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1、ExperimentReportonMolecularBiology,2012PlasmidDNAPreparationforRecombinationLinChengyuBio042010030007;Cooperator:YuanXiaowenExperimentDate:2012-03-15;SubmitDate:2012-03-181Introduction1.1BackgroundInformationPlasmidisasmall,independentlyreplicatingandcytoplasmicDNAmoleculethatcanbetransf
2、erredfromoneorganismtoanother.Itisakindofdouble-strandedandusuallycircularDNA,whosesizerangefrom1kbto200kb.Figure1SketchofplasmidnucleoidandcellBecauseofitsproperty,plasmidisoftenusedascloningvectorcarryingforeigngenesintothehostcells.Inordertomakeplasmidreadyforrecombination,severalprep
3、arativeprocessesshallbedone,includingplasmidDNAextraction,restrictionenzymedigestionandagarosegelelectrophoresis.1.2Objectives(1)Learnthecharacteristicsofplasmid.(2)PurifytheplasmidDNAbyalkalinelysismethod.(3)MeasuretheDNAconcentrationbyspectrophotometer.(4)Learnthecharacteristicsofrestr
4、ictionendonuclease,anduseEcoRIandXhoItoperformrestrictionenzymedigestion.(5)PerformagarosegelelectrophoresistoseparateDNAs,andcomparethedifferenceamongoriginalplasmids,singledigestion,anddoubledigestion.1.3Majorprinciples1.3.1PlasmidDNAextraction(1)Somespecialelements,includingheat,extre
5、mepH,organicsolutioncandenatureDNAandwillbringDNAintosediment;(2)Whendenaturingconditionsareeliminated,DNAwillberenatured,anditsspeedwilldependonthesizeandratiooverprotein;(3)TheisolationkithasspecialmaterialonthecolumnandcanspecificallybindwithDNAmolecules.Thus,plasmidscanbepurified;(4)
6、Double-strandDNAhasopticaldensityatwavelength260nm.ItsExperimentReportonMolecularBiology,2012concentrationcanbemeasuredbyspectrophotometer.1.3.2Restrictionenzymedigestion(1)TypeIIrestrictionendonucleasecanrecognizespecificnucleicacidsequenceandincisedoublestrainDNAinspecificsitesinthisse
7、quence.Figure2illustratethespecificsequenceEcoRIrecognizeanditscuttingsite;Figure2CuttingsiteofEcoRI(2)Mostplasmidshavemultiplecloningsites(MSC)whichisartificiallydesignforgeneticengineering(SeeFigure3).Restrictionendonucleasecandigestinthespecificsite,producestickyendsfo
