Inthissection,youwillfindtechniquesrelatedtooligonucleotides,suchasoligopurificationbyacrylamidegel,annealingtwooligostomakedoublestrandedDNAandvisualizationbyUVshadowingandmore...

Basics

·ResuspendingLyophilizedOligos(IDT)

·Preparationofoligonucleotideprimers(GSC)Prepareworkingprimersolutionfromcommercialsynthesizedoligos

·StorageandHandling(Genosys)Properstorageofyouroligonucleotidewillmaximizeitsshelf-life,allowingyoutogetthemostusefromyouroligo.Followingthesesimpledilutionandweight/volumeunitconversionguidelineswillenableyoutoworkeasilywithyourGenosysoligos.

AnnealingofOligos

·AnnealTwoOligostoMakeDouble-StrandedDNA(IDT)ItissometimesnecessarytomakedoublestrandedDNAfromsinglestrandedoligos.Whiletheannealingprocedureisverysimple,attentiontoafewdetailscangreatlyreducethepresenceofundesiredsinglestrandedmaterial.

·AnnealOligonucleotidestoMakeDouble-StrandedDNA(Genosys)

·ProtocolforAdapterProduction(LTI)Annealtwooligostomakeadaptor

OligoVisualization

·OligonucleotideVisualizationVisualizeoligosbyUVshadowingormethylenebluestaining

·UVShadowing(Ambion)UVshadowingisatechniqueforvisualizingnucleicacids,e.g.separatedinagel,usingshort-waveUVlight(254nm)andafluor-coatedTLCplate.ThismethodisforidentifyingnonisotopicprobesorunlabeledRNA(e.g.formicroinjection)duringgelpurificationsincetheotheralternativeofstainingwithEtBroracridineorangerequiressubsequentextractionofthedye.However,thismethodcanbeusedtovisualizeanynucleicacidpresentinsufficientamount(>0.3µg).

OligoPurification

·OligoPurificationonAcrylamideGels(PMCIResearch)

·IsolationofDNAfromAcrylamideGels(SchimentiLab)

·PAGEPurificationofOligonucleotides(LTI)ProtocolforPAGEPurificationofOligonucleotidesProtocolforPAGEPurificationofOligonucleotidesOligonucleotidescontaminatedbysignificantamountsofabortedsynthesisproducts(e.g.,n-1,...

·Oligonucleotidepurification(FrankBottoneJr.)Buanolprecipitationmethod

·ButanolPrecipitationofDeprotectedOligonucleotides(PMCIResearch)Crudeoligonucleotidescanbeprecipitatedafterdeprotectiondirectlyfromtheammoniasolutionwithouttheneedforaseparatesteptoremovetheammonia.Thisprocedureremovessmallmolecularweightimpuritiessuchastheammoniaitselfandthebyproductsofdeprotection(benzamide,isobutyramide).

OligoLabeling

·LabelingofOligonucleotides(PMCIResearch)Labeloligowithpolynucleotidekinase.

·Alkalinephosphataselabelingofoligos(Promega)Providesusefulguideforpreparingphosphataseconjugatedoligos

·5-EndOligoFluorescenceLabeling(AmershamPharmacia)Labelingofoligonucleotideswithasinglefluoresceinresidueatthe5end

·5endlabelingofoligonucleotideswithgamma-³²P-ATP(PetraKlaff)Labelingandpurificationmethodingreatdetail

OligoQuantitation,ConversionsandCalculations

• CommonConversionsofOligonucleotides(Fermentas)
• MeltingTemperatureofDuplexDNA&Oligonucleotides(Fermentas)

·MicrogramsofDouble-StrandedDNAtoPicomoles(Promega)

·PicomolesofDouble-StrandedDNAtoMicrogramsandNanograms(Promega)

·MicrogramsperMilliliterofOligonucleotides(Single-StrandedDNA)topicomolespermicroliter(Promega)

·PicomolesperMicroliterofOligonucleotides(Single-StrandedDNA)tomicrogramspermilliliter(Promega)

·MicrogramsofLinearDNAtoPicomolesofEnds(Promega)

·MolarRatioofInserttoVectorforLigations(Promega)

·ConversionFactorsforMetricPrefixes(Promega)

·OligonucleotideMeltingTemperatureCalculations(Promega)

·QuantitationofOligonucleotides(IDT)QuantitateyouroligobyUVSpectrophotometryorpolyacrylamidegel

·QuantitationofOligonucleotides(Genosys)UVQuantitation

·CalculationofMeltingTemperature(Genosys)

OligonucleotideCalculatorDetermineTm,MW,etc.ofyouroligonucleotide(requiresjavascriptenabled