拟南芥酵母文库构建

拟南芥酵母文库构建一.材料：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xx，未抽薹为准，250ug晚期花序：已开花的花序，250ugtotalRNA1mg经promegamRNAisolation试剂盒纯化沉淀浓缩成10ul。BDMatchmakerLibraryconstruction试剂盒第一链合成：SynthesizeFirst-StrandcDNAusinganOligo(dT)PrimerCombinethefollowingreagentsinasterile0.25-mlmicrocentrifugetube:3ulmRNA1.0lCDSIIIPrimer4.0lTotalvolumeMixcontentsandspinbriefly.Incubateat72Cfor2min.Coolonicefor2min.Spinbriefly.Addthefollowingtothereactiontube:2.0l5XFirst-StrandBuffer1.0lDTT(20mM)1.0ldNTPMix(10mM)1.0lMMLVReverseTranscriptase9.0lTotalvolumeMixgentlybytapping.Spinbriefly.Incubateat42Cfor10min.Add1.0lBDSMARTIIIOligonucleotide.Incubateat42Cfor1hr.Placethetubeat75Cfor10mintoterminatefirst-strandsynthesis.Coolthetubetoroomtemperature,thenadd1.0l(2units)RNaseH.Incubateat37Cfor20min.15.Anyfirst-strandreactionmixturethatisnotusedrightawayshouldbeplacedat-20C.First-strandcDNAcanbestoredat20Cforuptothreemonths.BDMatchmakerLibraryconstruction试剂盒dscDNA扩增：AmplifydscDNAbyLongDistaneePCR(LD-PCR)PreheatthePCRthermalcyclerto95C.2.2lFirst-StrandcDNA70lDeionizedH2010l10XBDAdvantage2PCRBuffer2l50XdNTPMix2l5'PCRPrimer2l3'PCRPrimer10l10XGC-MeltSolution2l50XBDAdvantage2PolymeraseMix100lTotalvolumeMixgentlybyflickingthetube.Centrifugebriefly.Capthetubeandplaceitinapreheated(95C)thermalcycler.Beginthermalcycling.Ifyouhaveahot-lidthermalcycler,usethefollowingprogram:?95C30sec?20cyclesa:95C10sec68C6minb?68C5min.0.25gofa1-kbDNAsizemarkerona1.2%agarose/EtBrgel.TypicalresultsobtainedwithHumanPlacentaPolyA+RNAareshowninAppendixA.IfyourPCRproductdoesnotappearasexpected,refertotheTroubleshootingGuide.7.ProceedwithSectionIorstoredscDNAat20Cuntiluse.胶电泳图：1234markerbp20001000750500250100第一批样品dscDNA7ul上样第一批样品第一链cDNA1ul上样第二批样品dscDNA7ul上样第二批样品第一链cDNA1ul上样5.0.1ug2000markerPurifydscDNAwithaBDCHROMASPIN?TE)0Column,结合两批材料样品沉淀浓缩成40ul。二.Constructing&ScreeningaTwo-HybridLibraryProtocolA构建：1.TransformyeaststrainAH109withdscDNAandpGADT7-Rec.?20ldscDNA(fromSectionIX」，Step16)?6lpGADT7-Rec(0.5g/I)?20lHerringTestesCarrierDNA,denatured**Transfer~50lofHerringDNAtoamicrocentrifugetubeandheatat100Cfor5min.Then,immediatelychilltheDNAbyplacingthetubeinanicebath.RepeatoncemorebeforeaddingtheDNAtothe15-mlreactiontube.A.Gentlymixbyvortexing.Add2.5mlPEG/LiAcSolution.Gentlymixbyvortexing.Incubateat30Cfor45min.Mixcellsevery15min.Add160lDMSO,mix,andthenplacethetubeina42Cwaterbathfor20min.Mixcellsevery10min.Centrifugeat700xgfor5min.Discardthesupernatantandresuspendin3mlofYPDPlusLiquidMedium.Incubateat30Cwithshakingfor90min.Centrifugeat700xgfor5min.Discardthesupernatantandresuspendin30mlofNaClSolution(0.9%).2.SelecttransformantsonSD—_euplates.Spread200loneach150-mmplate(150platestotal).Note:Tocheckthetransformationefficiency,spread100lofa1:10,1:100,1:1,000,and1:10,000dilutionon100-mmSD/-_euplates.Incubateplatesupsidedownat30Cuntilcoloniesappear(~3-6days).Calculatethetransformationefficiency.results:1:10,616个1:100,66个1:1,000,10个1:10,0001个》2.08x106transformants/3gpGADT7-RecHarvest(pool)transformants.Chillplatesat4Cfor3-4hr.Add10mlFreezingMediumtoeachplate.Usesterileglassbeadsandgentleswirlingtodislodgethecellsintotheliquid.Combineallliquidsinasterileflask.Mixwell.Checkthecelldensityusingahemacytometer.thecelldensity2x107cells/ml,。Aliquot(1-ml)andstoreat-80C.Todeterminethelibrarytiter,spread100lofa1:100,1:1,000,and1:10,000dilutionon100-mmSD/-Leuplates.Incubateat30Cuntilcoloniesappear(~2-3days).Countthecolonies(cfu)andcalculatethenumberofclonesinyourlibrary.Colonies:11.46x107个/ML,PCRColonyScreening:findthatitperformswellinyeastcellsamples,andbecauseitisoptimizedforapplicationsthatinvoIveIongertemplatesandrequirehighfidelity.1.PreheataPCRthermalcyclerto94C.TABLEXIII:ASSEMBLINGMASTERMIXESFORPCRCOLONYSCREENINGPOLYMERASEMIX(Mg2+includedinthebuffer)PCR-gradedeionizedH2O:451l10XAdvantage2PCRBuffer55l5'LDAmplimer(20M:11l3'LDAmplimer(20M):11l50XdNTPMix(10mMea.)11l50XAdvantage2PolymeraseMix11lTotal:550IIUsingasterilepipettetip,scrapeafewcellsfromacolonythatyouwishtoanalyze.Placethecellsinthebottomofaclean250-lPCRtube.Add24lofMasterMixtothetube.Gentlypipetteupanddowntodispersethecells.Capthetubeandplaceitinapreheatedthermalcycler.Beginthermalcycling,usethefollowingprogram:?94C3min?2530cycles:94C30sec68C3min?68C3min?Soakat15CAnalyzethePCRproductbpMarker123456789101112131415161718192021222320001000750500100