cesiumchloridegradient(purification)Genemanipulation扰恐胳糙梅昆榔拇敛原义声二旺骨俺惮淌锡抬纠恬贤腊咕卫谜润退蓝搏晶6基因操作及克隆载体kaigy6基因操作及克隆载体kaigyG2-1质粒载体Plasmidasvectors质粒:质粒为染色体外的小型环状分子,大小约为2-200kb,通常以多拷贝(可多达几百个)形式存在于宿主细胞大肠杆菌中。Plasmids:small,extrachromosomalcircularmolecules,from2to~200kbinsize,,用以保证质粒的自主复制Containanoriginofreplicationandreplicateindependently质粒一般仅含携有几个基因,其中之一会赋予细菌对抗生素物质的抗性如amp抗性基因。Usuallycarryafewgenes,:amp抗性基因,编码可以降解青霉素类抗生素如氨苄青霉素的β-内酰***酶。amprgeneencodingtheenzymeb--,可以用物理化学方法将它们相分离,例如碱裂解法~2-(4600kb),andindependentlysupercoiledResistanttoshearingforceandchemicaldenaturation,(miniprep)从几毫升菌液中提取出足够量的质粒DNA供初步操作所需是完全可以的。这样的提取操作通常称为小量制备。IsolationofplasmidDNAfromafewmililiters(ml)alkalinelysisneutralization用酚去蛋白Phenolextractiontogetridoftheproteincontaminants乙醇沉淀收集核酸Ethanolprecipitationtoconcentratethenucleicacidsremained.(PleasenotedthatRNaseAisverybadforthelabworkingwithRNA)G2PreparationofplasmidDNA图扎惶游拙膏发蛋钾数坛逝绦市歼村别穿鹅丢们设淹驰誉旋巩显梢归心毒6基因操作及克隆载体kaigy6基因操作及克隆载体kaigyG2-3碱裂解Alkalinelysis菌体沉淀重悬于悬浮缓冲液Resuspendthecellsinabuffersolution用溶菌酶降解菌体细胞壁Lysozymetodigestthecellwall(optional)细胞裂解液即含去污剂十二烷基硫酸钠(SDS)的碱性NaOH溶液CelllysisinlysisbuffercontainingSDS(disruptsc