T4 RNA Ligase 1 | Enzymatics

ProductDescription

T4RNALigasecatalyzestheATP-dependentligationofsingle-strandednucleicacids(RNAorDNA).

SourceofProtein
Arecombinant E.coli straincarryingtheT4RNALigasegenefrombacteriophageT4.

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

SuppliedWith 
B6050(10XT4RNALigaseBuffer)

10XT4RNALigaseBuffer(B6050)
500mMTris-HCl
100mMMgCl2
10mMATP
100mMDTT
pH7.8@25°C

UnitDefinition
Oneunitisdefinedastheamountofenzymerequiredtoligate50%of0.4µgofanequimolarmixoftwosinglestranded23baseRNAoligonucleotides(one5′-phosphorylated)in20µL1XT4RNALigaseBufferfollowinga30minuteincubationat37°C.

UnitCharacterizationAssay
Specificactivitywasmeasuredusinga2-foldserialdilutionmethod.Dilutionsofenzymeweremadein1XT4RNALigasereactionbufferandaddedto20µLreactionscontaining0.4µgofanequimolarmixoftwosingle-stranded23baseRNAoligonucleotides(one5′-phosphorylated)and1XT4RNALigaseBuffer.Reactionswereincubated30minutesat37°C,stopped,andanalyzedona15%TBE-UreagelstainedwithSYBR®GoldNucleicAcidGelStain(InvitrogenS-11494).

ProteinConcentration(OD280)Measurement
A2.0µLsampleofenzymewasanalyzedatOD280 usingaNanodropND-1000spectrophotometerstandardizedusinga2.0mg/mlBSAsample(PierceCat#23209)andblankedwithproductstoragesolution.Theobservedaveragemeasurementof3replicatesampleswasconvertedtomg/mLusinganextinctioncoefficientof49,070andmolecularweightof43,509Daltons.

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

Single-StrandedExonucleaseActivity
A50µlreactioncontaining10,000cpmofaradiolabeledsingle-strandedDNAsubstrateand10µLofenzymesolutionincubatedfor4hoursat37°Cresultedinlessthan5.0%releaseofTCA-solublecounts.

Double-StrandedExonucleaseActivity
A50µlreactioncontaining5,000cpmofaradiolabeleddouble-strandedDNAsubstrateand10µLofenzymesolutionincubatedfor4hoursat37°Cresultedinlessthan1.0%releaseofTCA-solublecounts.

Double-StrandedEndonucleaseActivity
A50µLreactioncontaining0.5µgofpBR322DNAand10µLofenzymesolutionincubatedfor4hoursat37°CresultedinnovisuallydiscernibleconversiontonickedcircularDNAasdeterminedbyagarosegelelectrophoresis.

E.coli 16SrDNAContaminationTest
Replicate5µLsamplesofenzymesolutionweredenaturedandscreenedinaTaqManqPCRassayforthepresenceofcontaminating E.coli genomicDNAusingoligonucleotideprimerscorrespondingtothe16SrRNAlocus.Theacceptancecriterionforthetestisthethresholdcyclecount(Ct)producedbytheaverageof3replicatenotemplatecontrolsamples.BasedonthecorrelationbetweenthenotemplatecontrolCt values,andstandardcurvedata,thedetectionlimitofthisassayis10copiesgenome/sample.

Non-SpecificRNAseAssay
Productwasscreenedfornon-specificRNAsecontaminationusingtheRNAseAlertkit,(IntegratedDNATechnologies),followingthemanufacturersguidelines.

Purity(SDS-PAGE)>99%SpecificActivity16,800U/mgSSExonuclease200UDSExonuclease200UDSEndonuclease200U=NoconversionE.coliDNAContamination200UNon-specificRNAse200Unonedetected

*Foradetailedsummaryofassayconditionsanddata,refertotheQualityControlsAnalysissection.

LimitationsofUse
Thisproductwasdeveloped,manufactured,andsoldforinvitrouseonly.Theproductisnotsuitableforadministrationtohumansoranimals.SDSsheetsrelevanttothisproductareavailableuponrequest.