Applications
Ampligase®ThermostableDNALigasecatalyzesNAD-dependentligationofadjacent3´-hydroxylatedand5´-phosphorylatedterminiinduplexDNAstructuresthatarestableathightemperatures.Derivedfromathermophilicbacterium,theenzymeisstableandactiveatmuchhighertemperaturesthanconventionalDNAligases.Itshalf-lifeis48hoursat65°Candgreaterthan1 hourat95°C.AmpligaseDNALigasehasbeenshowntobeactiveforatleast500thermalcycles(94°C/80°C)or16hoursofcycling.10Thisexceptionalthermostabilitypermitsextremelyhighhybridizationstringencyandligationspecificity.AmpligaseDNALigasehasnodetectableactivityinligatingblunt-endedDNAandhasnoactivityonRNAorRNA:DNAhybrids. UnitDefinition:OneunitofAmpligaseDNALigasecatalyzestheligationof50%ofthecossitesin1µgoflamBDaDNAin1minuteat45°Cin1XAmpligaseReactionBuffer. Note:OneunitofAmpligaseDNALigaseisequivalenttoatleast15ofthe"cohesiveendunits"or"nickligationunits"definedelsewhere.4 StorageBuffer:50%glycerolcontaining50mMTris-HCl(pH7.5),0.1MNaCl,0.1mMEDTA,1mMDTT,and0.1%Triton®X-100. Ampligase10XReactionBuffer:200mMTris-HCl(pH8.3),250mMKCl,100mMMgCl2,5mMNAD,and0.1%Triton®X-100. QualityControl:AmpligaseThermostableDNALigaseisassayedfortheabsenceofblunt-endligationactivitybyligatingSmaI-digestedbacteriophagelambdaDNAat45°C;ligationoccursonlyatcossitesasdeterminedbygelelectrophoresis.AmpligaseDNALigaseisfreeofdetectableexo-andendonucleaseandRNaseactivities. References
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Ampligase™isprovidedatastandardconcentrationof5U/μl,withtheexceptionofcatalognumbersA0102KandA110K(100U/μl).
AmpligaseDNALigaseKitcontains:AmpligaseDNALigase,10XReactionBuffer,andControlDNA.AmpligaseEnzyme&Buffercontain:AmpligaseDNALigaseand10XReactionBuffer(25μlBufferforeach50Uenzyme).
OneunitofAmpligase™isequaltoasmanyas15unitsofotherthermostableDNAligases.Pleasecomparecompetitiveunitdefinitions.
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