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LabelAmino-ModifiedOligonucleotideswithTideFluor™Dyes

Thefollowingprotocolhasbeenoptimizedforlabeling200µg(~6A260nmunits)ofaproprietaryoligonucleotide.Youneedmodifytheprotocoltogetthebestresultsforyourparticularapplicationbymultipleexperimentations.YOURAMINO-MODIFIEDOLIGOMUSTBETREATEDTOREMOVEAMMONIATHATRAPIDLYREACTSANDCONSUMESDYESUCCINIMIDYLESTERS.

1.      PrepareOligoSolution(SolutionA)

a.      Dissolveyouramino-modifiedoligo(~200µg)inatetraboratebuffer(100µL,pH8.5±0.5).

b.      Note1:Theoligonucleotidemustbesynthesizedwithanaminegrouponthe5’end.SeeAppenxidx1forthepurificationofamino-modifiedoligos.

c.      Note2:Avoidbuffersthatcontainprimaryamines,suchasTris,asthesecompeteforconjugationwiththeamine-reactivecompound.

2.      PrepareDyeSolution(SolutionB)

a.      Dissolve1mgdyeSEin100µLDMSO(>10mg/mLifpossible)bypipettingupanddown.Centrifugethesolutionstockonthesidesofthevialtothevialbottom.

b.     Note:preparetheDMSOdyesolutionbeforestartingtheconjugation.Extendedstorageofthedyesolutionmayreducethedyeactivity.Anysolutionscontainingthedyeshouldbekeptfromlight.WedonotrecommendthatyoustoretheDMSOdyesolutionforfutureuse.

3.      RunConjugationReaction

a.      Tothedyesolution(B,20-50µL)addtheoligosolution(A,100µL)withstirringorshaking(keepingthereactionmixturefromlight).

b.     Rotateorshakethereactionmixturefor4-6hoursatroomtemperatureonarotatororshaker.

c.      Note:Gentlyvortextapthevialevery10minutesforthefirsthourtoensurethatthereactionsolutionremainswellmixed.Donotmixviolently,asmaterialmaybeleftonthesidesofthevial.Aftersixhours,50–90%oftheamine-modifiedoligonucleotidemoleculesshouldbelabeled.Thereactionmightbeincubatedovernightifitismoreconvenient.However,overnightincubationwillnotresultinagreaterlabelingefficiencyinmostcases.

4.      PurifyDye-OligoConjugate

a.      Preliminarypurificationbyethanolprecipitationoflabeledoligonucleotide

                                                              i.     Add20µL(one-tenthreactionsolutionvolumeingeneral)of3MNaCland300µLcoldabsoluteethanol(twoandhalfreactionsolutionvolumevolumesingeneral)tothereactionvial.

                                                            ii.     Mixthesolutionwellandplaceitat–20°Cfor30minutes.

                                                           iii.     Centrifugethesolutioninamicrocentrifugeat10,000to15,000×gfor30minutes.

                                                          iv.     Note:Lossofsamplemayoccurifthecentrifugationisnotlongenough.

                                                            v.     Carefullyremovethesupernatant,rinsethepellet1-3timeswithcold70%ethanolanddrybriefly.

                                                          vi.     Note:Someunreactedlabelingreagentmayhaveprecipitatedoverthecourseofthereactionormaybestuckonthewallsofthereactionvial.Thismaterialshouldbecompletelyredissolvedbyextensivevortexmixingbeforecentrifugation.Redissolvingthelabelingreagentensuresthattheprecipitatedoligonucleotidewillbeminimallycontaminatedwithunreactedlabel.

b.      FinalPurificationbyHPLCorbygelelectrophoresis

                                                              i.      SeeAppendixI

LabelPeptideswithTideFluor™Dyes

Thefollowingprotocolhasbeenoptimizedforlabeling10mgofaproprietarypeptide(MW~2000)thatcontainsonlyasinglefreeaminogroup.YOUNEEDMODIFYTHEPROTOCOLTOARCHIETHEBESTRESULTSFORYOURPARTICULARAPPLICATIONBYMULTIPLEEXPERIMENTATIONS.

1.      PreparePeptideSolution(SolutionA)

a.      Dissolveyourpeptide(~10mg)inDMF(~1ml).

b.      Note1:Thepeptidemustbeneutralizedwithabasesuchastriethylamineorpotassiumcarbonate.

c.      Note2:Avoidbuffersthatcontainprimaryamines,suchasTris,asthesecompeteforconjugationwiththeamine-reactivecompound.

2.      PrepareDyeSolution(SolutionB)

a.      Dissolve5mgdyeSEin500µLDMF(>10mg/mLifpossible)bypipettingupanddown.

b.     Note:preparetheDMFdyesolutionbeforestartingtheconjugation.Extendedstorageofthedyesolutionmayreducethedyeactivity.Anysolutionscontainingthedyeshouldbekeptfromlight.WedonotrecommendthatyoustoretheDMFdyesolutionforfutureuse.

3.      RunConjugationReaction

a.      Tothedyesolution(B,500µL)addthepeptidesolution(A,1mL)withstirringorshaking(keepingthereactionmixturefromlight).

b.     Stirthereactionmixturefor4-6hoursatroomtemperature.

4.      PurifyDye-PeptideConjugate

a.      ThereactionsolutionwasconcentratedandpurifiedonaC18columntoaffordthedesiredconjugate.ThefractionswereanalyzedbyHPLC,andthefractionsof>97%puritywerepooledandlyophilized.

b.     Note1:HPLCPurificationConditions:TEABbuffer(triethylammoniumbicarbonate,0.25mmol,pH=7.0-8.0)wasusedasbufferAandacetonitrileasbufferB.TheHPLCwasrunfrom0%Bto30%Bin60min(flowrate:100mL/min).

c.      Note2:Avoidstronglightduringtheoperation.

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