APPLICATIONS

DESCRIPTION
TthPlusDNApolymeraseisisolatedfromtheThermusThermophilusstrain.TthPlusDNApolymeraseisasingle92kDapolypeptideshowinga5′-3′exonucleaseactivitybutlacking3′-5′exonucleaseactivity.Itcatalyzesthepolymerizationofnucleotidesintodouble-strandedDNAinthepresenceofMgCl₂.ItsefficiencyhasbeenshownmoreparticularlyonlargeDNAfragmentsupto12kb(usinglamBDaphageDNAasatemplate).TthPlusDNApolymeraseisalsocapableofcatalyzingthepolymerizationofDNAusingaRNAtemplateinthepresenceofMn²⁺.TheABIlityofTthPlusDNApolymerasetoreversetranscribeatelevatedtemperatures(70°C)minimizestheproblemsencounteredwithstrongsecondarystructuresinRNAsincetheyareunstableathigherreactiontemperatures.Highertemperaturesalsoresultinincreasedspecificityofprimerhybridizationandextension.IncoupledRT/PCRassays,TthPlusisabout50-100timesmoreefficientthanregularTaqDNApolymerase.

TthPlusDNApolymeraseisdeliveredwithaone-tube10xbuffer.Bothreactions(RTandPCR)arecarriedoutinthesamebufferandsametubewiththesameenzyme(TthPlus).The10xonetubebufferdoesnotcontainMn(OAc)₂.Mn(OAc)₂isprovidedextraandhavetobeaddedatafinalconcentrationof3.5mM.

SIZEandCONCENTRATION

500Units,5units/µl

FEATURES

UNITDEFINITION

Oneunitisdefinedastheamountofenzymethatincorporates10nmolesofdNTPsintoacid-insolubleformin30minutesat72°Cunderthefollowingreactionconditions:25mMTAPSbuffer(Tris-(hydroxymethyl)-methyl-amino-propanesulfonicacid,sodiumsalt)pH9.3(25°C),50mMKCl,2mMMgCl₂,1mMβ-mercaptoethanol,200µMdNTPsand10µgofcalfthymusDNAinafinalreactionvolumeof50µl.

STORAGEBUFFER

10mMK-phosphatebufferpH7.0(25°C),100mMNaCl,0.5mMEDTA,1mMDTT,50%glycerol(v/v),0,1mg/mlBSA.

STORAGETEMPERATURE

StoreTthPlusDNApolymerasepreferablyat-20°Cinaconstanttemperaturefreezer.

COMPONENT

-TthPlusDNApolymerase

-5xOneTubebuffer

-50mMMn(OAc)₂

Theoptimalexperimentalconditionsdependonthesystemusedandtheyshouldbeindividuallydetermined.TheMn²⁺concentrationsandtheenzymeamountarethelimitingfactorsforanaccurateresult.TrADItionally5unitsofenzymeandaMn-acetateconcentrationof3.5mMareusedforthereversetranscriptioninafinal50µlreactionvolume.

AdditionalInformation

Clickherefortechnicalinformation

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