EMSMutagenesis WARNING: EMSispowefulmutagenandasUSPectedcarcinogen.Wearglovesandworkinfumehood.Usedisposableplasticwareandinactivatemutagenbeforedisposalasoutlinedbelow. 1.GrowseveralplatesofwormsuntilmostanimalsareintheL4stage. 2.WashwormsoffplateinM9intoaplastic15mlconicaltube. 3.Spinthewormsinclinicalcentrifugeupto1700rpm.Donotusebrake.Aspiratesupernatant.Thewormpelletmaybeveryloosesobecarefulnottodisturbit. 4.AddM9tothewormpellet,inverttubeseveraltimes,andrepeatstep3. Resuspendwormpelletin2mlsofM9. 5.Inthehood,makeasolutionof0.1MEMS(ethylmetanesulfonate,alsoknownasmethanesulfonicacidethylester)byadding20ulofliquidEMSto2mlofM9bufferinaplastic50mlconicaltube.Gentlyagitateuntilthedenseoilyliquidhasdissolved. 6.Add2mlsofthesuspensionofwashedwormstothe2mlofEMSsolution(finalconcentration0.05MEMS).Incubate4hoursat20oC.Agitateoccasionallytoincreaseaeration.AftermutagenesiswashwormsfourtimeswithM9buffer.DistributewormstoNGMplates. 7.InactivateEMSsolutionsbymixingthemwithequalvolumeof"inactivatingsolution"[0.1MNaOH,20%w/vNa2S2O3(SodiumThiosulfate)]for24hours.Pipets/tubescontaminatedwithEMSshouldbesoakedininactivatingsolutionfor24hourspriortodisposal.