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AAT Bioquest/Helixyte&trade；绿色双链DNA定量试剂*200X二甲基亚砜溶液*/17598/10毫升
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 501/520 |
| MW | 661.17 |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | NucleicAcidDetection DNADetection |
| Related | LabelingCells FluorescenceImaging BiochemicalAssays |
1.PreparingtheHelixyteGreen™workingsolution:
1.1 PrepareanaqueousworkingsolutionoftheHelixyteGreen™bymakinga200-folddilutionoftheconcentratedDMSOsolutioninTE(10mMTris-HCl,1mMEDTA,pH7.5–8.0).Forexample,add50μLHelixyteGreen™to10mLTEtoprepareenoughworkingsolutiontoassay100samplesina200µLfinalvolume.Protecttheworkingsolutionfromlightbycoveringitwithfoilorplacingitinthedark.
Note1:Werecommendpreparingthissolutioninaplasticcontainerratherthanglass,asthedyemayadsorbtoglasssurfaces.
Note2:Forbestresults,thissolutionshouldbeusedwithinafewhoursofitspreparation.
2.PrepareserialdilutionsofdsDNAstandard(0to3ng/mL):
2.1 Preparea1mg/mLstocksolutionofdsDNA(suchascalfthymusDNAfromSigma)inddH2O.
2.2 Add10μLof1mg/mLdsDNAstocksolution(fromStep2.1)to998µLTEbuffertohave10μg/mLdsDNAsolution,andthenperform1:10and1:2serialdilutionstoget1000,100,50,25,12.5,6.25,3.125,and0ng/mL.
2.3 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:AddtheseriallydilutionsofdsDNAstandardsfrom0.1to1000ng/mLintowellsfromDS1toDS7induplicate.
3.RundsDNAassay:
3.1 Add100μLofdsDNAassaymixture(fromStep1.1)toeachwellofthedsDNAstandard,blankcontrol,andtestsamples(seeStep2.3)tomakethetotaldsDNAassayvolumeof200µL/well.
Note1:Fora384-wellplate,add25μLsampleand25μLofdsDNAassaymixtureperwell.
Note2:Forcurvetbasedassays,add1mLsampleand1mLofdsDNAassaymixturepercurvet.
3.2 Incubatethereactionatroomtemperaturefor5to10minutes,protectedfromlight.
3.3 MonitorthefluorescenceincreasewithafluorescencemicroplatereaderatEx/Em=490/525nm(cutoffat515nm).
3.4 Thefluorescenceinblankwells(withtheTEbufferonly)isusedasacontrol,andissubtractedfromthevaluesforthosewellswiththedsDNAreactions.TheDNAconcentrationsofthesamplesaredeterminedfromthestandardcurvegeneratedinDNAStandardCurve.
| 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.
