质粒提取与酶切电泳实验报告

分子实验报告2013030020华天瑞1PreparationofPlasmidDNA,RestrictionEnzymeDigestion,andAgaroseGelElectrophoresis2014/10/14-211Intro1.1ObjectiveTolearn•ThecharacteristicsofplasmidDNA•ThemethodofplasmidDNAmini-preparationbyalkalinelysisandthemeasurementofDNAconcentrationbyspectrophotometer•Thecharacteristicsofrestrictionendonuclease•HowtouseagarosegelelectrophoresistoseparateDNAsTounderstand:TheprinciplesofpurificationandquantificationofplasmidDNA1.2Principle1.2.1PlasmidandVectorPlasmidisasmall,independentlyreplicating,pieceofextrachromosomalcytoplasmicDNA(doublestrandedandusuallycircular)thatiscapableofautonomousreplicationandcanbetransferredfromoneorganismtoanother.VectorserveascarrierstoallowreplicationofrecombinantDNAinthehostcell,usuallyavectorcovers•Antibioticresistancegene:suchasampicillinresistantgene,kanamycineresistantgene,andetc.•Originofreplication(ori).•Multiplecloningsite(MSC)orpolylinker•Markergenes:suchasLacZgene.1.2.2AlkalineLysis(0.2molNaOH+1%SDS)SDSisakindofanionicdetergent.Itcanbreakbacterialcellsanddenatureproteins.Whenbacterialcellwallisbroken,theplasmidDNAandgenomicDNAwillbereleasedoutandbedenaturedinalkalienvironment.Whenthesolutionisneutralizedbyacidicreagent(suchasKAc),theplasmidDNAwillberenaturedrapidlyduetoitssmallersize.Aftercentrifugation,theplasmidDNAwillbeinsupernatant,whilethegenomicDNAwillstayinthesedimentatthebottomofthetubestogetherwithothercelldebris.1.2.3DNAConcentrationMeasurementBasedonthestrongabsorbanceofbasepairs(A-T,G-C)at260nmUV,theconcentrationofDNAcanbemeasuredbyspectrophotometry.Whendetectedunderneutralcondition,A260isusedtocalculatethenucleicacidconcentrationwhereastheratioofA260/A280canbeusedtoestimatethepurityofnucleicacid(1.8forpureDNA).1.2.4RestrictionEndonucleaseTypeIIREcutsdsDNAatspecificrestrictionsitesonspecificsequence,producingrestrictionfragments.1.2.5GelElectrophoresis分子实验报告2013030020华天瑞2Solidifiedagarosesolutionhascertainsizeofsmallporesofwhichthesizeisdecidedbyconcentration.IntheelectricfieldandbufferinneutralpH,negativelychargednucleicacidwillmigratetowardthepositivepole.DNAfragmentscanbeseparatedbydifferentmobilityingelelectrophoresis.1.2.6EB(Ethidiumbromide)EBcanbindwithDNAthroughinsertingintothebasepairsofDNAmolecule.ExcitedbyUV,theDNAbandsingelelectrophoresiswillemitredfluorescencewhichcanbedetectedeasily.TheminimalDNAquantitythatcanbetestedbythismethodisabout10ng.2MaterialsandReagents•E.coliDH5harboringpCMV-Myc-T10(SIPAR)•TIANprepMiniPlasmidKitP1:(1％Glucose,50mM/LEDTApH8.0,25mM/LTris-HClpH8.0)P2:0.2mM/LNaOH,1％SDSP3:5mol/LKac,pH4.8•plasmidpCMV-Myc-SIPAR•NEB1kbDNALadder•EcoRI,XhoI（Takara）•10×Hbuffer•agarose•TBE/TAEbuffer(1×)•EB(10mg/ml)•Loadingbuffer(3×):0.25%Bromophenolblue40％(W/V)sucroseor30％glycerol3Procedures3.1PreparationofPlasmidDNAa.Add500μlBufferBLtospincolumnCP3.Centrifugefor1minat12,000rpminatable-topmicrocentrifuge.Discardtheflow-throw,andplaceSpinColumnCP3intothecollectiontube.b.Harvest1.4mlbacterialcellsinamicrocentifugetubebycentrifugefor1minat12,000rpmfor1minat20℃,thenremoveallthetracesofsupernatant.Thenredowith1.4mlbacterialcellsinanothermicrocentifugetube.c.Resuspendpelletedbacterialcellsin250μlBufferP1d.Add250μlBufferP2andmixthoroughlybyinvertingthetube6-8timese.Add350μlBufferP3andmiximmediatelyandthoroughlybyinvertingthetube6-8timesf.Centrifugefor10minat12,000rpmg.ApplythesupernatantstotheSpinColumnCP3,centrifugefor1minat12,000rpmh.WashtheSpinColumnCP3byadding700μlBufferPWandcentrifugefor1minat12,000rpm.Discardtheflow-through,washagainwith500μlBufferPWandcentrifugefor1minat12,000rpm.i.Discardtheflow-throughandcentrifugefor2minat12,000rpmj.PlacetheSpinColumnCP3inaclean1.5mlmicrocentrifugetube.Add50μlEB,letstandfor4min,andcentrifugefor2minat12,000rpm.分子实验报告2013030020华天瑞33.2RestrictionEnzymeDigestionandAgaroseGelElectrophoresisa.EnzymedigestionofplasmidDNA（pCMV-Myc-SIPAR）Table1Plamid(ng)Buffer(μl)*EcoR1(μl)Xho1(μl)H2O(μl)Totalvolumn(μl)Ⅰ20120016.520Ⅱ20120.5016.020Ⅲ20120.50.515.520Digestionat37°Cwaterbathfor1hour.Add10μl3xloadingbuffertoeachtube,load15μlsampleforgelelectrophoresis.b.Add0.8gagaroseand100ml1XTAEintoaflask,microwaveagarosemeltsc.Insertcombintothemold.Positionthecomb0.5-1.0mmabovetheplated.Pourthewarmagarosesolution(65℃)intothemold,avoidairbubblese.Solifythegelunderroomtemperaturefor45min,thencarefullyremovethecombf.Placethegelintoelectrophoresischamberfullwith0.5×TAE/TBEg.Loadsample15μlmixturewithdisposablemicropipette.Changethemicropipetteeverytime.Add4μl1kbDNAladder(50ng/μl)asreference.h.Electrophoresisat100V,stopelectrophoresiswhenthebandofbromophenolblueisof4stringsawayfrombottomofthegeli.PlacethegelintoEBworkingsolution(0.5g/ml)tostainthegelfor3minj.Observationandphotography4ResultsandDiscussions4.1SpectrophotometryofDNAextractionTable2:SpectrophotometryresultsofDNAextractionA260A280A260/A280DNAconcentration0.4050.2141.904201ng/μlGenerallytheratioA260/A280ofpureDNAis1.8,smallerthantheresult.Meanwhile,theratioofA260/A230isrelativelyhigh(2.783),suggestingthattheamountofRNAissmall.Analysingbysynthesisthesetwofacts,theextracti