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LifeAmbionT7啟動(dòng)子轉(zhuǎn)錄試劑盒說(shuō)明書(shū)

USERGUIDE

mMESSAGEmMACHINEKit

HighYieldCappedRNATranscriptionKit

SP6,T7,andT3Kits

CatalogNumbersAM1340,AM1344,AM1348

PublicationNumber1340M

RevisionG

LifeAmbionT7啟動(dòng)子轉(zhuǎn)錄試劑盒說(shuō)明書(shū)

ForResearchUseOnly.Notforuseindiagnosticprocedures.

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LifeAmbionT7啟動(dòng)子轉(zhuǎn)錄試劑盒說(shuō)明書(shū)

Contents

■mMESSAGEmMACHINEKit......................................5

Introduction...........................................................................5

Background......................................................................5Materialsprovidedwiththekit......................................................6Materialsnotprovidedwiththekit...................................................6mMESSAGEmMACHINEKitProcedure..................................................8

PreparationoftemplateDNA.......................................................8Procedureoverview..............................................................10Cappedtranscriptionreactionassembly............................................11RecoveryoftheRNA..............................................................12Quantitationofreactionproducts...................................................13Troubleshooting......................................................................15

UseoftheControlTemplate.......................................................15Troubleshootinglowyield.........................................................16Multiplereactionproducts,transcriptsofthewrongsize..............................18■APPENDIXASupplementalInformation..........................19

Additionalprocedures.................................................................19

Analysisoftranscriptionproductsbygelelectrophoresis..............................19Optimizingyieldoflongtranscripts.................................................20Optimizingyieldofshorttranscripts................................................21Spincolumnpreparationanduse..................................................22Miniprepforisolatingtranscription-qualityplasmidDNA..............................23Recipes.............................................................................25RelatedproductsavailablefromLifeTechnologies........................................27Qualitycontrol........................................................................28

Functionaltesting................................................................28Nucleasetesting.................................................................28Proteasetesting.................................................................28■APPENDIXBSafety...........................................29

Chemicalsafety......................................................................29Biologicalhazardsafety...............................................................29Bibliography...................................................31

mMESSAGEmMACHINEKitUserGuide3

LifeAmbionT7啟動(dòng)子轉(zhuǎn)錄試劑盒說(shuō)明書(shū)

Contents

4DocumentationandSupport.....................................33ObtainingSDSs......................................................................33Obtainingsupport....................................................................33Limitedproductwarranty..............................................................33mMESSAGEmMACHINEKitUserGuide

LifeAmbionT7啟動(dòng)子轉(zhuǎn)錄試劑盒說(shuō)明書(shū)

mMESSAGEmMACHINEKitIntroduction

Materialsprovided

withthekit

Materialsnot

providedwiththe

kit

6ThemMESSAGEmMACHINEKitshouldbestoredinanon-frost-freefreezer.Keepallreagentsonicewhileusingthekit;thenucleotidesandenzymesareespeciallylabile.Reagentsareincludedfor25mMESSAGEmMACHINEreactions.AmountComponentStorage1.75mLNuclease-freeWateranytemp50LEnzymeMix(SP6,T7,orT3):–20CBuffered50%glycerolcontainingRNApolymerase,RNaseInhibitor,andothercomponents50L10XReactionBuffer(SP6,T7,orT3)–20Csalts,buffer,dithiothreitol,andotheringredients250L2XNTP/CAP(SP6,T7,orT3):aneutralizedbufferedsolution–20Ccontaining:SP6KitsT7andT3KitsATP10mM15mMCTP10mM15mMUTP10mM15mMGTP2mM3mMcapanalog8mM12mM100LGTP20mMinSP6Kits;30mMinT3andT7Kits–20C100LTURBODNase(2U/L)–20C10LpTRI-Xef,0.5mg/mL(ControlTemplate)–20C1mLAmmoniumAcetateStopSolution–20C5Mammoniumacetate,100mMEDTA1.4mLLithiumChloridePrecipitationSolution–20C7.5Mlithiumchloride,50mMEDTA1.4mLGelLoadingBufferII:a1–2XgelloadingsolutionforTBE–20Cpolyacrylamideandagarosegels95%formamide0.025%xylenecyanol,0.025%bromophenolblue18mMEDTA0.025%SDSComponentsarespecificallycalibratedforeachlotandkittype.Mixingcomponentsfromdifferentlots,orfromkitsfordifferentenzymes(SP6,T7,T3)willcompromiseRNAyield.StoreNuclease-freeWaterat–20C,4Corroomtemp.DNAtemplate:TheDNAtemplatemusthavethecorrectRNApolymerasepromotersite(T7,T3,orSP6)upstreamofthesequencetobetranscribed.Thesuggestedtemplateconcentrationis0.5g/LinwaterorTE(10mMTris-HCl(pH7–8),1mMEDTA).mMESSAGEmMACHINEKitUserGuide

