Product Class: Other

Thermostable RNase H
NEBU recombinant 50 No

Product Introduction

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 E. coli RNase H, but is active at much higher temperatures.

  • Recombinant enzyme supplied with 10X Reaction Buffer
  • Does not digest single or double-stranded DNA
  • Active and stable above 37°C over a broad temperature range, with activity increasing with temperature
  • Requires magnesium chloride for activity
  • Inactivated with proteinase K treatment or addition of excess EDTA
Catalog # Size Concentration
M0523S 250.0 units 5000 units/ml

Product Information

Description

Thermostable RNase H is an endoribonuclease that is functional at high temperatures and selectively hydrolyzes the phosphodiester bonds of an RNA strand in an RNA:DNA hybrid molecule, while leaving the DNA strand intact. In addition, Thermostable RNase H does not degrade single- or double-stranded RNA or DNA. While the Thermostable and E. coli RNase H homologs both exhibit these same endoribonucleolytic properties, the thermostable enzyme displays activity at higher temperatures with optimal activity above 65°C. These properties allow use in applications where higher assay temperatures are desired.

Product Source

An E. coli strain carrying a codon optimized plasmid encoding RNase H from the extreme thermophile Thermus thermophilus.
This product is related to the following categories:
RNases,
cDNA Synthesis & Reverse Transcriptases Products,
This product can be used in the following applications:
Transcription-Mediated and NASBA Amplification,
cDNA Synthesis,
PCR

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • M0523S     -20    
      Thermostable RNase H M0523SVIAL -20 1 x 0.05 ml 5,000 units/ml
      RNase H Reaction Buffer B0297SVIAL -20 1 x 1.5 ml 10 X

Properties & Usage

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

Application Features

  • Higher stringency RNA structure mapping and site-specific RNA cleavage
  • Removal of poly(A) tails from mRNA hybridized to oligo(dT)
  • Removal of mRNA during second strand cDNA synthesis
  • Component of isothermal amplification methods

Product Notes

  1. NOTE: The RNAse H Reaction Buffer contains MgCl2. When using Thermostable RNase H in the presence of the RNA:DNA hybrid of interest and other RNA species that are single-stranded (ie. total RNA) at elevated temperatures, it is advised to limit the reaction time and temperature to reduce metal-mediated breakdown of single-stranded RNA species.
  2. Thermostable RNase H can be inactivated by treatment with proteinase K or addition of excess EDTA.
  3. Optimal incubation temperature >65°C, up to 95°C.  Thermostable RNase H displays 3-4x more activity at 65°C compared to 37°C.

Protocols, Manuals & Usage

Protocols

  1. Cleavage of an RNA strand in an RNA-DNA hybrid with Thermostable RNase H (NEB #M0523)

FAQs & Troubleshooting

FAQs

  1. When I thaw the 10X RNase H Reaction Buffer, I see a white precipitate.  What is it and what should I do about it?
  2. What is the difference between E. coli RNase H (NEB #M0297) and Thermostable RNase H (NEB #M0523)?
  3. Which side of the RNA base does Thermostable RNase H cut?
  4. What is the optimal reaction temperature for Thermostable RNase H?
  5. Is Thermostable RNase H active in other buffers?
  6. Does Thermostable RNase H cut single-stranded RNA?
  7. Does Thermostable RNase H require co-factors for activity?
  8. Can Thermostable RNase H be inactivated by heat?
  9. What applications are Thermostable RNase H suitable for?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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