DAPI说明书.pdf

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1、DAPI4',6-Diamidine-2-phenylindoledihydrochlorideProductNo.A1001DescriptionFormula:C16H15N52HClMolecularweight:350.25g/molCAS-No.:[28718-90-3]Assay(HPLC):min.98%Meltingpoint:approx.330°CChloride:min.18.2%Nitrogen:min.18.0%UV-Spectrum:max.223nm,261nmand342nmmin.246nmand282nmStorage:+4°C,prot

2、ectedfromlightrecommendedstocksolution:1-5mg/mlinwater(insolubleinPBS!)recommendedworkingsolution:0.1µg/ml-200µg/ml(dilutedinmethanol)CommentDAPIisanexcellentdyeforthestainingofDNA.Originally,onlythespecificbindingtoAT-basepairswithoutanintercalationwasknown(2),butlateron,theintercalationinto

3、GC-basepairswasshown(3).ThemostpopularapplicationofDAPIisitsuseasareagenttodetectmycoplasmaorvirusDNA(e.g.vacciniainfectionor'unwanted'viralcontaminationofcellculturecells)inthecellculture.AppliChemrecommendsthefollowingstainingprocedure:Growcellsonacoverslipinacellculturedishtoreachapprox.70

4、%confluence.Pouroffthemediumfromthecells.Washthecoversliponcewith1µg/mlDAPIinmethanol.Incubatethecellsonthecoverslipat37°Cfor15minutesin1µgDAPI/mlinmethanol.Pouroffthestainingsolutionandwashthecoversliponcewithmethanol.Putitup-side-downonaslidewithPBSorglycerolasmountingmedium.Donotusewater.E

5、xaminethecellsunderamicroscope(excitation:365nm;emissionmaximumat450nm).ProlongedincubationwithDAPIincreasesthenuclearfluorescence,shorterincubationtimeleadstoaweakernuclearstaining,whichfacilitatestheexaminationofthecytoplasmicfluorescence.Solubility/StabilityDissolveDAPIindouble-distilledwa

6、tertoafinalconcentrationof1-5mg.Themaximumsolubilityinwaterisapprox.25mg/ml.DAPIisinsolubleinPBS.Donotuseanybuffers.Dilutethestocksolutionwithmethanoltoafinalconcentrationof1µg/ml.Solutionsarestableatroomtemperaturefor1-2weeks(4),at+4°Cupto6monthsandfrozenbetween6and12months(1mlaliquots).Ifth

7、esolutionbecomesturbid,DAPIishydrolyzed.DAPIbleachesquicklyincontactwithlight,evenifitisquitestableagainstUV-light.Incubateyoursamplesinthedark.Ifyoursamplesarestoredat+4°Cforoneday,fluorescenceisstabilzed.1ApplicationandLiterature(1)Asimplec