PROMEGA逆转录试剂盒说明书

PromegaCorporation·2800WoodsHollowRoad·Madison,WI53711-5399USATollFreeinUSA800-356-9526·Phone[phone]·Fax[phone]·(A)+isolatedRNA(1).TheReverseTranscriptionSystem(a)providestestedreagentstoefficientlyreversetranscribepoly(A)+mRNAortotalRNAin15minutes.Apolyadenylated1.2kbtranscriptisprovidedasacontroltemplateforthecDNAsynthesisreaction.cDNAsynthesizedwiththeReverseTranscriptionSystemcanbeuseddirectlyinPCR.CitationusingtheReverseTranscriptionSystem•Basgupta,P.etal.(2006)Nicotineinhibitsapoptosisinducedbychemotherapeuticdrugsbyup-regulatingXIAPandsurvivin.Proc.Natl.Acad.Sci.USA103,6332–7.RT-PCRwasperformedtomapthesubtypesofnicotinicacetylcholinereceptorsonA549cells.cDNAwassynthesizedusingtheReverseTranscriptionSystem.NorthernblottingtoassessXIAPandsurvivinexpressionwasperformed,andprobeswerelabeledusingthePrime-A-Gene®LabelingSystem.Apoptosiswasassessedinnicotine-stimulatedcellsusingaDeadEnd™TUNELAssay.Foradditionalpeer-reviewedarticlesthatciteuseoftheReverseTranscriptionSystem,visit:::[e-mail]rseTranscriptionSystemINSTRUCTIONSFORUSEOFPRODUCTA3500.PRINTEDINUSA.Revised3/09Part#TB0992.Preparea20μlreactionbyaddingthefollowingreagentsintheorderlisted(thisreactioncanbescaledupordown,dependingontheamountofRNA):*Thesuggestedmagnesiumconcentrationmaybeoptimizedforanygivensequencetoachievebetteryields.**Finalconcentrationofreactioncomponents:5mMMgCl2;1XReverseTranscriptionBuffer(10mMTris-HCl[pH9.0at25°C];50mMKCl;0.1%Triton®X-100);1mMeachdNTP;1u/μlRecombinantRNasin®RibonucleaseInhibitor;15u/μgAMVReverseTranscriptase(HighConc.);0.5μgOligo(dT)15PrimerorRandomPrimerspermicrogramRNA;50ng/μl1.2kbKanamycinPositiveControlRNA,poly(A)+mRNAortotalRNA.3.WhenusingOligo(dT)15Primer,incubatethereactionat42°Cfor15minutes.WhenusingRandomPrimers(randomhexamers),incubatethereactionatroomtemperaturefor10minutes,thenincubateat42°Cfor15minutes.Theadditionalincubationatroomtemperatureallowsextensionoftheprimerssothattheyremainhybridizedwhenthetemperatureisraisedto42°C.Note:TherearedifferenttemperaturerequirementsforthereversetranscriptionreactionwhenusingRandomPrimersthanwhenusingOligo(dT)15.4.Heatthesampleat95°Cfor5minutes,thenincubateat0–5°Cfor5minutes.ThiswillinactivatetheAMVReverseTranscriptaseandpreventitfrombindingtothecDNA.Forsecond-strandcDNAsynthesisoragarosegelanalysis,first-strandcDNAproductmaybeused.ForPCRamplification,proceedtoSection3.B.Alternatively,storethefirst-strandcDNAat–20°Cuntiluse.Notes:1.Priortosettingupthereaction,dispensethefollowingreagentsintoindividualtubesforuseasneededpriortoaddingtheRNA:water,buffer,dNTPs,MgCl2,RecombinantRNasin®RibonucleaseInhibitorandAMVReverseTranscriptase.Thisresultsinfewerpipettingstepsandimprovedaccuracy.PromegaCorporation·2800WoodsHollowRoad·Madison,WI53711-5399USATollFreeinUSA800-356-9526·Phone[phone]·Fax[phone]·μgofRNAperreaction.Includes:•1,500uAMVReverseTranscriptase(HighConc.)•2,500uRecombinantRNasin®RibonucleaseInhibitor(a)•50μgOligo(dT)15Primer(0.5μg/μl)•50μgRandomPrimers(0.5μg/μl)•5μg1.2kbKanamycinPositiveControlRNA(0.5μg/μl),10μl•320μldNTPMixture,10mM•1.4mlReverseTranscription10XBuffer•1.2mlMgCl2,25mM•13mlNuclease-FreeWaterStorageConditions:Storeallcomponentsat–20°C.3.ReverseTranscriptionProtocol3.A.ReverseTranscriptionReactionReversetranscriptionmaybeprimedwitheitherOligo(dT)15orRandomPrimers.ChooseOligo(dT)15whenprimingatthe3´poly(A)regionisdesired.ChooseRandomPrimerswhenprimingthroughoutthelengthoftheRNAisdesired.Oligo(dT)15isfrequentlyusedwhencDNAwillbeusedforcloningandRT