罗氏转染试剂说明书

×1mlStoreat–15to–25°C1.WhatthisProductDoesNumberofTestsUsingthestandardprocedure,1mlofX-tremeGENEHPDNATrans-fectionReagentcanbeusedtoperformupto10,000transfectionsin96-wellplates.FormulationX-tremeGENEHPDNATransfectionReagentisaproprietaryblendoflipidsandothercomponentssuppliedin80%ethanol,filteredthrough0.2mporesizemembrane,andpackagedinglassvials.Itdoesnotcontainanyingredientsofhumanoranimalorigin.StorageandStabilityStoreX-tremeGENEHPDNATransfectionReagentat–15to–25°C,withthelidtightlyclosed.Thereagentisstableuntiltheexpirationdateprintedonthelabelwhenstoredundertheseconditions.LX-tremeGENEHPDNATransfectionReagentremainsfullyfunc-tionalevenafterrepeatedopeningofthevial(atleastfivetimesoveratwo-monthperiod),aslongasthevialistightlyrecappedandstoredat-15to-25°C.LNotethattheshippingtemperatureofthisproductisdifferentfromthestoragetemperature.Thesedifferenttemperatureswillnotaffectproductperformanceorproductstability.SpecialHandlingNAfterremovingtheamountrequired,tightlyclosethevialwiththelidimmediatelyafteruse.NAlwaysbringthevialto+15to+25°CandmixX-tremeGENEHPDNATransfectionReagentpriortoremovingtheamountrequiredvortexingforonesecond.NDonotaliquotX-tremeGENEHPDNATransfectionReagent;storeintheoriginalglassvials.NMinimizethecontactofundilutedX-tremeGENEHPDNATrans-fectionReagentwithplasticsurfaces.NForuse,theminimumamountofX-tremeGENEHPDNATransfec-tionReagent:DNAcomplexis100µl.Complexformationatlowervolumescansignificantlydecreasetransfectionefficiency.NDonotusetubesormicroplatesmadeofpolystyreneforX-tremeGENEHPTransfectionReagent:DNAcomplexprepara-tion.Whennotabletoavoidpolystyrenematerials,makecertaintopipetthetransfectionreagentdirectlyintotheserum-freemedium(e.g.,Opti-Mem).NDonotusesiliconizedpipettetipsortubes.AdditionalEquipmentandReagentsRequiredAdditionalreagentsandequipmentrequiredtoperformtransfectionassaysusingX-tremeGENEHPDNATransfectionReagentinclude:➤StandardLaboratoryEquipment.Standardcellcultureequipment(e.g.,biohazardhoods,incuba-tors)StandardpipettesandmicropipettesVortexmixer➤ForPlasmidPreparationPurifiedplasmidstock(0.1–2.0µg/µl)insterileTE(10mMTris,1mMEDTA,pH8.0)bufferorsterilewaterGenopurePlasmidMidiKit*orGenopurePlasmidMaxiKit*toprepareplasmid➤ForVerificationofVectorFunctionAssayappropriatelyfortransfectedgeneG-418Solution*orHygromycinB*(optionalforstabletransfec-tionexperiments)➤ForTransfection-ComplexFormationOpti-MEMIReducedSerumMediumorserum-freemediumSterilepolypropylenetubesorround-bottom96-wellplates➤GrowingCellsSelectsubconfluentculturesinlogphaseforpreparationofcellculturesQuantifycellnumbertoreproduciblyplatethesamenumberofcellsApplicationX-tremeGENEHPDNATransfectionReagentisahighperformancetransfectionreagent,freeofanimal-derivedcomponents.BenefitsofX-tremeGENEHPDNATransfectionReagentinclude:�Designedtotransfectabroadrangeofeukaryoticcells,includinginsectcells,manycelllinesnottransfectedwellbyotherreagents,andhard-to-transfectcelllines(e.g.,HT-1080,K-562,HepG2).�Canbesuccessfullyusedinavarietyofapplications,suchasgeneexpressionanalysisandproteinproductionusingtransientlytrans-fectedcells,generationofstablecelllines,expressionofshRNAforgeneknockdownstudies,drugdiscoveryprograms,andtargetevaluation.Samplesanddetailedtransfectionprotocolsareavail-ableat�Producesminimalcytotoxicityorchangesinmorphologywhenade-quatenumbersofcellsaretransfected,eliminatingtherequirementtochangemediaafteraddingthetransfectioncomplex.�Suitablefortransientandstabletransfection.�Functionsverywellinthepresenceorabsenceofserum.Forlifescienceresearchonly.Notforuseindiagnosticprocedures.0511.064797740012:1,2:1,3:1and4:1ratiosofmicroliter(l)X-tremeGENEHPDNATransfectionReagenttomicrogram(g)DNA.Aratioof3:1ofmicroliter(l)X-tremeGENEHPDNATransfectionReagenttomicro-gram(g)DNAhasbeenshowntobeoptimalformanycelltypes.LLowercellconfluencieshavealsobeentestedsuccessfully.Therecommendedstartingconcentrationisa3:1.Formostcelltypes,theseX-tremeGENEHPDNATransfectionReagenttoDNAratiospro-videexcellenttransfectionefficiency.LFurtheroptimizationmayincreasetransfectionefficiencyinyourparticularapplication.Inadditiontovaryingtheratio,otherparam-etersmayalsobeevaluated,suchastheamountoftransfectioncomplexadded.Foradditionaloptimizationguidelines,seeSection3,Troubleshootingandvisit�Forbestresults,accuratelydeterminetheplasmidDNAconcentra-tionusing260-nmabsorption;estimatesofDNAbymeasuringgelbanddensityarenotrecommended.DetermineDNApurityusinga260nm/280nmratio(theoptimalratiois1.8).�PreparetheplasmidDNAsolutionus