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1、中国试剂网3.9.269重组DNA的分离、克隆与测序实验手册editedbyBruceA.RoeJudyS.CrabtreeandAkbarS.KhanDepartmentofChemistryandBiochemistryTheUniversityofOklahomaNorman,Oklahoma73019e-mail:broe@ou.eduphone:405325-4912fax:405325-7762August5,1995Aprintedversionofthisprotocolbookcanbeobtainedfrom:"DNAIsolationandSequenci
2、ng"(EssentialTechniquesSeries)byBruceA.Roe,JudyS.CrabtreeandAkbarS.KhanPublishedbyJohnWiley&Sons,ListPrice$49.95ISBN0-471-97324-0QP625.N89R641996JohnWiley&SonsIntroductionThismanualisacompilationofmanyoftheeverydaymethodsusedintheaveragemolecularbiologylaboratory,withemphasisonthetechniquesf
3、orlargescaleDNAsequencingprotocolsandDNAsequencingautomationtechniques.Themanualhasbeenwritteninaprotocolformat,withlittletheoreticaldiscussion.Fortheoryandadditionalinformation,usersofthismanualarereferredbacktotheoriginalliterature,ortoothertextualmanualssuchasthosepublishedbyManiatis(1)et
4、al.andGlover(2).中国试剂网3.9.269Thefollowingpersonsareacknowledgedforcontributingmethodsandsuggestionsduringtheassemblyofthismanual:StephanieChissoe,SandyClifton,DennisBurian,RickWilson,Din-PowMa,JamesWong,LeslieJohnston-Dow,ElaineMardis,ZhiliWang,KalaIyer,SteveToth,GoughayZhang,HuaQinPanandothe
5、rmembersoftheRoelaboratory,bothpastandpresent.1.Sambrook,J.,Fritsch,E.F.,andManiatis,T.,inMolecularCloning:ALaboratoryManual.ColdSpringHarborLaboratoryPress,NY,Vol.1,2,3(1989).2.Glover,D.M.DNACloningVolumeI:APracticalApproach.IRLPress,Oxford,1985.I.GeneralmethodsA.PhenolextractionofDNAsample
6、sPhenolextractionisacommontechniqueusedtopurifyaDNAsample(1).Typically,anequalvolumeofTE-saturatedphenolisaddedtoanaqueousDNAsampleinamicrocentrifugetube.Themixtureisvigorouslyvortexed,andthencentrifugedtoenactphaseseparation.Theupper,aqueouslayercarefullyisremovedtoanewtube,avoidingthepheno
7、linterfaceandthenissubjectedtotwoetherextractionstoremoveresidualphenol.Anequalvolumeofwater-saturatedetherisaddedtothetube,themixtureisvortexed,andthetubeiscentrifugedtoallowphaseseparation.Theupper,etherlayerisremovedanddiscarded,includingphenold
