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1、Invitrotranscriptionand InvitrotranslationInvitrotranscriptionInVitroTranscription目的:体外得到单一,大量的mRNA意义:探针------原位杂交siRNA------RNAiTranscript------体外翻译,蛋白表达InvitroTranscriptionT7T3EcoRIBamHIAntisense:CutwithEcoRIUseT-3polymeraseSense:CutwithBamHIUseT-7polymeraseVectorMaps
2、InvitroTranscriptionPlasmidwithT3,T7orSP6promotersLinearizationofplasmidDNAbyrestrictionenzymeInvitrotranscription:Plasmid buffer NTP labeledUTPRNApolymeraseDNAsedigestion,Phenol/ChloroformextractionandRNAprecipitataionGlovesshouldbewornatalltimesduringpreparationofinvi
3、troRNA.AllsolutionsshouldbeRNase-freei.e.madewithDEPCwaterifhome-madeorboughtspecificallytouseforRNAwork.InvitrotranscriptionreactionYouwillneed:Ribonucleaseinhibitor(RNasin) T7orT3RNAPolymerase2.5mMrNTPs5xT7/T3RNAPolymeraseBuffer(200mMTris.ClpH8.0,125mMNaCl,40mMMgCl2,1
4、0mMspermidine)100mMDTTRNase-freeDNaseIDEPC-treated,autoclavedInvitrotranscriptionreactionLinearizetheplasmidat,ornear,theterminusoftheinsertcDNAwithasuitablerestrictionendonuclease.2)ExtractplasmidDNAwithanequalvolumeofphenol/chloroformandanequalvolumeofchloroformpriort
5、oethanolprecipitation.3)LinearizedplasmidDNAisresuspendedinDEPC-treated,autoclavedTE,pH7.5ataconcentrationofapproximately200ng/ulpriortouse.Invitrotranscriptionreaction4)Thestandardreactionconditionsaresetupcontainingthefollowingquantities:2ul(400ng)linearizedplasmidDNA
6、 20Uribonucleaseinhibitor 2ul100mMDTT 4ul5xT3/T7RNApolymerasebuffer 4ul2.5mMNTPsSterile,DEPCtreatedH2Otoafinalvolumeof19.5ul 25U(0.5ul)T3/T7RNApolymeraseThereactionmixtureisincubatedat37°Cfor60minutes.Invitrotranscriptionreaction5)DNAtemplateisremovedbytheadditionof25UR
7、Nase-freeDNaseIandafurtherincubationfor30minutesat37°C.Thereactionmixtureisphenol/chloroformextractedtwice,ethanolprecipitated,vacuumdriedandresuspendedin25ulsterile,DEPC-treatedTE,pH7.5.6)RNAcanbeanalysedbeelectrophoresisusingdenaturingconditionsinanagarose/formaldehyd
8、egelsystem.InvitrotranscriptionreactionInVitroTranscription探针------原位杂交siRNA------RNAiTranscript------体外翻译,蛋白表
