New England Biolabs, M0523S, Thermostable RNase H

Thermostable RNase H specifically recognizes and cleaves the phosphodiester bonds of an RNA strand in an RNA-DNA hybrid while leaving the DNA strand intact. This thermostable nuclease exhibits the same enzymatic properties as Related Categories RNases,, cDNA Synthesis & Reverse Transcriptases Applications Transcription-Mediated and NASBA Amplification,, cDNA Synthesis,, PCR Specification Unit Definition One unit is defined as the amount of enzyme required to produce 1 nmol of ribonucleotides from 40 pmol of a fluorescently labeled 25 base pair RNA:DNA hybrid in a total reaction volume of 50 μl in 20 minutes at 50°C. Reaction Conditions 1X RNase H Reaction Buffer Incubate at ≥50°C 1X RNase H Reaction Buffer 50 mM Tris-HCl 75 mM KCl 3 mM MgCl2 10 mM DTT (pH 8.3 @ 25°C) Storage Buffer 50 mM Tris-HCl 100 mM NaCl 1 mM DTT 0.1 mM EDTA 0.1% Triton® X-100 50% Glycerol pH 7.5 @ 25°C Heat Inactivation No FAQ Q: When I thaw the 10X RNase H Reaction Buffer, I see a white precipitate.  What is it and what should I do about it? A: The white precipitate is DTT in the 10X buffer, and it will resuspend when thawed and mixed. The resuspended buffer is fully functional for its intended use. Q: What is the difference between E. coli RNase H (NEB #M0297) and Thermostable RNase H (NEB #M0523)? A: Both Thermostable RNase H and E. coli RNase H cleave RNA in a DNA:RNA hybrid molecule while leaving the DNA intact; they do not cleave single- or double-stranded RNA or DNA. However, Thermostable RNase H is active over a wider temperature range than the E. coli enzyme, with optimal activity at 65°C, and a range of activity from 37°C - 95°C. Q: Which side of the RNA base does Thermostable RNase H cut? A: Similar to E. coli RNase H, Thermostable RNase H acts as an endoribonuclease and cleaves the phosphodiester bond of RNA, generating 3´ hydroxyls and 5´ phosphates. Q: What is the optimal reaction temperature for Thermostable RNase H? A: Thermostable RNase H is active over a wide temperature range (37°C–95°C). Activity will depend on the Tm of the RNA:DNA hybrid substrate and the ability of the substrate to remain double-stranded at the selected reaction temperature. Q: Is Thermostable RNase H active in other buffers? A: Thermostable RNase H requires Mg2+ for activity, therefore buffers containing MgCl2 may be suitable for Thermostable RNase H reactions. The optimal reaction buffer is 1X RNase H Reaction Buffer, but we have tested NEBuffers 1–4, NEBuffers r1.1–r3.1 and rCutSmart® Buffer: Buffer 37°C 50°C 65°C >65°C RNase H Buffer +++ +++ +++ +++ NEBuffer 1 ++ ++ ++ ++ NEBuffer 2; +++ +++ ++ ++ NEBuffer 3 +++ +++ +++ + NEBuffer 4 ++ +++ +++ +++ NEBuffer r1.1 ++ ++ +++ - NEBuffer r2.1 ++ ++ ++ ++ NEBuffer r3.1 +++ +++ +++ - rCutSmart Buffer ++ ++ ++ + Q: Does Thermostable RNase H cut single-stranded RNA? A: No. Thermostable RNase H only cleaves the RNA strand in an RNA:DNA hybrid molecule. Q: Does Thermostable RNase H require co-factors for activity? A: Yes. Thermostable RNase H requires Mg2+ for activity, which is included in the 10X RNase H Reaction Buffer supplied with the enzyme. Q: Can Thermostable RNase H be inactivated by heat? A: No. Thermostable RNase H is stable at high temperatures (> 95°C) and cannot be inactivated by heat. Inactivation of the enzyme can be achieved by incubation with Proteinase K or addition of EDTA. Q: What applications are Thermostable RNase H suitable for? A: Thermostable RNase H can be used as a replacement for E. coli RNase H in assays such as RNA structure mapping, site-specific cleavage of RNA, isothermal amplification methods, removal of mRNA during second strand cDNA synthesis, and removal of poly(A) tails on mRNAs when hybridized to oligo(dT). Due to the increased stability of Thermostable RNase H at higher temperatures, assays requiring higher stringency may benefit from the use of this enzyme.