293T transfection protocolPEI transfection protocolStable clone selection protocolStable transfection protocolLinear DNA transfectionLinearize plasmid before transfection

Stable Transfection - Mirus Bio

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When generating a stable cell line, the transfected plasmid undergoes recombination during chromosomal integration. The recombination event can occur within any ...     Applications:StableCellLineGenerationStableCellLineGenerationSTEP1:AntibioticKillCurveSTEP2:StableTransfectionSTEP3:Selection/ExpansionProducts TransfectCellswithTargetPlasmidConstruct(s) Whileperformingthekillcurve(1week),optimizetransfectionconditionsinaT75flaskbytransfectingareporterplasmid(suchasaGFPencodingplasmid)intocellsathighconfluence,e.g.80%.Determinetheappropriatedoseofplasmid(5-15µg)andtransfectionreagent(15-45µl)inaT75flask.Observereportergeneexpression(GFP)andtoxicityatregulartimepointsoveratleasta48hourperiod,optimallyfor72hours.SpecifictipsonoptimizingDNAtransfectioncanbefoundatTipsfromtheBench-OptimizeDNATransfection.UsetheoptimalDNAandtransfectionreagentdosageforgeneratingstabletransfectants. Optional:Linearizeyourtargetplasmidsbeforetransfection.Whengeneratingastablecellline,thetransfectedplasmidundergoesrecombinationduringchromosomalintegration.Therecombinationeventcanoccurwithinanyregionoftheplasmid,includingthegeneexpressionorselectablemarkercassettesthatmightdisrupttheirfunction.Toincreasethelikelihoodthatrecombinationwilloccurinnon-essentialplasmidregions,suchasthebacterialrepliconorbacterialmarkergene,linearizetheplasmidwithrestrictionenzyme(s)thatcutwithinthesenon-essentialregions.Priortotransfection,purifythelinearizedDNAbyethanolprecipitation,sizeexclusionorcolumnpurification. Foreachindividualstablecelllinetobecreated,platecellsinthreeT75flasksandone6-welltissuecultureplateapproximately18–24hoursbeforetransfectionsuchthattheyreachhighconfluence(~60-80%)atthetimeoftransfection.Typicalcelldensityrangesareasfollows: Adherentcells:0.8–2.4×105cells/mlofcompletemedia Suspensioncells:3.2–4×105cells/mlofcompletemedia Leavethe6-welltissuecultureplateuntransfected.Thiswillserveasanuntransfectedcontrolandisimportantasareferenceduringtheselectionprocess(Step3). Transfecttheplatedcellswith5-15µgoftotalplasmidDNAperT75flask.Iftheantibioticselectionmarkerisonaseparateplasmidthanthegeneofinterest,thenmaintaina10:1ratioof“geneofinterest”plasmidover“antibioticselection”plasmid. Donotexposecellstotheselectionantibioticuntil48-72hoursposttransfectiontoavoidlowcellviability.Amediachangecanbeperformedat24hoursposttransfection,ifneeded. Theaboveprocedureworkswellforroutinelytransfectedcelltypes.Forhard-to-transfectcells,anothermethodtogeneratestablecelltransfectantsisvialentivirusorretrovirustransduction.Inthiscase,antibioticresistanceharboringvirusparticlesgeneratedaftertransfectionofproducercelltypessuchasHEK293Tareusedtotransducecellsthatcanthenbeselectedforvirusintegration.Detailsonvirusproductioncanbefoundhere.CopyLinkEnlargeImageTransIT®TransfectionReagentsareIdealforVirusProduction.AllTransIT®TransfectionReagentsarelowtoxicityanddonotrequireamediachange.Savetimeandmoneybyaddingformedcomplexesdirectlytocellsinmediacontainingserumandavoidingunnecessarymediachanges. STEP1:ANTIBIOTICKILLCURVE STEP3:SELECTIONANDEXPANSION Forstablecelllinegeneration,useMirus'highqualitycell-culturegradeselectionantibioticsthatareeasy-to-use: AllMirus'broadspectrumplasmidDNAtransfectionreagentscanbeusedforstablecelllinegeneration.Visittheproductpagesformoreinformationoneachproduct: TransIT-X2®DynamicDeliverySystem TransIT®-2020TransfectionReagent TransIT®-LT1TransfectionReagent Don'tSeeYourCellType?ConsultReagentAgent®TransfectionDatabase CitationDatabase:Checkifourreagentshavebeenusedbyotherresearcherstotransfectyourcelltype TechnicalSupport:Communicatedirectlywithatransfectionexpert Close PleaseWait... Yourrequestisprocessing

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