lipofectamineRNAiMAX英文说明书

Page4ScalingUporDownTransfectionsTotransfectcellsindifferenttissuecultureformats,varytheamountsofLipofectamine™RNAiMAX,RNAiduplex,cells,andmediumusedinproportiontotherelativesurfacearea,asshowninthetable.CulturevesselRel.surf.area1Vol.ofplatingmediumDilutionmediumreversetransfectionDilutionmediumforwardtransfectionRNAi(pmol)RNAi(nM)Lipofect-amine™RNAiMAX296-well0.2100µl20µl2x10µl0.12-61-500.1-0.3µl48-well0.4200µl40µl2x20µl0.24-121-500.2-0.6µl24-well1500µl100µl2x50µl0.6-301-500.5-1.5µl6-well52.5ml500µl2x250µl3-1501-502.5-7.5µl60mm105ml1ml2x500µl6-3001-505-15µl100mm3010ml2ml2x1ml12-6001-5015-35µl1Surfaceareasmayvarydependingonthemanufacturer.2IfthevolumeofLipofectamine™RNAiMAXistoosmalltodispenseaccurately,andyoucannotpooldilutions,prediluteLipofectamine™RNAiMAX10-foldinOpti-MEM®IReducedSerumMedium,anddispensea10-foldhigheramount(shouldbeatleast1.0µlperwell).DiscardanyunuseddilutedLipofectamine™RNAiMAX.CotransfectingDNAandRNAusingLipofectamine™RNAiMAXForcotransfectionsofplasmidDNAandStealth™RNAiorsiRNAintomammaliancells,werecommendusingLipofectamine™2000(Catalogno.11668-027),whichissuperiorforplasmidtransfections.IfyouwanttouseLipofectamine™RNAiMAXforyourcotransfections,performareversetransfectionasdescribedonpage2withthefollowingmodifications:1a:Add20ng(for24-wellformat)ofplasmidDNAtothedilutedRNAiduplex.2:Addcellssuchthattheywillbe80-100%confluent24hoursafterplating.PurchaserNotificationThisproductiscoveredbyoneormoreLimitedUseLabelLicenses(seetheInvitrogencatalogorourweb-site,).BytheuseofthisproductyouacceptthetermsandconditionsofallapplicableLimitedUseLabelLicenses.LimitedUseLabelLicenseNo.5LimitedUseLabelLicenseNo.27©2006InvitrogenCorporation.Allrightsreserved.Lipofectamine™RNAiMAXCat.No.13778-075Size:0.75mlCat.No.13778-150Size:1.5mlStoreat+4°C(donotfreeze)DescriptionLipofectamine™RNAiMAXisaproprietaryformulationspecificallydevelopedforthetransfectionofsiRNAandStealth™RNAiduplexesintoeukaryoticcells.Lipofectamine™RNAiMAXprovidesthefollowingadvantages:•Hightransfectionefficienciesinmanycelltypestominimizebackgroundexpressionfromuntransfectedcellsandmaximizeknockdown.•Minimalcytotoxicitytoreducenon-specificeffectsandcellularstress.•GenerallyrequireslowconcentrationsofRNAiduplexestoobtainhighknockdownlevels,furtherminimizingnon-specificeffects.•Abroadpeakofoptimaltransfectionactivitywithminimalcytotoxicity,allowingachievementofhighknockdownlevelsdespitedifferencesincelldensity,minorpipettinginaccuracies,andothervariations.ImportantGuidelinesforTransfection•Reversetransfection(page2)andforwardtransfection(page3)protocolscanbeusedformostcelllinestested.Cell-typespecifictransfectionprotocolsareavailableat•WerecommendOpti-MEM®IReducedSerumMedium(Cat.No.31985-062)todiluteRNAiduplexesandLipofectamine™RNAiMAXbeforecomplexing.•Donotaddantibioticstomediaduringtransfectionasthiscausescelldeath.•Testserum-freemediaforcompatibilitywithLipofectamine™RNAiMAX.•Toassesstransfectionefficiency,werecommendusingaKIF11Stealth™SelectRNAi,asdescribedinAssessingTransfectionEfficiency(page2).•Use10nMRNAiduplexandindicatedprocedureasastartingpoint;optimizetransfectionsasdescribedinOptimizingTransfections(page3).QualityControlLipofectamine™RNAiMAXistestedforabsenceofmicrobialcontaminationwithbloodagarplates,Sabarauddextroseagarplates,andfluidthioglycolatemedium,forabsenceofRNAseactivity,andfunctionallybytransfectionofStealth™RNAiandappropriatecontrolsintoareportercellline.PartNo.:13778.PPSRev.Date:11Jan2006Page4ScalingUporDownTransfectionsTotransfectcellsindifferenttissuecultureformats,varytheamountsofLipofectamine™RNAiMAX,RNAiduplex,cells,andmediumusedinproportiontotherelativesurfacearea,asshowninthetable.CulturevesselRel.surf.area1Vol.ofplatingmediumDilutionmediumreversetransfectionDilutionmediumforwardtransfectionRNAi(pmol)RNAi(nM)Lipofect-amine™RNAiMAX296-well0.2100µl20µl2x10µl0.12-61-500.1-0.3µl48-well0.4200µl40µl2x20µl0.24-121-500.2-0.6µl24-well1500µl100µl2x50µl0.6-301-500.5-1.5µl6-well52.5ml500µl2x250µl3-1501-502.5-7.5µl60mm105ml1ml2x500µl6-3001-505-15µl100mm3010ml2ml2x1ml12-6001-5015-35µl1Surfaceareasmayvarydependingonthemanufacturer.2IfthevolumeofLipofectamine™RNAiMAXistoosmalltodispenseaccurately,andyoucannotpooldilutions,prediluteLipofectamine™RNAiMAX10-foldinOpti-MEM®IReducedSerumMedium,anddispensea10-foldhigheramount(shouldbeatleast1.0µlperwell).DiscardanyunuseddilutedLipofectamine™RNAiMAX.CotransfectingDNAandRNAusingLipofectamine™RNAiMAXForcotransfectionsofplasmidDNAandStealth™RNAiorsiRNAintomammaliancells,werecommendusingLipofectamine™2000(Catalogno.11668-027),whichissuperiorforplasmidtransfections.IfyouwanttouseLipofectamine™RNAiMAXforyourcotransfections,performareversetransfectionasdescribedonpage2withthefollowingmodifications:1a:Add20ng(for24-wellformat)ofplasmidDNAtothedilutedRNAiduplex.2:Addcellssuchthattheywillbe80-100%confluent24hoursafterplating.PurchaserNotificationThisproductiscoveredbyoneormoreLimitedUseLabelLicenses(seetheInvitrogencatalogor