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VeraSeqULtraDNApolymeraseisanengineered,ultra-thermostable,uracil-literatepolymerasedesignedtomaximizethespeed,accuracy,andlengthofDNAsynthesisduringsequencingtemplatepreparation.Theresultisanovelenzymethatcanreadthroughuracil,extendakilobaseofsequencein15secondsandhasanaccuracy25timeshigherthanTaqDNAPolymerase.
SourceofProtein
Arecombinant E.coli straincarryingtheengineeredVeraSeqULtragene.
Suppliedin
20mMTris-HCl
100mMKCl
1mMDTT
0.1mMEDTA
Stabilizer
50%glycerol
pH7.4@25°C
SuppliedWith:
5XVeraSeqBufferII(B7102)
5XVeraSeqGCBuffer(B7130)
UnitDefinition
Oneunitisdefinedastheamountofenzymerequiredtoincorporate10nmolesofdNTPsintoacid-insolubleformat74°Cin30minutes.
UnitCharacterizationAssay
Specificactivitywasmeasuredusinga2-foldserialdilutionmethod.Dilutionsofenzymeweremadein1Xreactionbufferandaddedto50µLreactionscontainingactivatedcalfthymusDNA;25mMTAPS(tris-[hydroxymethyl]-methyl-amino-propanesulfonicacid,sodiumsalt),pH9.3at25°C;50mMKCl;2mMMgCl2;1mMβ-mercaptoethanol;200µMeachdATP,dGTP,dTTP;and100µM[3H]-dCTP(0.075Ci/mmole).Reactionvesselsweremixedandincubatedat74°Cfor10minutes.
ProteinConcentration(OD280)Measurement
TheenzymewasanalyzedatOD280 usingaNanodropspectrophotometerstandardizedwitha2.0mg/mlBSAsampleandblankedwithproductstoragesolution.Theobservedaveragemeasurementof3replicatesampleswasconvertedtomg/mLusinganextinctioncoefficientof134,130andmolecularweightof97,754Daltons.
SDS-Page(PhysicalPurityAssessment)
Aconcentratedsampleofenzymewasloadedonadenaturing4-20%Tris-GlycineSDS-PAGEgelflankedbyabroad-rangeMWMarkeranda1:100dilutionofthesample.Followingelectrophoresis,thegelwasstainedandthesamplescomparedtodeterminephysicalpurity.Theacceptancecriteriaforthistestrequiresthattheaggregatemassofcontaminantbandsintheconcentratedsampledonotexceedthemassoftheproteinofinterestbandinthedilutesample.Comparisonbetweentheconcentratedandthedilutedsamplesisusedtoevaluatepercentpurity.
Double-StrandedEndonucleaseActivity
AreactioncontainingplasmidDNAincubatedwithenzymesolutionfor4hoursat37°CresultedinnovisuallydiscernibleconversiontonickedcircularDNAasdeterminedbyagarosegelelectrophoresis.
E.coli16SrDNAContaminationTest
ReplicatesamplesofenzymeweredenaturedandscreenedinaTaqManqPCRassayforthepresenceofcontaminatingE.coligenomicDNAusingoligonucleotideprimerscorrespondingtothe16SrRNAlocus.Theacceptancecriterionforthetestisthethresholdcyclecount(Ct)producedbytheaverageof3replicatenotemplatecontrolsamples.BasedonthecorrelationbetweenthenotemplatecontrolCtvalues,andstandardcurvedata,thedetectionlimitofthisassayis<10copiesgenome/sample.
| VeraSeq™ULtraDNAPolymerase | |
|---|---|
| PartNumber | P7520L |
| Price | $363 |
| Concentration | 2,000U/ml |
| UnitSize | 500U |
| SDS | Availableonrequest |
| StorageTemperature | -25to-15°C |
|---|---|
| Test | Specification |
| Purity(SDS-PAGE) | >95% |
| SpecificActivity | 100,000U/mg |
| DSEndonuclease | 120U=noconversion |
| E.coliDNAContamination | 150U<10copies |
*Foradetailedsummaryofassayconditionsanddata,refertotheQualityControlsAnalysissection.
ThisproductislicensedfromBio-RadLaboratories,Inc.,underU.S.Pat.Nos.6,627,424,7,541,170,7,560,260andcorrespondingpatentsinothercountriesforusesolelyinDNAsequencing,DNAmicro-array,andconventionalPCRapplications,includingpre-amplificationstepsthatarerequiredforsuchapplications,inthelifescienceresearchfieldsbutnotreal-timePCRordigitalPCR.In-vitrodiagnosticsrightsareavailable,pleasecontactEnzymaticsformoreinformation.
LimitationsofUse
Thisproductwasdeveloped,manufactured,andsoldforinvitrouseonly.Theproductisnotsuitableforadministrationtohumansoranimals.SDSsheetsrelevanttothisproductareavailableuponrequest.