分子细胞遗传学常用实验技术(精)

分子细胞遗传学常用技术分子细胞遗传学常用技术分子细胞遗传学常用技术分子细胞遗传学常用技术20072007年8月#TOC\o"1-5"\h\zEDTA10mMKCl100mMSucrose0.5MSpermidine(亚精胺)4.0mMSpermine(精胺)I.OmMMercaptoethanol0.1%YoupreparealargestockandstoreditinarefrigeratorFilterthroughnylonmesh:170,125,48and22口msequentially,intocold(onice)50mlcentrifugetubes(Nylonfiltersobtainedfrom:TetkoInc,P.OBox346,Lancaster,NY,14086,tele914-941-7767)usingcooledfunnel(youmayskipthe22pmfilterbecausethisstepwilllooselotsofnucleibutmakethenucleicleaner).Add1mlNIBcontaining10%(V/V)TritonX-100andgentlymixthefiltrate.Centrifugeat2000xgfor10minat4°C.Decantthesupernatantandresuspendthepelletin200to1mlNIBdependingontheamountofnucleiharvested(concentration〜5x106iuclei/ml,canbecheckedbystainingwithDAPIandexaminingunderamicroscope).mix1:1with100%glycerolandstoreat-20°C.ExtensionofDNAfibersExtendingthefibersisacriticalstepinFiber-FISH.Thereareseveralmethodsofextendingthefibers.Wehavechosenthemethodofdraggingwithacoverslipasitseemstogivethemostuniformresults.Whendraggingitisimperativethatitbedoneslowlyandsmoothly.Wealsousepoly-L-lysineslidesobtainedfromSigma.TheseslidesaretreatedsoastopromotetheadhesionofoneorbothendsoftheDNAmolecule.Silinatedslidescanalsobeusedbutseemtogeneratetoomuchbackgroundsignal.Also,thecalibrationsofthemethodshouldbecheckedoccasionallybyusingBACsorcosmidsofaknownlength.Ourcalibrationis2.87kb/pm.Note:Identifythenucleiportioninthenucleistock.Thenucleitendtosettleneartheverybottomofthetubeandinmyexperiencethesettlingprocesscantakeadayorlonger.Theverybottomofthetubemayappearverywhiteandthenucleioftentimessitrightabovethisbottomfilm.Thecolorofthenucleiisvariableacrossspeciesandsamples,butverycleannucleiisnormallyagray/whitecoloration.Layersabovethenucleitendtocontaindebrisandanythingfloatingaboveinthestoragesolutionisdebris.Somepeopleliketomixthenucleistockpriortoslidepreparationbygentlyinvertingtheeppendorftubeseveraltimes.Thisisnormallyavoidedasitmixesthedebriswiththenuclei.Mixthenucleusstockbygentlyinvertingtheeppendorftubeseveraltimes.Centrifuge10-10plnucleisuspension(1〜5pl/slidedependingontheconcertration)at3000〜3600rpmfor5min.Pipettheglycerolout.ResuspendthenucleiinPBS(thefinalvolumeis2plperslide).PBS:ComponentVolumePBS:ComponentVolume10mM10mM140mMSodiumphosphatepH7.0NaClPipet2口1suspensionacrossoneendofacleanpoly-L-lysineslides(Sigma,Poly-Prep,Cat#P0425)andairdryfor5〜10min.Pipet10~13口ISTElysisbufferontopofthenucleiandincubatefor4min.STE:ComponentVolumeSTE:ComponentVolumeSDS0.5%EDTA5mMTrispH7.0100mMSlowlydragthesolutiondowntheslidewiththeedgeofacleancoverslipheldjustabovethesurfaceoftheslide.Airdryfor10min.Fixinfresh3:1100%EtOH:glacialaceticacidfor2min.Bakeat60°Cfor30min.SlidescanbeuseddirectlyinFISHwithoutfurtherpreparation,orstoredinabox.Probeapplication:ProbeispreparedasdescribedinJiang(1996)and10mlisappliedtotheslide,coveredwitha22x22mmcoverslipandsealedwithrubbercement.Afterthecementdriestheslideisplacedinan80°Covenfor3minindirectcontactwithaheatedsurface,thenfor2mininawetchamberpre-warmedinthe80°Coven.Thewetchamber,withtheslides,isimmediatelytransferredto37°Cchamberovernight.Fordifficultprobes,thehybridizationtimecanbeseveraldaysinthe37°Cwetchamber.ThreelayerdetectionThreelayerdetectiongivesmuchstrongersignalthandoesthesinglelayermethodweuseformetaphasepreparations.Also,theblockingstepusing4Mbufferseemstohelpreducesomeofthebackgroundnoise.DrybovinemilkfromSigmaworksthebestinthe4Mbuffer,othersubstitutes(i.e,Carnationdrymilk)tendtoreducetheamountofsignal.The4Mshouldbepreparedatapplicationstrengthandstoredin-20°Cfreezer.TheTNBbuffercanbepreparedat5Xandstoredat-20°Caswell.Thewashsolutions,TNT,canbepreparedat20Xand10X,respectively,andstoredatroomtemperature.Immergein2XSSC,2〜5minatRT.Doffthecoverslip.ComponentTime(total)2.washin2XSSCatRT5minwashin2XSSCat42°C10minwashin2XSSCatRT5min3.washin1XTNTatRT5min4.FITC-AvidininTNB30min(45minisOK)washthreetimesin1XTNTatRT5(15)min5.Biotinanti-Avidin&mouseanti-diginTNB30minwashthreetimesin1XTNTatRT5(15)min6.FITC-Avidin&diganti-mouseinTNB30minwashthreetimesin1XTNTatRT5(15)min7.Rodamineanti-diginTNB30minwashthreetimesin1XTNTatRT5(15)min

notes:1.All30minincubationsperiodsareat37°C.antibodies:Volume1口1per100口Volume1口1per100口1buffer0.5口Iper100口1buffer0.5口Iper100pIbuffer1plper100^1buffer1〜2plper100^1bufferFITC-Avidin,Biotinanti-Avidinmouseanti-digdiganti-mouseRodamineanti-digsolution:4M:3to5%nonfatdrymiIk[Sigma,Cat#M7409]in4xSSCTNB:0.1MTris-HClpH7.5,0.15MNaCl,0.5%blockingreagent(BoehringerMannheim)TNT:0.1MTris-HCl,0.15MNaCl,0.05%Tween20,pH7.5PBS:0.13MNaCl,0.007MNa2HPO4,0.003MNaH2PO4BACFiber-FISHPriortopreparingslides:1Labelappropriateprobeswithbiotinanddig(seeProbeLabellingprotocol).Mini-prepBACDNA(Alkalinelysismethod;use20Uwaterforresuspension).Silanize22x22coverslipsbydippinginSigmacotefor10min,thenairdry.Slidepreparation:Preparewet-chamberat37°C,turnslidewarmerupto60°C.DiluteBACDNA(w/cutP20pipettips)toappropriatelevel(Weliketodilute1UlBACinto9Ulwater).Addall10UlofdilutedBACtoPoly-Prepslide(Sigma#P0425).Add15UlofFISHlysisbuffer*toBACdrop.Allowdroptospread.Letthissitatroomtemperaturefor~5min.Addwatertotheslideifitdries.Gentlyplace(“drop”)asilanizedcoverslipdirectlyovertheliquid(lowertheslidewithforceptstoavoidbubbles).Transferslidestoslidewarmer.Allowslidesto“bake”for15min.Atthispointoneshouldseetheliquidbegintorecede.Placeslidesin3:1(EtOH:GlacialAceticAcid),wait1min.Gentlyshakeslidetopromoteremovalofthecoverslip.Oncecoverslipfallsoff,transferslidestonewcontainerof3:1andincubatefor1:30.Transferslidesbacktoslidewarmerforanadditional15min.Addprobe,denature,anddetectasinNuclearFiber-FISHprotocol.FISHlysisbuffer:2%Sarkosyl0.25%SDS50mMTris(pH7.4)50mMEDTA(pH8.0)DEPC-trsatedwater6.55Xtailincibiiff