LifeAmbionT7啟動(dòng)子轉(zhuǎn)錄試劑盒說(shuō)明書(shū)

mMESSAGEmMACHINEKitIntroduction

(optional)Labelednucleotide(s):[α-32P]UTPor[α-32P]CTPcanbeaddedtothe

reactionasatracertofacilitatequantitationoftheRNAsynthesized.Anyspecificactivityisacceptable.

(optional)ForpurificationofthesynthesizedRNA:

–Buffer-orwater-saturatedphenol/chloroform

–Isopropanol

–SpinColumns

mMESSAGEmMACHINEKitUserGuide7

LifeAmbionT7啟動(dòng)子轉(zhuǎn)錄試劑盒說(shuō)明書(shū)

mMESSAGEmMACHINEKit

mMESSAGEmMACHINEKitProcedure

mMESSAGEmMACHINEKitProcedurePreparationoftemplateDNALinearizedplasmidDNA,andPCRproductsthatcontainanRNApolymerase8promotersitecanbeusedastemplatesforinvitrotranscriptionwiththemMESSAGEmMACHINEKit.Ingeneral,anyDNAwithapromotersite,thatispureenoughtobeeasilydigestedwithrestrictionenzymescanbeusedforinvitrotranscription.Figure1PhagePolymerasePromoters:MinimalSequenceRequirementsT7+1TAATACGACTCACTATAGGGAGAThe+1base(inbold)isthefirstbaseincorporatedintoRNAduringSP6+1transcription.TheunderlineshowstheATTTAGGTGACACTATAGAAGNGminimumpromotersequenceneededforefficienttranscription.T3+1AATTAACCCTCACTAAAGGGAGA-17+6TemplatesizeThemMESSAGEmMACHINEKitisdesignedtofunctionbestwithtemplatesthatcodeforRNAtranscriptsinthe0.3to5kbrange.Thekitcanbeusedtoproduceshorter,orlongerRNA,butmodifythereactionasdescribedinsection“Optimizingyieldofshorttranscripts〞onpage21.OrientationIfsenseRNAisneeded,itisimportanttotranscribeusingtheRNApolymerasecorrespondingtothephagepromoteratthe5',oramino-terminalsideofthecodingregionoftheprotein(usingpromoter1inthediagrambelow).Ifthetemplateconsistsofaplasmid,itshouldbelinearizedinthepolylinkerattheopposite(3'orcarboxy-terminalside)oftheprotein-codingregion.Antisense(mRNA-complementary)transcriptswillbesynthesizediftheRNApolymerasecorrespondingtotheRNAphagepromoteratthe3',orcarboxy-terminalsideofthecodingregionoftheproteinisused(usingpromoter2inthediagrambelow).5'ATGAAAAAA3'promoter1promoter23'5'TranscriptionusingtheRNApolymerasecorrespondingtopromoter1willmakesenseRNA(thesamesequenceasthemRNA).IftheRNApolymeraseforpromoter2isused,antisenseRNAwillbetranscribed.mMESSAGEmMACHINEKitUserGuide

LifeAmbionT7啟動(dòng)子轉(zhuǎn)錄試劑盒說(shuō)明書(shū)

mMESSAGEmMACHINEKit

mMESSAGEmMACHINEKitProcedure

ProcedureoverviewFigure2TranscriptionreactionassemblyandincubationPreparationoftemplateDNACappedtranscriptionreactionassembly

