RNAi-Feeding-Protocol

AHRINGERLABCloneinformationandRNAiFeedingProtocolVectorandinserts:GenomicfragmentsobtainedbyPCRwereclonedintotheTimmonsandFirefeedingvector(L4440),whichisamodifiedversionofBluescriptwithaT7promoteroneachsideoftheMCSdrivingtranscriptionofeachDNAstrand(Nature,395,854).InformationabouttheL4440vector(includingsequenceinformation)canbefoundat~kimlab/primers.12-22-99.htmlandaredisplayedvisuallyinWormBase().ClonesareavailablefromMRCgeneservice().Bacteria:GenomicfragmentsclonedintoL4440weretransformedintoHT115(DE3),anRNaseIII-deficientE.colistrainwithIPTG-inducibleT7polymeraseactivity(Gene,263,103-112).ThestrainisavailablefromtheCaenorhabditisGeneticsCenter().Thegenotypeisasfollows:F-,mcrA,mcrB,IN(rrnD-rrnE)1,lambda-,rnc14::Tn10(DE3lysogen:lavUV5promoter–T7polymerase)(IPTG-inducibleT7polymerase)(RNaseIIIminus).ThisstraingrowsonLBor2xYTplates(andisresistanttotetracycline,seebelow),andcompetentcellscanbemadeusingstandardtechniques.Noteontetracycline:TheTn10transposoninterruptingthernc14genecarriesatetracyclineresistancegene.Therefore,bacteriashouldbesubjectedtotetracyclineselection(12.5g/ml)tomaintaintheRNasedeficiency.However,thetransposonisquitestable,aswehavenotlostitintheabsenceofselection.Usingourprotocol(seebelowandKamathetal.GenomeBiology,2,1-10)inclusionoftetracyclineduringfeedingsignificantlydecreasedtheRNAieffectforseveralgenestested,sowerecommendthatitnotbeusedincultureorinNGMplatesduringfeedingusingthemethodbelow.However,usingthemethodofTimmons,etal(Gene,263,103-112),animprovementinfeedingresultsbyincludingtetracyclinewasreported.NGMMedia:Forfeedingplates,usestandardNGMagarplusthefollowingingredients:Carbenicillinto25g/mlfinalconcentrationIPTGto1mMfinalconcentrationPlatesarepoured4-7daysbeforeseeding.FeedingProtocol(fromKamathetal(2001)GenomeBiology,2,1-10):1.Pickandgrowbacteria6-12hoursinLB+50g/mlampicillin(untilafairlydensecultureisobtained),thenseeddropwiseontoNGMagarplatesincludingadditivesabove.Usequitealotofbacteriatoinoculatetheculture.(DonotaddIPTGortetracyclinetotheliquidculture,asthiswillreducetheRNAieffect.).2.Letdryandinduceovernightatroomtemperature.3.Thefollowingday,transferL3-L4-stagehermaphroditesontofirstplate,minimizingtheamountofOP50bacteriatransferred(weusuallywashwormsinM9bufferandthenaliquotthemdirectlyontoplates).Leave72hoursat15oC(or36-40hoursat22oC)forRNAitotakeeffect,thenreplicaplatesingleadultsontoanotherplateseededwiththesamebacteria.After24hours,removetheadultfromthereplicaandscoretheprogenyforphenotypes.Alternatively,aliquotembryosorlarvaeontothefeedingplatesandscorethemlater.4.Noteontemperature:Wehaveobservedthatsomegenesgivedifferentphenotypesat15oCvs22oC;thus,itmaybeworthwhiletotestagivengeneusingbothconditions.Ifyouhaveanyquestions,pleasecontactJulieAhringer([e-mail]).