2.TotheT+Ctubes,add15microlitersofhydrazine,mixwellandincubateatroomtemperaturefor10min.Fillthetubewithbutanol,vortexverywell(makesurethereisnotaseparatewaterandbutanolphase)andspininthemicrofugefor5min.Removebutanol,brieflyspinagainandremoveanyremainingbutanol.Dryinspeedvac.ResUSPendin150microliters1/10dilutedpiperidine.Storethesereactionsonicewhilecompletingtheothersequencingreactions.Usepiperidineinthefumehood.

3.TotheG+Atubes,add1.0microliter1MNa/HFormatepH2(useNaOHtoadjustthepHof1Mformicacid).Mixwellandincubate30minat37degrees.Add150microliters1/10dilutedpiperidineandstoreonice.

4.TotheA+Ctube,add1.0microliter30%NaOH.Incubate90degreesfor15min.ThisstepworksbestifperformedinaPCRtubeandincubatedinaPCRinstrumentwithaheatedlidtopreventevaporationduringthereaction.Afterthereactioniscomplete,transfertoa1.5mlmicrofugetube.Add150microliters1/10dilutedpiperidineandstoreonice.

5.Afteralltheabovereactionsarecomplete,incubatetubesat90degreesfor30min.Putaweightontopofthetubestopreventopening.ThisstepcleavestheDNAatthepointofmodification.Cooltubesandspinbeforeopening.

6.Add150microliters70%ethanoltotheA+Creactions.Fillalltubeswithbutanolandvortexverywelltomixphases.Spin5minutesinthemicrofugeandremovethesupernatants.Brieflyrespintoremoveallremainingbutanol.

7.Resuspendpelletsin150microlitersof1%SDS.Fillwithbutanolandprecipitateagain.Afterremovingsupernatant,brieflyspinandremoveanyremainingbutanol.DryinspeedvacandquantitaterADIoactivity.Resuspendinaconvenientvolumeofformamidesequencingdye.

ForsequencingofshortDNAs(lessthan50bp)increasethetimeofhydrazinereactionto12-15min,increasetheFormatereactionto50min,andincreasetheNaOHreactionto25min.

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