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RibonucleaseHfromE.coli
RibonucleaseHisanendonucleasewhichspecificallydegradestheRNAmoietyofaDNA:RNAhybrid,withoutaffectingtheunhybridizedRNA.Itdoesnotaffectdouble-strandedRNAorDNA.Itisprovidedina50%glycerolstoragebuffer.ItisalsoavailableinaTrehaloseBuffer(Suitableforlyophilization).Concentration1000U/mL.
Pleaseinquireforcustomconcentrationsandbulkquantities.
Applications:
Enablingthesynthesisofsecond-strandCDNAbyremovaloftheRNA
UsedinconjunctionwithAMVRTandT7RNAPolymeraseinamplificationofRNA
UnitDefinition:
OneunitofRNaseHisthatamountofenzymewhichcatalysistheproductionofonenmolofacid-solublenucleotidein20minutesat37ºCusingthefollowingreactionconditions:
40mMTrisHCl,pH7.5
1.0µM[3H]-poly(rA):24µMpoly(dT)
4.0mMMgCl2
1.0mMDTT
30µg/mLBSA
4.0%glycerol
StorageBufferConditionsofRNaseHinGlycerol:
20:0mMTrisHCl,pH7.5
300mMKCl
20.0mMMagnesiumAcetate
7.0mMEDTA
1.0mMDithiothreitol
50%Glycerol
0.2%TritonX100
StorageBufferConditionsofRNaseHinTrehalose.Sameasabove,exceptfor1.0MTrehaloseinsteadofGlycerol.
QualityControl:
DNaseActivity:
One-halfµgofHaefragmentsofPhiX-174DNAisincubatedat37ºCwith2.5unitsofRNaseHfor3hours,andthenelectrophoresedinanativeagarosegelsimultaneouslywithcontrolpositiveDNase1reactions.Nomorethantheequivalentof2.5X10E-4unitofDNase1isdetected.
RibonucleaseActivity:
OnemicrogramofanRNALadderisincubatedfor2hoursat37ºCwith4.0unitsofRNaseH,andthenelectrophoresedinanativeagarosegelsimultaneouslywithcontrolpositiveRNase1Areactions.Nomorethantheequivalentof8X10-8unitofRNase1Aisdetected.
SpecificActivity:
ThespecificactivityoftheE.coliRNaseHisnolessthan300,000unitspermg.
References:
Sambrook,J.,Fritsch,E.F.,andManiatis,T.(1989)MolecularCloning:ALaboratoryManual,(2ndEd.),8.64–8.65
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