遗传实验参考实验1

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1、Experiment1PrpnArAtinnnfnlA^midDNAExperiment2I1vl1vl“11111^411111PRestrictionEnzymeDigestionandAgaroseGelElectrophoresisDate:2012.03.08(Thu)/2012.03.15(Thu)Place:RenhuanBuildingRoom2062012.03.18Dr.BioOl2-22010080086SeheefJeongReportingDatePreparedfor

2、ClassGroupStudentNumberNameObjectives•Learnthecharacteristicsofplasmid.•LearntopurifytheplasmidDNAbyalkalinelysismethod.•LearntomeasuretheDNAconcentrationbyspectrophotometer.•Learnthecharacteristicsofrestrictionendonucleaseandagarosegelelectrophoresi

3、stoseparateDNAs.TheoreticalBackgroundPlasmidAsmall,independentlyreplicating,pieceofextrachromosomalcytoplasmicDNAthatiscapableofautonomousreplicationandcanbetransferredfromoneorganismtoanother.Itisakindofdouble-strandedandusuallycircularDNA.Described

4、largelyfrombacteriaandprotozoa・Someplasmidsarecapableofintegratingintothehostgenome.Anumberofartificiallymodifiedplasmidsareusedascloningvectors.RestrictionEnzymeRestrictionenzymesareenzymesisolatedfrombacteriathatrecognizespecificsequencesinDNAandth

5、encuttheDNAtoproducefragments,calledrestrictionfragments.RestrictionenzymesplayaveryimportantroleintheconstructionofrecombinantDNAmolecules,asisdoneingenecloningexperiments.Anotherapplicationofrestrictionenzymesistomapthelocationsofrestrictionsitesin

6、DNA.ItisanimportanttoolinDNArecombinationandalsowidelyusedinstudiesofDNAprimarystructure,recombinantDNAtechnologyandotherfieldsofmoleculargeneticsandmolecularbiology.MaterialsEquipmentUsedPowersupply,Electrophoresischamber,UVtransilluminator,Gelimage

7、system,Disposablegloves,PipetteReagentsExperiment1E.coliDH5aharboringpCMV-Myc-TIO⑸PAR)Suitablehoststrains:DH5fHB101,andothergeneraIpurposestrains.Selectablemarker:plasmidconfersresistaneetoampicillin(100mg/ml)toE.colihosts.Ecolireplicationorigin:pUCC

8、opynumber:~500plasmidrapidisolationkitRNaseSolution1・GETbuffer:50mmol/Lglucose,lOmmol/LEDTA,25mMTris-HCIpH8.0Solution2-0.2mol/LNaOHz1%SDSSolution3-KAc(3MzpH=4.8):60ml5mol/LKAcz11.5mlaceticacid,28.5mLH2OBindingbuffer-GuanidinepH7.2Washingbuffer-80mMKA