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1、RapidExtractionofHighQualityDNAfromWholeBloodStoredat4ºCforLongPeriodMaterialsandMethodsStandardchemicalsThismethodusesstandardchemicalsthatcanbeobtainedfromanymajorsupplier;weusedchemicalssuppliedbySigmaCo.asfollow:·EDTA(0.5M),pH8.0:Add186.1grofanhydrous
2、EDTAto800mlofdistilledwater.AdjustpHto8.0withNaOHpellets.Makeupto1literwithdistilledwater.Autoclaveat15p.s.i.for15min.·1MTris-HC1,pH7.6:Dissolve121.1grofTrisbasein800mlofdistilledwater.AdjustpHwithconcentratedHCl.Allowmixturetocooltoroomtemperaturebeforef
3、inallycorrectingpH.Makeupto1literwithdistilledwater.Autoclaveat15p.s.i.for15min.·PreparationofRedbloodcelllysisbuffer:0.01MTris-HClpH7.6,320mMsucrose,5mMMgC12,1%TritonX100.Add10mlof1MTris,109.54grofsucrose,1.01grMgC12,adjustpHto8.0andfinallyadd10mlofTrito
4、nX-100to800mlofdistilledwater,andmakeupto1literwithdistilledwater.Autoclaveat15p.s.i.for10min.Sugarsathightemperaturecancausecaramelization(browning),whichdegradesthesugars[5].·PreparationofNucleiclysisbuffer:0.01MTris-HC1,11.4mMsodiumcitrate,1mMEDTA,1%so
5、diumdodecylsulphate(SDS).Take10mlof1MTris-HC1(pH7.6),3.75grofanhydrousEDTA(pH8.0),10grSDS,2.94grofsodiumcitrate,andadjustpHto8.0.Makeupto1literwithdistilledwater.Autoclave15minat15p.s.i.·TEBuffer,pH8.0:Take5mlof1MTris-HCl,pH7.6,2mLof0.5MEDTA,pH8,andmakeup
6、to1literwithdistilledwater.AdjustpHto8.0andautoclave15minat15.p.s.i.·Chloroformprechilledto4°C.·Ethanol(100%)prechilledto-20°C.ProcedureofDNAExtractionBeforestartingDNAextraction,liquidbloodvenogectsshouldbeshakegentlybyrotatingbloodmixer(vortex)1.Pour500
7、µlofbloodintoa1.5mleppendorftubeandadd1000µlofredcelllysisbuffer.2.Shakemicrofugetubegently(uptohomogenizing),thenspinfor2minutesat7000rpm.3.Discardsupernatantandrepeatsteps1-3twoorthreemoretimestoremovehemoglobin.Itisimportanttobreakdownthepelletbyvortex
8、ingandrinsesitwellinredbloodcelllysisbufferinordertocleanthewhitebloodcellsfromresidualofhemoglobin.4.Placingthetubeontissuepaperforfewsecondsdownward.Becarefulfromcross-contaminationbetweendifferentsamples.5.Add400
