MicroRNA and siRNA Cloning Method英文学习材料

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1、MicroRNAandsiRNACloningMethodBartelLabProtocolRevisedMay,2002RNACloningMethodFlowchartSynthesizep17.91xPurifytotalRNAfromSynthesizeImpA.Purify.17.92,17.93DNAcells/organism/tissue.oligos.Deprotect.AnalyzebyTLC.Checkqualityondenaturinggel,EtBrstaining.Gelpurify17.9232and17.93.UsefreshPto

2、labelRNAPrecipitateandmarkers18.113(18mer)quantitate.and44.12(24mer).Gelpurifyandprecipitate.Adenylatep17.91x.GelpurifyApp17.91x(UVshadow–topband).Precipitateandquantitate.RuntotalRNAwithhotRNAmarkers.VisualizebyUVshadowingandphosphorimaging.Cutoutgelslicecontainingbothhotmarkers.Elute

3、andprecipitateCarryout3'EndLigationofApp17.91xwithgel-purifiedsmallRNApool.Checkligationefficiencyfromhotmarkersreaction.Gelpurifyandprecipitate.Carryout5'endligationwith17.93RacceptoroligoandT4Order17.93RfromRNALigase.CheckligationDharmaconInc.Deprotectefficiencyfromhotmarkers.andgelp

4、urify17.93R.Gelpurifyandprecipitate.Quantitate.RTwith15.22.DigestwithBanI.ConcatamerizewithT4PCRamplifywith17.92Checkdigestcompletion.DNALigase.Purifyand17.93Dfor20-25Heatinactivate,phenollongconcatamersonancycles.Phenolextractextractandprecipitate.agarosegel.Precipitate.andprecipitate

5、Pickcolonies.DoPCRManuallyinspectscreenofcolonieswithFillinproductswithchromatograms.Topovectorprimers.Analyzesequences.PurifyPCRproductsforTaq.TATOPOclone.sequencing.Submit.Pleasereference:Lau,etal.Science2001294:858-62Page1of9MicroRNAandsiRNACloningMethodBartelLabProtocolRevisedMay,2

6、002ProceduralOutline:1.OligoDesign2.ImpASynthesis3.SynthesizingOligos4.OligonucleotideAdenylation5.SmallRNAPurification6.3'AdaptorLigation7.5'AdaptorLigation8.RT-PCR9.Concatamerization10.CloningintoTopovector11.ScreenandSequence·OligosDesignOligosforRNACloning[UnderlineisBanIsite(G¯GYR

7、CC)]3'EndDonorOligo(requires5'Phosphorylationand3'Modification)17.91:pCTGTAGGCACCATCAAx(x:DMT-O-C3–CPG)5'EndAcceptorOligo17.93R:ATCGTaggcacctgaaa(RNA/DNAversion,lowercaseRNA)3'RTPrimerOligo15.22:ATTGATGGTGCCTAC5'PCRoligoo17.93D:ATCGTAGGCACCTGAAA(DNAversion,calcTm=42C)3'PCRoligoo17.92