jetPRIME转染试剂说明

Polyplus-transfectionS.A.-Bioparc-850BdS.Brant-67400Illkirch-France-Phone:[phone]-Fax:[phone]Polyplus-transfectionInc.-1251AveoftheAmericas-34thfl.-New-York-NY10020-USA®invitroDNA&siRNAtransfectionreagentPROTOCOLDESCRIPTIONjetPRIME®isanovelpowerfulmoleculebasedonapolymerformulationmanufacturedatPolyplus-transfection®.jetPRIME®ensureseffectiveandreproducibleDNAandsiRNAtransfectionintomammaliancells.jetPRIME®isextremelyefficientonawidevarietyofcelllines.Thispowerfulreagentonlyrequireslowamountsofnucleicacidpertransfection,henceresultinginverylowcytotoxicity.1TransientDNAtransfectionprotocol..........................................................................21.1Cellseeding..........................................................................................................................................21.2DNAtransfectionprotocol...................................................................................................................21.3Virusproductioninadherentcells.......................................................................................................51.4Optimizationguidelines.......................................................................................................................52siRNAtransfectionprotocol.......................................................................................62.1Cellseeding..........................................................................................................................................62.2siRNAtransfectionprotocol.................................................................................................................73DNA&siRNAcotransfectionprotocol........................................................................73.1Cellseeding..........................................................................................................................................73.2DNA&siRNAcotransfectionprotocol.................................................................................................84TransfectionofCRISPR/Cas9.....................................................................................95StableDNAtransfection.............................................................................................96Troubleshooting.......................................................................................................107Productinformation.................................................................................................11}CPT114vJ(March2015)2jetPRIME®DNA&siRNATransfectionReagent1TRANSIENTDNATRANSFECTIONPROTOCOL1.1CELLSEEDINGForoptimalDNAtransfectionconditions,werecommendusingcellswhichare60to80%confluentatthetimeoftransfection.Typically,forexperimentsin6-wellplates,200000cellsareseededperwellin2mlofcellgrowthmedium24hpriortotransfection.Forothercultureformats,refertoTable1.jetPRIME®isstableinpresenceofserumandantibioticsthereforeyoumayuseserumandantibioticcontainingmediumduringtheentireexperiment.Table1.Recommendednumberofcellstoseedthedaybeforetransfection.CulturevesselNumberofadherentcellstoseedSurfaceareaperwell(cm2)Volumeofmediumperwelltoseedthecells(ml)96-well7500-100000.30.124-well50000-800001.90.512-well80000-1500003.816-well/35mm150000-2500009.4260mm/flask25cm2250000-80000025-285100mm/flask75cm21x106-2x10675-78.510150mm/flask175cm22x106-5x106153-175201.2DNATRANSFECTIONPROTOCOLThefollowingconditionsaregivenperwellofa6-wellplate.Forothercultureformat,pleaserefertoTable2.1.Dilute2µgDNAinto200µljetPRIME®buffer(supplied).Mixbyvortexing.2.Add4µljetPRIME®,vortexfor10s,spindownbriefly.3.Incubatefor10minatRT.4.Add200µloftransfectionmixperwelldropwiseontothecellsinserumcontainingmedium,anddistributeevenly.5.Gentlyrocktheplatesbackandforthandfromsidetoside.6.Ifneeded,replacetransfectionmediumafter4hbycellgrowthmediumandreturntheplatestotheincubator.7.Analyzeafter24horlater.Polyplus-transfectionS.A.-Bioparc-850BdS.Brant67400IllkirchFrance-Phone:[phone]-Fax:[phone]Polyplus-transfectionInc.-1251AveoftheAmericas-34thfl.-New-York-NY10020-USA®DNA&siRNATransfectionReagentTable2.DNAtransfectionguidelinesaccordingtothecellculturevesselperwellCultureVesselVolumeofjetPRIME®Buffer(µl)AmountofDNA(µg)VolumeofjetPRIME®reagent(µl)Volumeofgrowthmedium(ml)96-well*50.10.2-0.30.124-well500.51-1.50.512-well750.81.6-2.416-well/35mm20024-6260mm/flask25cm220048-125100mm/flask75cm25001020-3010150mm/flask175cm210002040-6020*Prepareamastermixofminimum50µltoallowaccuratepipettingandhomogenouspreparationofthecomplexesNOTE:jetPRIME®buffermustbeusedforsuccessfultransfection.Browseourcelltransfectiondatabasetofindtheoptimizedconditionsaccordingtoyourcellline.“DNATransfectionusingjetPRIME®”onYouTube!=G39wNXaZPX4Standardconditions1:2DNAtojetPRIME®ratio(w/v)for1µgofDNAuse2µlofjetPRIME®}C