Forresearchuseonly.Notforhumanoranimaltherapeuticordiagnosticuse.Lipofectamine®2000ReagentCat.nos.11668-030Size:0.3mLStoreat4°C(donotfreeze)11668-0270.75mL11668-0191.5mL11668-50015mLPub.PartNo.11668.2k.ppsPub.No.MAN0000995Rev.Date20July2012Description•Lipofectamine®2000TransfectionReagentisaproprietaryformulationfortransfectingnucleicacids(DNA,RNA,andmRNA)intoawiderangeofeukaryoticcells.•Nucleicacid-Lipofectamine®2000complexescanbeaddeddirectlytocellsinculturemedium,inthepresenceorabsenceofserum/antibiotic.•Itisnotnecessarytoremovecomplexesorchange/addmediumaftertransfection.ImportantGuidelinesforTransfection•UseOpti-MEM®IReducedSerumMedium(Cat.no.31985-062)todiluteLipofectamine®2000TransfectionReagentandnucleicacids.•TheamountofLipofectamine®2000Reagentrequiredforsuccessfultransfectionvariesdependingonthecelltypeandpassagenumber.StartanynewtransfectionbytestingtherecommendedfourconcentrationsofLipofectamine®2000Reagenttodetermineanoptimumamount.•Preparehigh-qualityplasmidDNAat0.5–5μg/μLindeionizedwaterorTEbuffer.AGFP(greenfluorescentprotein)plasmidcanbeusedtodeterminetransfectionefficiency.•Foradditionalinformation,andprotocolsontransfectingsuspensioncellsrefertothemanualatwww.lifetechnologies.com/transfection.5Day0Day1Day2–4CellsLipofectamine®2000ReagentDNASeedcellssotheyare70–90%confluentatthetimeoftransfection.Dilute4amountsofLipofectamine®2000ReagentinOpti-MEM®Medium.DiluteDNAinOpti-MEM®Medium.AdddilutedDNAtoeachtubeofdilutedLipofectamine®2000Reagent(1:1ratio).Incubate.AddDNA-reagentcomplextocells.Visualize/analyzetransfectedcells.PlasmidTransfectionTransfectcellsaccordingtothefollowingtable.Thetransfectionisdesignedfor1DNAamountcombinedwith4differentamountsofLipofectamine®2000.Foreachlipidreagentamount,thepreparedmixisenoughtohavetriplicates(96-well),duplicates(24-well),andsinglewell(6-well)transfections,andaccountforpipettingvariations.Component96-well24-well6-wellAdherentcells1–4x1040.5–2x1050.25–1x106Opti-MEM®Medium25µLx450...
