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AAT Bioquest/Sunnyvale Red™ SE *Superior 6-ROX Replacement*/394/5 mg
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 576/605 |
| MW | ~650 |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | OligoSynthesis DyeLabelingReagents |
| Related | Rhodamines Amine-ReactiveProbes |
| Spectrum | AdvancedSpectrumViewer |
Theprotocolhasbeenoptimizedforlabeling100μgofan5′-amine–modifiedoligonucleotide,15to25basesinlength.Someadjustmentstotheprotocolmaybenecessaryforgreatlyshorterorlongeroligonucleotides.Thereactionmaybescaledupordownaslongastheconcentrationofeachcomponentisnotchanged.
1. Purifytheamine-modifiedoligonucleotide(ifnecessary).Dissolvetheoligonucleotidein100μLdH2Oandextractthreetimeswithanequalvolumeofchloroform.Precipitatetheoligonucleotidebyaddingone-tenthvolume(10μL)of3MNaClandtwoandahalfvolumes(250μL)ofcoldabsoluteethanol.Mixwellandplaceat≤–20°Cfor30minutes.Centrifugethesolutioninamicrocentrifugeat~12,000gfor30minutes.Carefullyremovethesupernatant,rinsethepelletonceortwicewithcold70%ethanolanddryundervacuum.DissolvethedrypelletindH2Otoachieveafinalconcentrationof25μg/μL.Thisamine-modifiedoligonucleotidestocksolutionmaybestoredfrozenat≤–20°C.
a. [Note:Toensurethattheoligonucleotideisfreeofinterferingcompounds,especiallyamines(suchastriethylamineorTris)andammoniumsalts,westronglyrecommendextractingandprecipitatingthesamplepriortoinitiatingthelabelingreaction.]
2. Preparelabelingbuffer.Makea0.1Msodiumtetraboratebufferbydissolving0.038gofsodiumtetraboratedecahydrateforeverymLofwater.AdjustpHwithHClto8.5.
a. [Note:Thislabelingbuffershouldbemadeascloseaspossibletothetimeoflabeling.Alternatively,itmaybedividedintosmallaliquotsandfrozenimmediatelyforlong-termstorage.Exposureofthissolutiontoairforalongtimewillresultincarbondioxideabsorption,whichwillchangethepHofthebuffer.]
3. PrepareSunnyvaleRed™SEDMSOStockSolution.AllowSunnyvaleRed™SEtocometoroomtemperaturebeforeopeningthevial.Dissolve300μgofSunnyvaleRed™SEin10μLDMSObypipettingupanddown,washingthesidesofthevial.Use200μgSunnyvaleRed™SEforlabeling100μgofoligonucleotide.
a. [Note:ItisimportantthatSunnyvaleRed™SEbefreshlypreparedforeachlabelingreactionasitisnotstableinsolution.]
4. Starttheconjugation.TothevialcontainingSunnyvaleRed™SElabelingreagentinDMSO,add7μLdH2O,75μLlabelingbuffer(stepb),4μLofa25μg/μLoligonucleotidestocksolution(stepa).Placethevialonashakeroscillatingatlowspeedorgentlyvortexmixortapthevialeveryhalfhourforthefirsttwohourstoensurethatthereactionremainswellmixed.Donotmixviolently,asmaterialmaybeleftonthesidesofthevial.Aftersixhours,50–90%oftheamine-modifiedoligonucleotidemoleculesshouldbelabeled.
a. [Note:Thereactionmixturemayhaveagrainyappearance,butthisshouldnotadverselyaffecttheconjugation.Thereactionmaybescaledupordownaslongastheconcentrationofeachcomponentisnotchanged.Donotaddmoredyethanrecommended,asexcessdyewillnotimprovethelabelingefficiencyandmaymakethepurificationmoredifficult.]
5. PurifyingtheSunnyvaleRed™-LabeledOligonucleotide.Followingthereaction,thelabelingmixturecontainslabeledoligonucleotide,unlabeledoligonucleotide,andunincorporateddye.Precipitatethereactionmixturewithethanolasfollows:Addonetenthvolumeof3MNaClandtwoandahalfvolumesofcoldabsoluteethanoltothereactionvial.Mixwellandplaceat≤–20°Cfor30minutes.Centrifugethesolutioninamicrocentrifugeat~12,000×gforafull30minutes.Thelabeledoligonucleotidecanbefurtherpurifiedbypreparativegelelectrophoresisorreverse-phaseHPLC.
a. [Note:Lossofsamplemayoccurifthecentrifugationisnotlongenough.Carefullyremovethesupernatant,rinsethepelletonceortwicewithcold70%ethanolanddrybriefly.Ifthelabeledoligonucelotidebecomescompletelydry,itwillbedifficulttoredissolve.]
6. HPLCPurification:Useastandardanalytical(4.6×250mm)C8column.Dissolvethepelletfromtheethanolprecipitationin0.1MTEAA(triethylammoniumacetate).Loadthedissolvedpelletontothecolumnin0.1MTEAAandrunalinear5–65%acetonitrilegrADIentover30minutes.Thisgradientisa2%increaseinacetonitrileperminute.Theunlabeledoligonucleotidewillmigratefastest,followedbythelabeledoligonucleotideandfinallythefreedye.
7. GelEelectrophoresisPurification:Topurifythelabeledoligonucleotidebygelelectrophoresis,poura0.5mm–thickpolyacrylamideslabgel.Foroligonucleotideslessthan25basesinlength,use19%acrylamide,foroligonucleotides25–40bases,15%acrylamide,andforoligonucleotides40–100bases,12%acrylamide.ResUSPendthepelletfromethanolprecipitationin200μLof50%formamide,andincubateat55°Cfor5minutestodisruptanysecondarystructure.Loadthewarmedoligonucleotideontothegel(youmayneedtouseseveralwells)andloadanadjacentwellwith50%formamideplus0.05%bromophenolblue.Thebromophenolbluewillmigrateatapproximatelythesamerateastheoligonucleotide.Runthegeluntilthebromophenolblueindicatordyeistwo-thirdsofthewaydownthegel.RemovethegelfromtheglassplatesandplaceonSaranWrap.LaythegelonafluorescentTLCplate.LocatethelabeledandunlabeledoligonucleotidesbyIlluminationwithahandheldUVsource.Fluorophore-labeledoligonucleotideswillshowfluorescencewhenilluminatedwithUVlight.Cutoutthebandcontainingthelabeledoligonucleotideandpurifybythe“crush-and-soak”methodorothersuitablemethod.
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