RemoveunwantedRNAbydigestingwiththisheatlABIle,non-sequencespecificribonuclease
- Complete:UnlikeRNaseA,RNaseIdoesnothaveabasepreferenceanddigestsbetweenalldinucleotidepairsinsingle-strandedRNA
- HeatLabile:RNaseIiseasilyinactivatedbyheating70°Cfor15minutes
RNaseIdegradessingle-strandedRNAtonucleoside3´monophosphatesvia2´,3´cyclicmonophosphateintermediatesbycleavingbetweenalldinucleotidepairs,2,3unlikeRNaseA,whichcleavesonlyaftercytosineanduridine.Inaddition,theenzymeiscompletelyinactivatedbyheatingat70°Cfor15minutes,eliminatingtherequirementtoremovetheenzymepriortomanysubsequentprocedures.
Applications
- RemovalofRNAfromDNApreparations.1
- RNaseprotectionassaystodetectsingle-basepairmismatchesinRNA:RNAandRNA:DNAhybrids.1
UnitDefinition:OneunitofRNaseIdegrades100ngofE.coliribosomalRNApersecondintoacid-solublenucleotidesat37°Cunderstandardassayconditions.
DilutionandStorageBuffer:50%glycerolcontaining50mMTris-HCl(pH7.5),100mMNaCl,and0.1mMEDTA.
QualityControl:RNaseIisfreeofdetectableexo-andendodeoxyribonucleaseactivities.
References
- Johnson,M.(1996)EpicentreForum3(4),7.
- Shen,V.andSchlessinger,D.(1982)TheEnzymesXV(PartB),501.
- delCardayré,S.B.andRaines,R.T.(1995)Anal.Biochem.225,176.
Figure1.RemovalofRNAfromplasmidDNApreparations.PlasmidDNAwaspreparedfrom1.5mLofanovernightculture,sUSPendedin50µLTEbuffer(10mMTris-HCl(pH8),1mMEDTA),andtreatedwithRNaseA,RNaseI,ornoenzyme.FiveµLofeachreactionwereresolvedbyagarosegelelectrophoresisandvisualizedbystainingwithethidiumbromide.Lane1,noRNase;Lane2,1.5UofRNaseI;Lane3,20µg/mLRNaseA;MW,1-kbladder.ArrowdenotessmallundigestedRNA. |