| 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.
AAT Bioquest AAT Bioquest是一家位于美国的生物公司，前身为ABD Bioquest，总部位于加利福尼亚州。专门从事光学检测技术十多年，一直致力于光谱学检测领域技术的创新和突破。其独特的光学检测技术，综合了化学、生物学和信息学等各个领域的研究，引领了比色、荧光和发光技术新一代光学探针的浪潮。AAT Bioquest在全球拥有强大的经验丰富的专业分销商网络，为从小型研究机构到《财富》500强企业的各类客户提供卓越的产品和定制服务。
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试剂盒里有详细的说明书，告诉你样品需要多少量，每个试剂需要加入多少量，和详细的实验步骤，一般买来就可以用，不用人教。
所以你问一个样需要多少量是没法回答的，测定过程是要加很多种试剂的。
加入荧光标记探针，巧妙地把核算扩增、杂交、光谱分析和实时检测技术结合在一起，借助于荧光信号来检测PCR产物。一方面提高了灵敏度，另一方面还可以做到PCR每循环一次就收集一个数据，建立实时扩增曲线，准确地确定CT值，从而根据CT值确定起始DNA的拷贝数，做到真正意义上的DNA定量。另外由于CT值是一个完全客观的参数，CT值越小，模版DNA的起始拷贝数越小。因此，利用CT值确定DNA拷贝数实时PCR方法比普通终点定量方法更加准确
LOQ-定量限LOD-检测限
至少可以说同一种蛋白定量试剂盒并不适用于所有细胞
蛋白定量试剂盒有分Bradford，BCA，Lowry等，每一种对不同的细胞都有独特的适用范围和灵敏度。所以需要多种蛋白定量试剂盒对比检测才能让结果准确
如果要是直接注射，就不知道了
现在跑tricine－sds－page电泳老是跑不好，
我刚做了bradford定量，
觉得差异与空菌对照还是挺明显的，
但是这个方法只能初略的估计，
还是不能精确定量，
做HPLC我现在还没有条件，
所以我想能不能用BCA或者lowry法先进行定量啊？
这种方法如果做出来结果的话，
能不能发个sci之类的文章啊？
希望高手们指点一下！
谢谢了
