Enzymatics/TaqIT/P7620L/5,000 U

TaqIT

ProductDescription

TaqITisanexonucleasedeficientderivativeofTaqDNApolymerase.TaqITlacksthefirst280aminoacidsofnativeTaqpolymerasethatcontainthe5′-3′exonucleasedomain.ThisdeletionmakesTaqITslightlymoreThermostableandhasslightlygreaterfidelitythanfulllengthTaq.LikeTaqpolymerase,TaqIThasnoinherent3′-5′exonucleaseactivity.

SourceofProtein
ArecombinantE.colistraincarryingtheTaqITgenefromthethermophilicorganismThermusAquaticusYT-1.

UnitDefinition
1unitisdefinedastheamountofenzymethatwillincorporate10nmolofdNTPintoacid-insolublematerialin30minutesat75°C.

Molecularweight
62,407Daltons

QualityControlAnalysis

UnitActivityismeasuredusinga2-foldserialdilutionmethod.Dilutionsofenzymeweremadeinareduced-glycerol(5%)containingTaq-BDNAPolymerasestoragesolutionandaddedto50µLreactionscontainingCalfThymusDNA,25mMTAPS(pH9.3),50mMKCl,1mMDTT,3H-dTTPand100µMdNTPs.Reactionswereincubated10minutesat75°C,plungedonice,andanalyzedusingthemethodofSambrookandRussell(MolecularCloning,v3,2001,pp.A8.25-A8.26).

ProteinConcentrationisdeterminedbyOD₂₈₀absorbance.

PhysicalPurityisevaluatedbySDS-PAGEofconcentratedanddilutedenzymesolutionsfollowedbysilverstaindetection.Purityisassessedbycomparingtheaggregatemassofcontaminantbandsintheconcentratedsampletothemassoftheproteinofinterestbandinthedilutedsample.

Single-StrandedExonucleaseisdeterminedina50µLreactioncontainingarADIolabeledsingle-strandedDNAsubstrateand10µLofenzymesolutionincubatedfor4hoursat37°C.

Double-StrandedExonucleaseisdeterminedina50µlreactioncontainingaradiolabeleddouble-strandedDNAsubstrateand10µLofenzymesolutionincubatedfor4hoursat37°C.

Double-StrandedEndonucleaseisdeterminedina50µLreactioncontaining0.5µgofplasmidDNAand10µLofenzymesolutionincubatedfor4hoursat37°C.

E.coli16SrDNAContaminationisevaluatedusing5µLreplicatesamplesofenzymesolutiondenaturedandscreenedinaTaqManqPCRassayforthepresenceofcontaminatingE.coligenomicDNAusingoligonucleotideprimerscorrespondingtothe16SrRNAlocus.

Suppliedin:
20mMTris-HCl
222mM(NH₄)₂SO₄
10mM2-mercaptoethanol
0.1mMEDTA
0.1%Brij58
50%glycerol
pH8.6@25°C

Suppliedwith:
10XTaqITReactionBuffer(B7620):
500mMTris-HCl
160mM(NH₄)₂SO₄
35mMMgCl₂
0.25%Brij58
pH9.2@25⁰C

ProductInformation

TaqIT
PartNumber	P7620L
Price	$503
Concentration	50,000U/mL
UnitSize	5,000U
SDS	Availableonrequest

ProductSpecification*

StorageTemperature	-25⁰Cto-15⁰C
Test	UnitsTested	Specification
SDSPurity	n/a	>99%
SpecificActivity	n/a	42,000U/mg
SSExonuclease	500	<1.0%Released
DSExonuclease	500	<1.0%Released
DSEndonuclease	500	NoConversion
E.coliDNAContamination	500	<10copies

*Foradetailedsummaryofassayconditionsanddata,refertotheQualityControlsAnalysissection.

References

ThefidelityofTaqpolymerasecatalyzingPCRisimprovedbyanN-terminaldeletion.BarnesWM.Gene.1992.112(1):29-35.
ComparativethermaldenaturationofThermusaquaticusandEscherichiacolitype1DNApolymerases.KarantzeniI,RuizC,LiuCC,LicataVJ.BiochemJ.2003374(Pt3):785-92.

LimitationsofUse
Thisproductwasdeveloped,manufactured,andsoldforinvitrouseonly.TheproductisnotsuitableforadmiNISTrationtohumansoranimals.SDSsheetsrelevanttothisproductareavailableuponrequest.