jetPRIME转染试剂说明_精品文档.pdf

jetPRIME转染试剂说明_精品文档.pdf

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Polyplus-transfectionS.A.-Bioparc-850BdS.Brant-67400Illkirch-France-Phone:

+33390406180-Fax:

+33390406181Polyplus-transfectionInc.-1251AveoftheAmericas-34thfl.-New-York-NY10020-USAwww.polyplus-jetPRIMEinvitroDNA&siRNAtransfectionreagentPROTOCOLDESCRIPTIONjetPRIMEisanovelpowerfulmoleculebasedonapolymerformulationmanufacturedatPolyplus-transfection.jetPRIMEensureseffectiveandreproducibleDNAandsiRNAtransfectionintomammaliancells.jetPRIMEisextremelyefficientonawidevarietyofcelllines.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.11CPT114vJ（March2015）2jetPRIMEDNA&siRNATransfectionReagent1TRANSIENTDNATRANSFECTIONPROTOCOL1.1CELLSEEDINGForoptimalDNAtransfectionconditions,werecommendusingcellswhichare60to80%confluentatthetimeoftransfection.Typically,forexperimentsin6-wellplates,200000cellsareseededperwellin2mlofcellgrowthmedium24hpriortotransfection.Forothercultureformats,refertoTable1.jetPRIMEisstableinpresenceofserumandantibioticsthereforeyoumayuseserumandantibioticcontainingmediumduringtheentireexperiment.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.Dilute2gDNAinto200ljetPRIMEbuffer（supplied）.Mixbyvortexing.2.Add4ljetPRIME,vortexfor10s,spindownbriefly.3.Incubatefor10minatRT.4.Add200loftransfectionmixperwelldropwiseontothecellsinserumcontainingmedium,anddistributeevenly.5.Gentlyrocktheplatesbackandforthandfromsidetoside.6.Ifneeded,replacetransfectionmediumafter4hbycellgrowthmediumandreturntheplatestotheincubator.7.Analyzeafter24horlater.Polyplus-transfectionS.A.-Bioparc-850BdS.Brant67400IllkirchFrance-Phone:

+33390406180-Fax:

+33390406181Polyplus-transfectionInc.-1251AveoftheAmericas-34thfl.-New-York-NY10020-USAwww.polyplus-3jetPRIMEDNA&siRNATransfectionReagentTable2.DNAtransfectionguidelinesaccordingtothecellculturevesselperwellCultureVesselVolumeofjetPRIMEBuffer（l）AmountofDNA（g）VolumeofjetPRIMEreagent（l）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*Prepareamastermixofminimum50ltoallowaccuratepipettingandhomogenouspreparationofthecomplexesNOTE:

jetPRIMEbuffermustbeusedforsuccessfultransfection.Browseourcelltransfectiondatabasetofindtheoptimizedconditionsaccordingtoyourcellline.http:

/www.polyplus-thevideo“DNATransfectionusingjetPRIME”onYouTube!

http:

/Standardconditions1:

2DNAtojetPRIMEratio（w/v）for1gofDNAuse2lofjetPRIMECPT114vJ（March2015）4jetPRIMEDNA&siRNATransfectionReagentDNATransfectionin6-wellplatesPolyplus-transfectionS.A.-Bioparc-850BdS.Brant67400IllkirchFrance-Phone:

+33390406180-Fax:

+33390406181Polyplus-transfectionInc.-1251AveoftheAmericas-34thfl.-New-York-NY10020-USAwww.polyplus-5jetPRIMEDNA&siRNATransfectionReagent1.3VIRUSPRODUCTIONINADHERENTCELLSjetPRIMEisidealforvirusproduction,especiallyretrovirus,AAVandlentivirus,inadherentcells（ex:

HEK-293T）.Forcotransfectionofmultipleplasmids,thetotalDNAamountperwell/plateshouldnotexceedtheDNAamountindicatedinTable2.Theratiotouseforeachplasmiddependsonthesizeoftheplasmids,theplasmidconstructsandthedesiredexpressionlevelofeachplasmid.Pleaseadjusttheratiosaccordingtoyourapplication.Eachplasmidshouldrepresentatleast10%ofthetotalDNAamountperwell/plate.Thefollowingconditionsaregivenper100mmdish.Forothercultureformat,pleaserefertoTable2.Foroptimization,pleaserefertoTable3.1.Dilute10gtotalDNAamountinto500ljetPRIMEbuffer（supplied）.Mixbyvortexing.2.Add20ljetPRIM