1.“Thawthefrozenreagents〞onpage11

2.“Assembletranscriptionreactionatroomtemp〞onpage11

3.“Mixthoroughly〞onpage11

4.“Incubateat37C,1hr〞onpage11

5.“(optional)Add1LTURBODNase,mixwellandincubate15minat37C〞on

page12

RecoveryoftheRNA

1.“MEGAclearKit〞onpage12

2.“Lithiumchlorideprecipitation〞onpage12

3.“Spincolumnchromatography〞onpage12

4.“Phenol:chloroformextractionandisopropanolprecipitation〞onpage13

10mMESSAGEmMACHINEKitUserGuide

LifeAmbionT7啟動(dòng)子轉(zhuǎn)錄試劑盒說(shuō)明書(shū)

mMESSAGEmMACHINEKit

mMESSAGEmMACHINEKitProcedureCapped

transcriptionreactionassembly1.ThawthefrozenreagentsPlacetheRNAPolymeraseEnzymeMixonice,itisstoredinglycerolandwillnot

befrozenat–20C.

Vortexthe10XReactionBufferandthe2XNTP/CAPuntiltheyarecompletelyinsolution.Oncethawed,storetheribonucleotides(2XNTP/CAP)onice,butkeepthe10XReactionBufferatroomtemperaturewhileassemblingthereaction.

Allreagentsshouldbemicrofugedbrieflybeforeopeningtopreventlossand/orcontaminationofmaterialthatmaybepresentaroundtherimofthetube.

2.Assembletranscriptionreactionatroomtemp

Thespermidineinthe10XReactionBuffercancoprecipitatethetemplateDNAifthereactionisassembledonice.

Addthe10XReactionBufferafterthewaterandtheribonucleotidesarealreadyinthetube.

Thefollowingamountsareforasingle20Lreaction.Reactionsmaybescaledupordownifdesired.

IMPORTANT!ThefollowingreactionsetupisrecommendedwhentheRNA

producedwillbe300basesto5kbinlength.Fortranscriptslongerorshorterthanthis,considerthesuggestionsinsections“Optimizingyieldoflongtranscripts〞3.Mixthoroughly

Gentlyflickthetubeorpipettethemixtureupanddowngently,andthen

microfugetubebrieflytocollectthereactionmixtureatthebottomofthetube.

4.Incubateat37C,1hr

Typically,80%yieldisachievedaftera1hrincubation.Formaximumyield,werecommenda2hrincubation.SinceSP6reactionsaresomewhatslowerthanT3andT7reactions,theyespeciallymaybenefitfromthesecondhourofincubation.

Asecondhourofincubationisrecommendedforsynthesisof300base

transcriptsandforinefficientlytranscribedtemplates.(Seesections“Optimizingyieldoflongtranscripts〞onpage20and“Optimizingyieldofshorttranscripts〞onpage21foroptimizingyieldfromtemplatescodingRNAoutsidethe0.3–5kbrange.

mMESSAGEmMACHINEKitUserGuide11

LifeAmbionT7啟動(dòng)子轉(zhuǎn)錄試劑盒說(shuō)明書(shū)

