Nucleofection
来自 : 蚂蚁淘
ThisisanextractoftheAmaxaBiosystemsprotocolVs.09-2005optimizedforusewiththeUC06cellline.Itissuggestedthatyoutryall5programs(A-23,A-27,A-13,A-12,B-16)witheachNucleofectionsolutioninitiallytodeterminetheoptimalprogramandsolutionforyourspecifichESstrain.
- CollecthESCasfornormalcultureprotocolusingCollagenase.WashthecellswithPBSafterharvest,addCellDissociationBuffer(Gibco),andtrituratetogeneratesmallclumpsofapproximately50cells(donotovertriturate).WashagainwithPBS.
- Prepare2ugcircularDNAfortransientexpressionor10uglinearizedDNAforstablecloneselectionforeachsampleinavolume≤5μl.
- Pre-warmthesupplementedCellLineSolutionVtoroomtemperature.
- Pre-warmone500μlaliquotofgrowthmediumpersample(inasterilemicrofugetube)at37°C.
- Prepare6-wellplateswith2mlculturemediumandpre-incubatetheplatesinahumidified37°C/5%CO2incubator.Platesshouldbeplatedwith4x105MEFcells/wellantibiotic-resistantfeedercells.
- Countthecellsanddeterminecelldensity(cells/ml).Placetheappropriatenumberofcellsinacentrifugetube,andcentrifugethecellsat100xgfor10minutes.Use2x106cellsfortransientexperimentsandupto5x106cellsforstableexpression.
- ResUSPendthepreparedcellsinroomtemperatureNucleofectorCellLineSolutionVtoafinalconcentrationof1-2x106cells/100μl.Performeachsampleseparatelytoavoidstoringthecellslongerthan15mininNucleofectorSolution.
- Mix100μlofcellsuspensionwiththeappropriateamountofDNAasdescribedabove.
- Transferthesampleintoanamaxa-certifiedcuvette.
- SelecttheappropriateNucleofectorprogram—A-27orB-16.(A-27givesbettersurvival,andB-16givesbettertransferefficiency.)
- Toavoiddamagetothecells,use500μlofmediatoremovethesamplefromthecuvetteimmediatelyaftertheprogramhasfinished.Gentlytransfer500μlofthepre-warmedrecoverymediafromthemicrofugetubetothecuvette.Gentlyremovethecellsuspensionfromthecuvette,carefullyreturnittothemicrofugetube,andplaceitinthe37°Cincubator.Itiscriticaltobegentlewiththecellsatthispoint,astheycanbeeasilydamagedbytheshearstressesgeneratedbypipetting.Donotmixthecellsuspensionbyrepeatedpipetting.
- After5minutes,transferthesamplestotheprepared6-wellplates.Transientgeneexpressioncanbeobservedwithin12–18hoursafternucleofection.
- Waitatleast48hoursbeforeselectionforstableorhomologousrecombinationtransfectants.
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