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EMS Mutagenesis
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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.

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