mMESSAGEmMACHINEKitTroubleshooting

Troubleshooting

lowyield

162.ExpectedyieldfromthecontrolreactionTheyieldfromthecontrolreactionforT7andT3Kitsshouldbe20–30gofRNA,and15–25gwiththeSP6Kits.Ifa[32P]NTPwasaddedtothetranscriptionreactionasatracer,approximately15%oftheradiolabelshouldbeincorporated.3.Whattodoifthecontrolreactiondoesn’tworkasexpectedIftheyieldofRNAfromthecontrolreactionislow,somethingmaybewrongeitherwiththeprocedureorthekit,orthequantitationisinerror.a.DoublechecktheRNAquantitationToconfirmthatthequantitationiscorrect,verifytheyieldbyanindependentmethod.ForexampleifTCAprecipitationwasusedtoassessyield,tryalsorunninganaliquotofthereactiononanagarosegel.b.TrythepositivecontrolreactionagainIftheyieldisindeedlowbytwodifferentmeasurements,theremaybeatechnicalproblemwiththewaythekitisbeingused.Forexample,thespermidineinthe10XReactionBuffermaycauseprecipitationofthetemplateDNAifitisnotdilutedbytheotheringredientspriortoaddingtheDNA.(Thisisthereasonthatthewaterisaddedfirst.)Repeatthereaction,followingtheprocedurecarefully.Ifthingsstilldon’tgowell,contactTechnicalServicesformoreideas.TheamountofRNAsynthesizedinastandard20LmMESSAGEmMACHINEreactionshouldbe15–20gandmayexceed30g;however,thereisagreatdealofvariationinyieldfromdifferenttemplates.Iftheyieldislow,thefirststepintroubleshootingthereactionistousethepTRI-XefcontroltemplateinastandardmMESSAGEmMACHINEreaction.1.NeithermytemplatenorthecontrolreactionworksDoublecheckthatyouhavefollowedtheprocedureaccurately,andconsidertryingthecontrolreactionasecondtime.Ifthekitcontrolstilldoesn’twork,itisanindicationthatsomethingmaybewrongwiththekit,callourTechnicalSupportgroupformoreideas.2.Thecontrolreactionworks,butmytemplategiveslowyieldIfthetranscriptionreactionwithyourtemplategeneratesfull-length,intactRNA,butthereactionyieldissignificantlylowerthantheamountofRNAobtainedwiththepTRI-Xefcontroltemplate,itispossiblethatcontaminantsintheDNAareinhibitingtheRNApolymerase.Amixingexperimentcanhelptodifferentiatebetweenproblemscausedbyinhibitorsoftranscriptionandproblemscausedbythesequenceofatemplate.Includethreereactionsinthemixingexperiment,usingthefollowingDNAtemplates:1.1LpTRI-Xefcontroltemplate2.experimentalDNAtemplate(0.5gplasmidor2–6LPCRproduct)3.amixtureof1and2Assesstheresultsofthemixingexperimentbyrunning0.5–1Lofeachtranscriptionreactiononadenaturinggelasdescribedinsection,“Additionalprocedures〞onpage19.mMESSAGEmMACHINEKitUserGuide

LifeAmbionT7啟動(dòng)子轉(zhuǎn)錄試劑盒說(shuō)明書(shū)

mMESSAGEmMACHINEKitTroubleshooting

Multiplereaction

products,

transcriptsofthe

wrongsize1.ReactionproductsproduceasmearwhenrunonadenaturinggelIftheRNAappearsdegraded(e.g.smeared),removeresidualRNasefromtheDNAtemplatepreparationbeforeinvitrotranscription.DothisbydigestingtheDNAprepwithproteinaseK(100–200g/mL)inthepresenceof0.5%SDSfor3018minat50C,followthiswithphenol/chloroformextraction.TheRNaseInhibitorthatispresentinthetranscriptionreaction,canonlyinactivatetraceRNasecontamination.LargeamountsofRNasecontaminationwillcompromisethesizeandamountoftranscriptionproducts.2.Reactionproductsrunasmorethanoneband,orasasinglebandsmallerthanexpecteda.SampleisnotadequatelydenaturedinthegelIftheamountofRNAproducedisacceptable,butthesizeoftheproductisunexpected,considerthattheRNAmayberunningaberrantlyduetosecondarystructure.SometimestheRNAwillrunastwodistinctbandsonanativeagarosegel,butwhenthesameRNAisrunonadenaturinggel,itwillmigrateasasinglebandoftheexpectedsize.b.PrematureterminationoftranscriptionIfdenaturinggelanalysisshowsthepresenceofmultiplebandsorofasinglebandsmallerthantheexpectedsize,theremaybeproblemswithprematureterminationbythepolymerase.Possiblecausesofthisaresequenceswhichresemblethephagepolymeraseterminationsignals,stretchesofasinglenucleotides,andGC-richtemplates.Differentphagepolymerasesrecognizedifferentterminationsignals,sousingadifferentpolymerasepromotermayhelp.Terminationatsinglepolynucleotidestretchescansometimesbealleviatedbydecreasingthereactiontemperature(Krieg,P.A.1990).Wesuggesttesting30C,20Cand10C.However,decreasingthereactiontemperaturewillalsosignificantlydecreasetheyieldofthereaction.Thereisareportthatsingle-strandedbinding(SSB)proteinincreasedthetranscriptionefficiencyofaGCrichtemplate(AzizandSoreq,1990).3.Reactionproductsarelargerthanexpecteda.PersistentsecondarystructuremMESSAGEmMACHINEproductsoccasionallyrunas2bands;1largerthantheexpectedsize,and1attheexpectedsize.ThismayoccurwithtranscriptsfromthepTRI-Xefcontroltemplate,evenwhentheRNAisdenaturedduringtheelectrophoresis.Thisphenomenonoccursbecauseofpersistentsecondarystructure.Toverifythis,thebandthatmigratesattheexpectedsizecanbeexcisedfromthegelandruninaseconddenaturinggel.IftheRNArunsasadoubletinthesecondgelalso,itisagoodindicationthatthelargerbandissimplyanartifactofelectrophoresis.b.CirculartemplateLonger-than-expectedtranscriptionproductswillbeseenifanyofthetemplatemoleculesarecircular.Thisistypicallycausedbyincompletedigestionofaplasmidtemplate.SincetheRNApolymerasesareextremelyprocessive,evenasmallamountofcirculartemplatecanproducealargeamountofRNA.mMESSAGEmMACHINEKitUserGuide

