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AAT Bioquest/Helixyte™ Green Fluorimetric dsDNA Quantitation Kit *Optimized for Microplate Readers*/17650/200 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 501/520 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | NucleicAcidDetection DNADetection |
| Related | LabelingCells FluorescenceImaging BiochemicalAssays |
1.PreparingHelixyteGreen™workingsolution
1.1 PrepareHelixyteGreen™workingsolutionbyadding50µLofHelixyteGreen™(ComponentA)into10mLofAssayBuffer(ComponentB). Protecttheworkingsolutionfromlightbycoveringitwithfoilorplacingitinthedark.
Note1:Werecommendpreparingthissolutioninaplasticcontainerratherthanglass,asthedyemayadsorbtoglasssurfaces.
Note2:Forbestresults,thissolutionshouldbeusedwithinafewhoursofitspreparation.
2.PrepareserialdilutionsofdsDNAstandard(0to10µg/mL):
2.1 Add10μLof100μg/mLdsDNAstocksolution(ComponentC)to190µLofAssaybuffer(ComponentB)tohave5μg/mLdsDNAsolution,andthenperform1:3serialdilutionstoget1667,556,185,61.7,20.6,6.85,2.3,and0ng/mL.
2.2 AdddsDNAstandardsandDNAcontainingtestsamplesintoa96-wellsolidblackmicroplateasdescribedinTables1and2.
Table1.LayoutofdsDNAstandardsandtestsamplesinasolidblack96-wellmicroplate*
BL | BL | TS | TS | …. | …. |
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DS1 | DS1 | …. | …. | …. | …. |
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DS2 | DS2 |
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DS3 | DS3 |
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DS4 | DS4 |
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DS5 | DS5 |
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DS6 | DS6 |
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DS7 | DS7 |
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*Note:DS=dsDNAStandards;BL=BlankControl;TS=TestSamples
Table2.Reagentcompositionforeachwell*
dsDNAStandard | BlankControl | TestSample |
Serialdilutions*(100μL) | TE:100μL | 100μL |
*Note:AddtheseriallydilutionsofdsDNAstandardsfrom2.3to1667ng/mLintowellsfromDS1toDS7induplicate.
3.RundsDNAassay:
3.1 Add100μLofHelixyteGreen™workingsolution(fromStep1.1)toeachwellofthedsDNAstandard,blankcontrol,andtestsamples(seeStep2.2)tomakethetotaldsDNAassayvolumeof200µL/well.
Note:Fora384-wellplate,add25μLsampleand25μLofHelixyteGreen™workingsolutionperwell.
3.2 Incubatethereactionatroomtemperaturefor5to10minutes,protectedfromlight.
3.3 MonitorthefluorescenceincreasewithafluorescencemicroplatereaderatEx/Em=490/525nm(cutoffat515nm).
3.4 Thefluorescenceinblankwells(withtheTEbufferonly)isusedasacontrol,andissubtractedfromthevaluesforthosewellswiththedsDNAreactions.TheDNAconcentrationofthesamplearedeterminedfromthestandardcurvegeneratedinDNAStandardCurve.
| References&Citations |  PrinterFriendlyVersion |
1. PetersonEJ,KireevD,MoonAF,MidonM,JanzenWP,PingoudA,PedersenLC,SingletonSF.(2013)InhibitorsofStreptococcuspneumoniaesurfaceendonucleaseEndAdiscoveredbyhigh-throughputscreeningusingaPicoGreenfluorescenceassay.JBiomolScreen,18,247.
2. ChenY,SonnaertM,RobertsSJ,LuytenFP,SchrootenJ.(2012)ValidationofaPicoGreen-basedDNAquantificationintegratedinanRNAextractionmethodfortwo-dimensionalandthree-dimensionalcellcultures.TissueEngPartCMethods,18,444.
3. MorenoLA,CoxKL.(2010)QuantificationofdsDNAusingtheHitachiF-7000FluorescenceSpectrophotometerandPicoGreendye.JVisExp.
4. DraganAI,Casas-FinetJR,BishopES,StrouseRJ,SchenermanMA,GeddesCD.(2010)CharacterizationofPicoGreeninteractionwithdsDNAandtheoriginofitsfluorescenceenhancementuponbinding.BiophysJ,99,3010.
5. DraganAI,BishopES,Casas-FinetJR,StrouseRJ,SchenermanMA,GeddesCD.(2010)Metal-enhancedPicoGreenfluorescence:applicationtofastandultra-sensitivepg/mlDNAquantitation.JImmunolMethods,362,95.
6. ABIodunOO,GbotoshoGO,AjaiyeobaEO,HappiCT,HoferS,WittlinS,SowunmiA,BrunR,OduolaAM.(2010)ComparisonofSYBRGreenI-,PicoGreen-,and[3H]-hypoxanthine-basedassaysforinvitroantimalarialscreeningofplantsfromNigerianethnomedicine.ParasitolRes,106,933.
7. DraganAI,BishopES,Casas-FinetJR,StrouseRJ,SchenermanMA,GeddesCD.(2010)Metal-enhancedPicoGreenfluorescence:applicationfordouble-strandedDNAquantification.AnalBiochem,396,8.
8. HoldenMJ,HaynesRJ,RabbSA,SatijaN,YangK,BlasicJR,Jr.(2009)FactorsaffectingquantificationoftotalDNAbyUVspectroscopyandPicoGreenfluorescence.JAgricFoodChem,57,7221.
9. IkedaY,IwakiriS,YoshimoriT.(2009)DevelopmentandcharacterizationofanovelhostcellDNAassayusingultra-sensitivefluorescentnucleicacidstain"PicoGreen".JPharmBiomedAnal,49,997.
10. RenX,XuQH.(2009)Label-freeDNAsequencedetectionwithenhancedsensitivityandselectivityusingcationicconjugatedpolymersandPicoGreen.Langmuir,25,43.
