Manta1.0DNAPolymerase(HighConcentration)

ProductDescription

Manta1.0DNAPolymerase(exo-)isathermophilicDNApolymerasedeficientinbothproofreading(3′→5′)andnick-translation(5′→3′)nucleaseactivities.TheproteinwasoriginallycharacterizedanditscrystalstructuresolvedbyLorenaBeese(1).

SourceofProtein
Arecombinant E.coli straincarryingtheManta1.0DNAPolymerase(exo-)polymerasegene.

Suppliedin
10mMTris-HCl
50mMKCl
1.0mMDTT
0.1mMEDTA
0.1%TritonX-100
50%glycerol
pH7.5@25°C

SuppliedWith
B7140(10XPCRBufferII)

10XPCRBufferll(B7140):
200mMTris-HCl
100mM(NH4)2SO4
100mMKCl
20mMMgSO4
1.0%TritonX-100
pH8.8@25°C

UnitDefinition
1unitisdefinedastheamountofpolymeraserequiredtoconvert10nmolofdNTPsintoacidinsolublematerialin30minutesat65°C.

QualityControlAnalysis

UnitCharacterizationAssay
Unitactivitywasmeasuredusinga2-foldserialdilutionmethod.Dilutionsofenzymebatchweremadein1Xreactionbufferandaddedto50µLreactionscontainingCalfThymusDNA,1XPCRBufferII, 3H-dTTPand100µMdNTPs.Reactionswereincubated10minutesat65°C,plungedonice,andanalyzedusingthemethodofSambrookandRussell(MolecularCloning,v3,2001,pp.A8.25-A8.26)

ProteinConcentration(OD280)Measurement
A2.0µLsampleofenzymewasanalyzedatOD280 usingaNanodropND-1000spectrophotometerstandardizedusinga2.0mg/mlBSAsample(PierceCat#23209)andblankedwithproductstoragesolution.Theobservedaveragemeasurementof3replicatesampleswasconvertedtomg/mLusinganextinctioncoefficientof52,770andmolecularweightof66,215Daltons.

SDS-Page(PhysicalPurityAssessment)
2.0µLofconcentratedenzymesolutionwasloadedonadenaturing4-20%Tris-GlycineSDS-PAGEgelflankedbyabroad-rangeMWMarkerand2.0µLofa1:100dilutionofthesample.Followingelectrophoresis,thegelwasstainedandthesamplescomparedtodeterminephysicalpurity.Theacceptancecriteriaforthistestrequiresthattheaggregatemassofcontaminantbandsintheconcentratedsampledonotexceedthemassoftheproteinofinterestbandinthedilutesample,confirminggreaterthan99%purityoftheconcentratedsample.

ContaminationTests

Single-StrandedExonucleaseActivity
A50µLreactioncontaining10,000cpmofaradiolabeledsingle-strandedDNAsubstrateand10µLofenzymesolutionincubatedfor4hoursat37°Cresultedinlessthan5.0%releaseofTCA-solublecounts.

Double-StrandedExonucleaseActivity
A50µLreactioncontaining5,000cpmofaradiolabeleddouble-strandedDNAsubstrateand10µLofenzymesolutionincubatedfor4hoursat37°Cresultedinlessthan1.0%releaseofTCA-solublecounts.

Double-StrandedEndonucleaseActivity
A50µLreactioncontaining0.5µgofpBR322DNAand5µLofenzymesolutionincubatedfor4hoursat37°CresultedinnovisuallydiscernibleconversiontonickedcircularDNAasdeterminedbyagarosegelelectrophoresis.

E.coli 16SrDNAContaminationTest
Replicate5µLsamplesofenzymesolutionweredenaturedandscreenedinaTaqManqPCRassayforthepresenceofcontaminating E.coli genomicDNAusingoligonucleotideprimerscorrespondingtothe16SrRNAlocus.Theacceptancecriterionforthetestisthethresholdcyclecount(Ct)producedbytheaverageof3replicatenotemplatecontrolsamples.BasedonthecorrelationbetweenthenotemplatecontrolCt values,andstandardcurvedata,thedetectionlimitofthisassayis<10copiesgenome/sample.

 

ProductInformation

Manta1.0DNAPolymerase(HighConcentration)
PartNumberP7140-HC-L
Price$363.00
Concentration400,000U/ml
UnitSize100,000U
SDSAvailableonrequest

ProductSpecification*

StorageTemperature-25to-15°C
TestSpecification
Purity(SDS-PAGE)>99%
SpecificActivity400,000U/mg
SSExonuclease4000U<5.0%released
DSExonuclease4000U<1.0%released
DSEndonuclease4000U=noconversion
E.coliDNAContamination4000U<10copies

*Foradetailedsummaryofassayconditionsanddata,refertotheQualityControlsAnalysissection.

Notes

Specificactivitymeasuredunderaboveconditionsisapproximately3XhigherthancitedbyBeeseetal.whenusingactivatedcalfthymusDNAasasubstrate.

References

  1. Kiefer,etal.Structure15January1997.5,95-108.

LimitationsofUse
Thisproductwasdeveloped,manufactured,andsoldforinvitrouseonly.Theproductisnotsuitableforadministrationtohumansoranimals.SDSsheetsrelevanttothisproductareavailableuponrequest.