LifeAmbionT7啟動(dòng)子轉(zhuǎn)錄試劑盒說(shuō)明書(shū)

A

Additionalprocedures

Analysisof

transcription

productsbygel

electrophoresisSupplementalInformation1.AgaroseorAcrylamide?ThesizeofmMESSAGEmMACHINEreactionproductscanbeassessedbyrunninganaliquotofthereactiononanagaroseorpolyacrylamidegel.Transcriptslargerthanabout1.5kbshouldberunonagarosegels,whereas

polyacrylamidegels(4–5%)arebetterforsizingsmallertranscripts.Since

secondarystructureinthetranscriptmaycauseaberrantmigrationand/or

multiplebands,thegelshouldberununderdenaturingconditions.Foragarosegels,thismeansglyoxalorformaldehydegels,preparedandrunaccordingtostandardprocedures(MolecularCloning,ALaboratoryManual,1989).

Instructionsforpreparingandrunningdenaturingacrylamidegelsaresuppliedinsection“Denaturingacrylamidegelmix〞onpage25.

2.Samplepreparation

TogetgoodresolutionoftheRNA,load~1gpergellane.Fordenaturing

polyacrylamidegelsaddanequalvolumeofGelLoadingBufferIItoeach

sample,andheatfor3–5minat80–90C.(GelLoadingBufferIIcannotbeusedwithglyoxalagarosegelsanditwillnotcompletelydenaturesamplesrunonformaldehydeagarosegels.Usealoadingbufferspecificallyformulatedforthetypeofagarosegelyouplantorun.)

TostaintheRNAwithethidiumbromideduringelectrophoresisdooneofthefollowing:

a.Add0.5g/mLethidiumbromidetothegelmix

b.Add0.5g/mLethidiumbromidetotherunningbuffer

c.Add10g/mLethidiumbromidetotheRNAsamples(andgelloading

buffer)beforeloadingthegel.

(Becausesingle-strandednucleicacidsbindethidiumlessefficientlythandouble-strandednucleicacids,thefluorescenceofRNAsamplesonadenaturingagarosegelwillbelessintensethanthesameamountofDNA.)

3.Visualizingreactionproducts

a.Ethidiumbromidestainedsamples

ViewethidiumbromidestainedgelsonaUVtransilluminator.Ideallythere

willbeasingle,tightbandattheexpectedmolecularweight.See

section“Multiplereactionproducts,transcriptsofthewrongsize〞onpage

18fortroubleshootingsuggestionsifthisisnotwhatappearsonyourgel.

b.Radioactively-labeledtranscripts

mMESSAGEmMACHINEKitUserGuide19

LifeAmbionT7啟動(dòng)子轉(zhuǎn)錄試劑盒說(shuō)明書(shū)

AAppendixASupplementalInformationAdditionalprocedures

Optimizingyieldof

longtranscripts

20Ifthetranscriptionreactioncontainedaradiolabelednucleotidetracer(e.g.[α-32P]UTP),theRNAcanbevisualizedbyautoradiography.AgarosegelsshouldbedriedbeforeexposingtoX-rayfilm,butthin(0.75mmthick)polyacrylamidegelsmaybetransferredtofilterpaper,coveredwithplasticwrap,andexposed