Ligatesingle-strandedadenylatedDNAorRNAoligostoRNAtargetsforavarietyofapplications.
Ligatesingle-strandedadenylatedDNAorRNAoligostoRNAtargets- UsethisenzymetoprepareCDNAlibrariesforsmall-RNAtranscriptomeanalysissuchasRNA-Seq
- PerformlinkerligationformiRNAcloningapplications
T4RNALigase2,DeletionMutant,alsoknownasT4Rnl2(1-249),isusedtoligatesingle-strandedadenylatedDNAorRNAoligonucleotidestosmallRNAsforcloningornext-generationRNAsequencing.Thepreadenylated5´endsofDNAorRNAareligatedtothe3´endsofRNA.Unlikethefull-lengthenzyme,T4Rnl2(1-249)isunabletoadenylatethe5´endofthesubstrateinthepresenceofATP.However,itcanuseapreactivateddonor(App-DNAorApp-RNA)andjoinittothe3´endofanacceptor;thus,performingtheligationreactionintheabsenceofATPwhichpreventscircularizationandotherundesirablebimolecularreactions.
Theenzymeisavailablein2,000and10,000-Unitsizesataconcentrationof200U/µl.Theenzymeissuppliedwitha10XReactionBuffer.
UnitDefinition:Oneunitistheamountofenzymerequiredtogive50%ligationofa22-merRNAtothepreadenylatedendofa17-merDNAwhenbotholigosareannealedtoacomplementary39-merDNAstrandin30minutesat37°Cunderstandardassayconditions.
StorageBuffer:50%glycerolcontaining50mMTris-HCl(pH7.5),0.1MNaCl,0.1mMEDTA,1mMdithiothreitol(DTT),and0.1%Triton®X-100.
T4RNALigase2,DeletionMutant,10XReactionBuffer:500mMTris-HCl(pH7.5),20mMMgCl2,and10mMDTT.
ActivityAssay:Theunitdefinitionassayisperformedinareactioncontaining50mMTris-HCl(pH7.5),2mMMgCl2,1mMDTTand0.4µgofequimolarmixof22-mer,17-merand39-meroligonucleotides,andvaryingamountsofenzyme.
QualityControl:T4RNALigase2,DeletionMutant,isfreeofdetectableDNAexo-andendonuclease,andRNaseactivities.
ORDERINFORMATION
T4RNALigase2,DeletionMutantisprovidedat200U/μl.
Contents:T4RNALigase2DeletionMutant,10XReactionBuffer.
