质粒的提取及酶切.ppt

**质粒DNA的提取及酶切IsolationofPlasmidDNAandEnzymaticDigestion一、实验目的(ExperimentalPurpose)**Aftertheexperimentisfinished,studentsshouldbeabletoisolateplasmidDNAbythealkaline-detergentmethod.掌握碱去垢剂法提取质粒DNA的方法。PlasmidsaresmallcircularDNAmoleculeswhichreplicateindependentlyofthehostgenomeandencodeantibioticresistance.TheyarethecommonestvectorsforcarryingclonedDNA.Somesmallplasmidsusethehost’senzymestoreplicate.Largerplasmidmaycarrygenesthatencodetheirownreplicativeenzymes.质粒是存在于几乎所有细菌中染色体之外的外状DNA分子。质粒通常携带有染色体上所不存在的基因，并表现出一些有用的性状，如抗生素抗性，耐受重金属等。质粒拥有自己的复制原点，因此可以不依赖于染色体而进行独立复制。一些小的质粒利用宿主细胞的酶进行复制，而较大的质粒则自身携有编码与复制有关的酶。Plasmidsmaybestringent(lowcopynumber)orrelaxed(highcopynumber).质粒分为：严紧型(低拷贝数)和松弛型(高拷贝数)Plasmidsarefrequentlyusedascloningvectorsandalkalinelysisisoneofthemostwidelyusedmethodsfortheprepar-ationofplasmidDNA.质粒通常用作克隆载体，而碱裂解法是制备质粒DNA最为常用的方法之一。**二、实验原理(ExperimentalPrinciple)本方法是依据共价闭合环状质粒DNA与染色体DNA在变性和复性之间存在差异进行的。WhensuspendedinasolutionofNaOHandsodiumdodecylsulfate(SDS),thecellsarelysedbythehigh(alkaline)pH.Additionally,theproteinandchromosomeDNAwillbedenatured,andprecipitatetogetherwithcelldebris.ThismethodisbasedonthedifferentratesofdenaturationandrenaturationofcovalentlyclosedcircularplasmidDNAandchromosomalDNA.当细胞悬浮于NaOH和十二烷基酸钠(SDS)溶液中时，在高pH(碱)的作用下细胞发生裂解，此外，蛋白质和染色体DNA发生变性与细胞碎片一起沉淀下来。Inthepresenceofanacidsolution(potassiumacetate)andthencentrifuged,theplasmidDNAwerekeptinthesupernatant.Aftertheadditionofanalkalinesolution,theplasmidDNAdenaturationalsooccurred,butthecloseproximityofthetwochainsremain.Whenaddedtotheacidicsolution,eachofthetwochainsofplasmidannealedwithitscomplementarystrand,therebyformingtheoriginalstate.加入中和溶液(乙酸钾)溶液后离心，则质粒DNA则留在上清液中。当加入碱溶液时质粒DNA也发生变性，但其两条链仍然靠得很近，就像一条链上的两