ProductDescription

E.coliRibonucleaseH(RNaseH)isanendonucleasethatspecificallydegradestheRNAofDNA-RNAhybrids,withoutaffectingDNAorunhybribizedRNA.ThishighlypurifiedpreparationissuitedforuseinRNAAmplificationreactions.Thisenzymepreparationispreparedinastoragebuffercontaining1.0MTrehalose.

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