拟南芥酵母文库构建

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1、拟南芥酵母文库构建一．材料：1．totalRNAtrizol法提取1mgRNA（两批材料）：拟南芥7天幼苗全株：250ug14天幼苗全株：250ug早期花序：已刚看见花蕾，未抽薹为准，250ug晚期花序：已开花的花序，250ug2．totalRNA1mg经promegamRNAisolation试剂盒纯化沉淀浓缩成10ul。3．BDMatchmakerLibraryconstruction试剂盒第一链合成:SynthesizeFirst-StrandcDNAusinganOligo(dT)Primer1.Combineth

2、efollowingreagentsinasterile0.25-mlmicrocentrifugetube:3ulmRNA1.0ìlCDSIIIPrimer4.0ìlTotalvolume2.Mixcontentsandspinbriefly.3.Incubateat72°Cfor2min.4.Coolonicefor2min.5.Spinbriefly.6.Addthefollowingtothereactiontube:2.0ìl5XFirst-StrandBuffer1.0ìlDTT(20mM)1.0ìldNTPM

3、ix(10mM)1.0ìlMMLVReverseTranscriptase9.0ìlTotalvolume7.Mixgentlybytapping.Spinbriefly.8.Incubateat42°Cfor10min.9.Add1.0ìlBDSMARTIIIOligonucleotide.10.Incubateat42°Cfor1hr.11.Placethetubeat75°Cfor10mintoterminatefirst-strandsynthesis.12.Coolthetubetoroomtemperature

4、,thenadd1.0ìl(2units)RNaseH.13.Incubateat37°Cfor20min.15.Anyfirst-strandreactionmixturethatisnotusedrightawayshouldbeplacedat–20°C.First-strandcDNAcanbestoredat–20°Cforuptothreemonths.4.BDMatchmakerLibraryconstruction试剂盒dscDNA扩增：AmplifydscDNAbyLongDistancePCR(LD-P

5、CR)1.PreheatthePCRthermalcyclerto95°C.2.2ìlFirst-StrandcDNA70ìlDeionizedH2O10ìl10XBDAdvantage2PCRBuffer2ìl50XdNTPMix2ìl5'PCRPrimer2ìl3'PCRPrimer10ìl10XGC-MeltSolution2ìl50XBDAdvantage2PolymeraseMix100ìlTotalvolume3.Mixgentlybyflickingthetube.Centrifugebriefly.4.Ca

6、pthetubeandplaceitinapreheated(95°C)thermalcycler.5.Beginthermalcycling.Ifyouhaveahot-lidthermalcycler,usethefollowingprogram:•95°C30sec•20cyclesa:95°C10sec68°C6minb•68°C5min.6.Whenthecyclingiscomplete,analyzea7-ìlaliquotofthePCRproductfromeachsamplealongside0.25ì

7、gofa1-kbDNAsizemarkerona1.2%agarose/EtBrgel.TypicalresultsobtainedwithHumanPlacentaPolyA+RNAareshowninAppendixA.IfyourPCRproductdoesnotappearasexpected,refertotheTroubleshootingGuide.7.ProceedwithSectionIorstoredscDNAat–20°Cuntiluse.胶电泳图：1234markerbp20001000750500

8、2501001.第一批样品dscDNA7ul上样2.第一批样品第一链cDNA1ul上样3.第二批样品dscDNA7ul上样4.第二批样品第一链cDNA1ul上样5.0.1ug2000marker5.PurifydscDNAwithaBDCHROMASPIN™TE-400Column，结合两批材料样品沉淀