Product Class: Other

RtcB Ligase
NEBU cloned at NEB recombinant 37

Product Introduction

  • This is an Enzyme for Innovation (EFI). EFI is a project initiated by NEB to provide unique enzymes to the scientific community in the hopes of enabling the discovery of new and innovative applications. These enzymes have interesting properties and unique specificities.
  • RtcB Ligase from E. coli joins single stranded RNA with a 3´-phosphate or 2´,3´-cyclic phosphate to another RNA with a 5´ -hydroxyl (1,2).
  • Ligation requires both GTP and MnCl2 and proceeds through a 3´-guanylate intermediate (3).
  • For substrates with a 2´,3´-cyclic phosphate end, hydrolysis to a 3´-phosphate precedes 3´ end activation with GMP and ligation.
Catalog # Size Concentration
M0458S 25.0 reactions 15 µM

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Product Information

Description

Product Source

RtcB Ligase is expressed as a His-tagged fusion in E. coli.
This product is related to the following categories:
RNA Ligation
This product can be used in the following applications:
RNA Cloning

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • M0458S     -20    
      RtcB Ligase M0458SVIAL -20 1 x 0.025 ml 15 µM
      RtcB Reaction Buffer B0790AVIAL -20 1 x 0.5 ml 10 X
      MnCl2 B1761SVIAL -20 1 x 1 ml 10 X
      GTP N2080AVIAL -20 1 x 0.05 ml 10 mM

Properties & Usage

Reaction Conditions

1X RtcB Reaction Buffer
Supplement with 0.1 mM GTP and 1 mM MnCl2
Incubate at 37°C

1X RtcB Reaction Buffer
50 mM Tris-HCl
75 mM KCl
3 mM MgCl2
10 mM DTT
(pH 8.3 @ 25°C)

Storage Buffer

50 mM NaCl
10 mM Tris-HCl
0.1 mM EDTA
1 mM DTT
50% Glycerol
pH 7.4 @ 25°C

Application Features

  • Ligate ssRNA or ssDNA with a 3´-phosphate or a 2´,3´-cyclic phosphate to the 5´-OH of ssRNA
  • Circularization of ssRNA with compatible ends, as above
  • Detection of 3´-phosphorylated or 5´ -OH RNA using labeled probes

Product Notes

  1. For difficult substrates, addition of PEG 8000 up to 15% can improve ligation yields.

References

  1. Naoko, T., Shuman, S. (2011). J. Biol. Chem. 286, 7727-7731.
  2. Naoko, T. et al. (2011). J. Biol. Chem. 286, 43134-43143.
  3. Chakravarty, A. K. et al (2012). Proc. Natl. Acad. Sci. USA. 109, 6072-6077.

Protocols, Manuals & Usage

Protocols

  1. Typical Protocol for RNA Ligation (NEB #M0458)

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. Does secondary structure affect ligation by RtcB Ligase?
  2. Can RtcB Ligase ligate DNA?
  3. What is the optimum reaction temperature for RtcB Ligase?
  4. Can RtcB Ligase be used in other NEBuffers?
  5. How can I increase ligation efficiency?
  6. Can magnesium, or another divalent cation, be substituted for manganese?
  7. Does PEG improve ligation efficiency?
  8. Can I purchase large amounts of an existing Enzyme for Innovation?
  9. What are Enzymes for Innovation?

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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