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1、DoublecolorISHprotocolformedakafish使用DNA或者RNA针來探檢测号其互补的另一条磁在細菌或其他真垓细胞中的位?LReagents吐沿Forfixation:lxPBS(DEPCtreated,orautoclavedlhr),pH7.5,addTween20to0.1%andsterilefilter(0.2pm,nitrocellulose).4%PFA:dissolve4%paraformaldehydeinPBS(DEPCtreated)bystirringandheatingto65°C,adddropwise1MNaO
2、Huntilthesolutiongetsclear(checkpH=7.5,important!!!),cooltoroomtemperatureandstoreat4°C(forupto4weeks,or-20°Cformonths(Forembryos:after2daysfixation,storedin50%Formalmide/PBS-20°Ctilldechroined)ProceduresSamplepreparation1)Freshtissues(2mmthickslices)aretakenout,punctuatewithneedleort
3、akeoffthemembrane,2)fixin4%PFA(DEPCPBS,pH7.5)overnight3)25%methanol10-20min4)50%methanol10-20min5)75%methanol10-20min6)100%methanol120min或storeat・20度forseveralmonths7)75%methanol10-20min8)50%methanol10-20min9)25%methanol10-20min10)PBS2X10min11)30%sucrose(DEPC-treatedPBS)overnight12)OC
4、Tover120min13)4-5umfortestis,8-10umforovary14)incubateat37度4horovernight15)storeat・80度1.RNAprobepreparationl)linearize10jagoftemplate(0.5・lkbbetter)withasuitableenzymeallowingastranscription(bluntor5-primeoverhangshouldbepreferredtoavoidsnapbackeffects,Xhol,BamHl,Notl,EcoRlcanbeused,S
5、acIIandsaclshouldbeavoided)2)Controlforacompletedigestonanagarosegel.3)Purifyingtemplatefromenzymeanddigestionbufferbyphenol(苯酚):chloroform(氯仿)extraction,2-propanolprecipitation(异丙醇沉淀),dissolvedinDEPCtreatedH2O.2)Addinthefollowingordertoatotalvolumeof20pl(10glisOkandtemplatecanbedoubl
6、ed,whichcanproducemoreprobe!IfusePCRproducts,200ngisok)linearizedtemplate(PCRproducts)1pg(lpl)10mMNTPwithDig-UTP/Fluorescein-UTP2pl1OxTranscriptionbuffer2pIDEPCH2Oad13plRNA-Polymerase2pl(EnzymeMix:Buffered50%glycerolcontainingRNApolymerase,RNaseInhibitor,andothercomponents)NOTE：(1)模板和
7、Transcriptionbuffer先混匀后再加RNA-Polymerase(2)RNA-Polymerase中己经含有T7和Rnasin,所以不需再加Rnasin3)Incubatefor2hrsat37°Candcheckbyelectrophesis(电泳可不做)(If10ulthenadd10ulDEPCH2Oandlulforchecking)4)AddlulmRNAtranscriptionbuffer(ornot)andlulTurboRNase-freeDNase,incubateat37°Cfor15minutes5)Purifytheprob
8、ebyLi
