SteveHahn

Lastupdated9/30/98

ThisDyemeasuresDNAconcentrationusingafluorescentdyewhichbindsonlydoublestrandedDNA.ThefluorescenceofthisdyeisgreatlyamplifiedwhenboundtoDNA.1.TurnontheBeacon2000instrumentandallowittowarmupfor30minto1hrbeforetakinganymeasurements.2.Togenerateastandardcurve,aliquotthefollowingamountsofDNAtoEppendorftubes:30ng,20ng,10ng,5ng,2.5ng,1ng,0.5ng.Alsoneededare2tubeswithnoDNA.Onetubewillreceivedyeandtheotherwillreceivenodye(theblank).TheDNAcanbedilutedinTE,butthemaximumvolumeaddedtotheassaytubesshouldbe5microlitersorless.3.AliquotamountsoftheunknownDNAtotubesintherangeofthestandardcurve(preferablyinthemidpointoftherange).4.Add200microlitersofPanVeraDNAassaybuffertoeachtubeandmixwell(Important:theDNAassaybuffermustbeused,H20orotherbufferswillnotwork).5.PrepareandlabelglasstubesforfluorescencemeasurementintheBeacon2000.6.Add6microlitersPanVeraDNAassaydyetoeachreactionandvortex(thedyecanbeaddedtoallthetubesandthenallsamplesread;thevaluesdonotsignificantlychangewithin10minofdyeaddition).7.Measurethetotalintensityvaluewithin~10minofaddingthedye.Useprogram#3(staticmeasurementwithsingleblankvalueat23degrees).8.Thestandardcurveshouldbelinear.DeterminetheunknownDNAconcentrationsfromthestandardcurve.

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