质粒提取与酶切电泳实验报告

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1、PreparationofPlasmidDNA,RestrictionEnzymeDigestion,andAgaroseGelElectrophoresis2014/10/14-211Intro1.1ObjectiveTolearn•ThecharacteristicsofplasmidDNA•ThemethodofplasmidDNAmini-preparationbyalkalinelysisandthemeasurementofDNAconcentrationbyspectrophotometer•Thecharacteristicsofrestrictionendo

2、nuclease•HowtouseagarosegelelectrophoresistoseparateDNAsTounderstand:TheprinciplesofpurificationandquantificationofplasmidDNA1.2Principle1.2.1PlasmidandVectorPlasmidisasmall,independentlyreplicating,pieceofextrachromosomalcytoplasmicDNA(doublestrandedandusuallycircular)thatiscapableofautono

3、mousreplicationandcanbetransferredfromoneorganismtoanother.VectorserveascarrierstoallowreplicationofrecombinantDNAinthehostcell,usuallyavectorcovers•Antibioticresistancegene:suchasampicillinresistantgene,kanamycineresistantgene,andetc.•Originofreplication(ori)・•Multiplecloningsite(MSC)orpol

4、ylinkcr•Markergenes:suchasLacZgene.1.2.2AlkalineLysis(0.2molNaOH+1%SDS)SDSisakindofanionicdetergent.Itcanbreakbacterialcellsanddenatureproteins・Whenbacterialcellwallisbroken,theplasmidDNAandgenomicDNAwillbereleasedoutandbedenaturedinalkalienvironment.Whenthesolutionisneutralizedbyacidicreag

5、ent(suchasKAc),theplasmidDNAwillberenaturedrapidlyduetoitssmallersize.Aftercentrifugation,theplasmidDNAwillbeinsupernatant,whilethegenomicDNAwillstayinthesedimentatthebottomofthetubestogetherwithothercelldebris.123DNAConcentrationMeasurementBasedonthestrongabsorbanceofbasepairs(A-T,G-C)at26

6、0nmUV,theconcentrationofDNAcanbemeasuredbyspectrophotometry.Whendetectedunderneutralcondition,A260isusedtocalculatethenucleicacidconcentrationwhereastheratioofA260/A280canbeusedtoestimatethepurityofnucleicacid(1.8forpureDNA).124RestrictionEndonucleaseTypcIIREcutsdsDNAatspecificrestrictionsi

7、tesonspecificsequence,producingrestrictionfragments.1.2.5GelElectrophoresisSolidifiedagarosesolutionhascertainsizeofsmallporesofwhichthesizeisdecidedbyconcentration.IntheelectricfieldandbufferinneutralpH,negativelychargednucleicacidwillmigratetowardthepo