Biomiga-EZgene-EndoFree-Plasmid-Miniprep-Kit

Page1BiomigaEZgeneTMEndoFreePlasmidMiniprepKitContentsIntroduction………………………………………………………….2ImportantNotes………………….………………………………….2StorageandStability………………………………………………...4BeforeStarting……………………………………………….……..5KitContents………………………………………………....………5SafetyInformation………………………………………….……….5EZgene™EndoFreePlasmidMiniprepSpinProtocol…………......6A:RemovalofEndotoxinduringPlasmidPurification....……….6B:RemovalofEndotoxinafterPlasmidPurification....…………9EZgene™EndoFreePlasmidMiniprepSpin/VacuumProtocol……10无内毒素质粒小提试剂盒简明步骤………………………………11PurificationofLow-Copy-NumberPlasmidandCosmid…………..14TroubleShootingGuide...........................................................……...15RelatedProduct……………………………………………………...16Page2BiomigaEZgeneTMEndoFreePlasmidMiniprepKitIntroductionKeytothiskitisourproprietaryDNAbindingsystemsthatallowthehighefficientbindingofDNAtoourmatrixwhileproteinsandotherimpuritiesareremovedbywashbuffer.NucleicacidsareeasilyelutedwithsterilewaterorElutionBuffer.Plasmidisolatedwithtraditionalprotocolnormallycontainshighlevelofendotoxins(lipopolysaccharidesorLPS).Fortransfectionofendotoxinsensitivecelllinesormicroinjection,theendotoxinsshouldberemovedbeforetheapplications.TheEzgene™endofreesystemusesaspeciallyformulatedbufferthatextractstheendotoxinfromtheplasmidDNA.Tworoundsofextractionwillreducetheemdotoxinlevelto0.1EU(Endotoxin)perµgofplasmidDNA.TheendofreeplasmidminiprepkitprovidesanefficientendotoxinremovalstepintothetraditionalpurificationproceduretoproducetransfectiongradeplasmidDNAThiskitisdesignedforfastandefficientpurificationofplasmidDNAfrom3to12mLofE.coliculture.TheminiprepcolumnhasaplasmidDNAbindingcapacityof80µg.ThepurifiedendofreeDNAisreadyfordownstreamapplicationssuchastransfectionofendotoxin-sensitivecelllines,primaryculturedcellsormicroinjection.ImportantNotesPlasmidCopyNumbers:TheyieldofplasmidDNAdependsontheoriginofthereplicationandthesizeoftheplasmid.Theprotocolsareoptimizedforhighcopynumberplasmidpurification.Forlowcopynumberplasmids,boththeculturevolumeandthebuffervolumeneedtobescaledup2times.PleasecontactourcustomerserviceforfurtherinformationandreferencetheTable1forthecommonlyusedplasmids,Table1Commonlyusedplasmids.PlasmidOriginCopyNumbersExpectedYield(µgper10mL)pSC101pSC10150.5-0.75pACYCP15A10-121-1.5pSuperCospMB110-201-2.5pBR322pMB115-201.5-2.5pGEMRMutedpMB1300-40030-40pBluescriptRColE1300-50030-50pUCMutedpMB1500-70050-70Page3BiomigaEZgeneTMEndoFreePlasmidMiniprepKitHostStrains:Thestrainsusedforpropagatingplasmidhavesignificantinfluenceonyield.HoststrainssuchasTop10andDH5ayieldhigh-qualityplasmidDNA.endA+strainssuchasJM101,JM110,HB101,TG1andtheirderivatives,normallyhavelowplasmidyieldduetoeitherendogenousendonucleasesorhighcarbohydratesreleasedduringlysis.WerecommendtransformplasmidtoanendA-strainiftheyieldisnotsatisfactory.PleasereferenceTable2fortheendAinformation.Table2endAstrainsofE.Coli.EndA-StrainsofE.ColiDH5αDH1DH21JM106JM109SK2267SRBXLOTOP10DH10BJM103JM107SK1590MM294Stbl2™XL1-BlueBJ5182DH20JM105JM108SK1592Select96™Stbl4™XL10-GoldEndA+StrainsofE.ColiC600JM110RR1ABLE®CCJ236KW251P2392BL21(DE3)HB101TG1TB1ABLE®KDH12S™LE392PR700BL21(DE3)pLysSJM101JM83TKB1HMS174ES1301M1061Q358BMH71-18AllNMstrainsAllYstrainsOptimalCellMass(OD600xmLofCulture):ThisprocedureisdesignedforisolatingplasmidgrowninstandardLBmedium(LuriaBertani)for12-16hourstoadensityofOD6002.0to3.0.IfrichmediumsuchasTBor2xYTareused,makesurethecelldensitydoesn’texceed3.0(OD600).AhighratioofbiomassoverlysisbuffersresultinlowDNAyieldandpurity.Theminicolumnhasanoptimalbiomassof30-45.Forexample,iftheOD600is3.0,theoptimalculturevolumeshouldbe10-15mL.CultureVolume:Useaflaskortube4timesbiggerinvolumnthantheculturemediumtosecureoptimalconditionforbacteriagrowth.Don’texceedthemaximumculturevolumesuggestedintheprotocol.Incompletelysisduetooveramountofbacterialcultureresultsinloweryieldandlesspurity.StorageandStabilityBufferA1shouldbestoredat4°ConceRNaseAisadded.Allothermaterialscanbestoredatroomtemperature(22-25°C).TheGuaranteedshelflifeis12monthsfromthedateofpurchase.Page4BiomigaEZgeneTMEndoFreePlasmidMiniprepKitBeforeStartingAlternativeendotoxinremovalproceduresareprovided.ProtocolAremovesendotoxinduringthepurificationofplasmidDNA,whileProtocolBremovesendotoxinafterthepurificationofplasmidDNA.Prepareallcomponentsandgetallnecessarymaterialsreadybyexaminingthisinstructionbookletandbecomefamiliarwitheachstepsandpayspecialattentiontothefollowings.ImportantRNaseA:Itisstableforhalfofyearunderroomtemperature.SpindownRNaseAvialbriefly.AddtheRNaseAsolutiontoBufferA1andmixwellbeforeuse.Add8mL(PD1212-00)or60mL(PD1212-01)or96mL(PD1212-02)96-100%ethanoltoeachDNAWashBufferbottlebeforeuse.BufferB1precipitatesbelowroomtemperature.Itiscriticaltowarmupthebufferat50°Ctodissolvetheprecipitatesbeforeuse.KeepthecaptightlyclosedforBufferB1afteruse.Ensuretheavailabilityofcentrifugecapableof13,000rpm.Carryoutallcentrifugationsatroomtemperature.Materialssuppliedbyusers96-100%ethanol1.5mLand2.0mLpyrogenfreemicrocentrifugetubesHighspeedmicrocentrifugeVacuumman