Product Class: Other

Thermostable 5´ App DNA/RNA Ligase
NEB1 Gray property icon for cloned at NEB recombinant 65 Gray property icon for heat inactivation no

Product Introduction

Thermostable 5´ App DNA/RNA Ligase ligates a 5´ pre-adenylated 3´-blocked DNA linker to the 3´-OH of RNA and ssDNA.

  • Isolated from a recombinant source
  • Supplied with 10X reaction buffer
  • Tested for the absence of DNases and RNases
  • Not sure which ligase to choose? Refer to our DNA and RNA Ligase Properties Chart

Bioz Badge Exists : True
Catalog # Size Concentration
M0319S 10 reactions 20 µM
M0319L 50 reactions 20 µM

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Product Information

Description

Thermostable 5´ App DNA/RNA Ligase is a point mutant of catalytic lysine of RNA ligase from Methanobacterium thermoautotrophicum (1). This enzyme is ATP independent. It requires a 5´ pre-adenylated linker for ligation to the 3´-OH end of either RNA or single stranded DNA (ssDNA). The enzyme is also active in ligation of RNA with 2´-O-methylated 3´ end to 5´-adenylated linkers (1). The optimal temperature for ligation reaction is 60–65°C (2). The mutant ligase is unable to adenylate the 5´-phosphate of RNA or ssDNA, which reduces the formation of undesired ligation products (concatemers and circles).

The ability of the ligase to function at 65°C might reduce the constraints of RNA secondary structure in RNA ligation experiments.

Product Source

Thermostable 5´ App DNA/RNA Ligase is expressed as His-tag fusion in E. coli.
This product is related to the following categories:
RNA Ligation,
DNA Ligases,
This product can be used in the following applications:
Non-Cloning Ligation

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • NEB # Stored at (°C)
    M0319S -20
    Component Name Component # Stored at (°C) Amount Concentration
    Thermostable 5´ App DNA/RNA Ligase M0319SVIAL -20 1 x 0.02 ml 20 µM
    NEBuffer™ 1 B7001SVIAL -20 1 x 1.25 ml 10 X
    MnCl2 B0787AVIAL -20 1 x 0.5 ml 50 mM
  • NEB # Stored at (°C)
    M0319L -20
    Component Name Component # Stored at (°C) Amount Concentration
    Thermostable 5´ App DNA/RNA Ligase M0319LVIAL -20 1 x 0.1 ml 20 µM
    NEBuffer™ 1 B7001SVIAL -20 1 x 1.25 ml 10 X
    MnCl2 B0787AVIAL -20 1 x 0.5 ml 50 mM

Properties & Usage

Reaction Conditions

1X NEBuffer™ 1
Incubate at 65°C

1X NEBuffer™ 1
10 mM Bis-Tris-Propane-HCl
10 mM MgCl2
1 mM DTT
(pH 7 @ 25°C)

Storage Buffer

50 mM KCl
10 mM Tris-HCl
0.1 mM EDTA
1 mM DTT
50% Glycerol
pH 7.5 @ 25°C

Heat Inactivation

No

Application Features

  • Ligate a 5´ pre-adenylated 3´-blocked DNA linker to the 3´-OH of RNA and ssDNA.

Product Notes

  1. For optimal ligation of ssDNA to adenylated DNA linkers, we recommend using NEBuffer 1 supplemented with manganese. For ligation of ssRNA to adenylated DNA linkers, just use NEBuffer 1.
  2. DNA to AppDNA ligation occurs with reduced efficiency, therefore we recommend extending the incubation time overnight.
  3. We recommend using a thermocycler or heated top to reduce evaporation.
  4. For inactivation of the enzyme we recommend Proteinase K treatment or heat-kill at 90°C for 3 minutes
  5. Proteinase K treatment is recommended to release RNA bound by the ligase; or you may see a smear on a gel.

References

  1. Zhelkovsky, A. M., McReynolds, L. A. (2012). BMC Mol. Biol.. 13, 24.
  2. Torchia, C., Takagi, Y. and Ho, C. K. (2008). Nucleic Acids Res.. 36, 6218-6227.

Tools & Resources

Selection Charts

Web Tools

FAQs & Troubleshooting

FAQs

  1. How can you synthesize an adenylated DNA linker?
  2. Can I use the wild type Mth RNA ligase included in the 5’ DNA adenylation kit instead of the Thermostable 5’AppDNA/RNA ligase for library construction?
  3. How much Thermostable 5’AppDNA/RNA ligase do I need to add to a reaction?
  4. Why do you recommend adding MnCl2 to the DNA ligation but not the RNA reaction?
  5. Does the Thermostable 5’ AppDNA/RNA ligase ligate dsRNA or dsDNA?
  6. Will PEG or increased time enhance the amount of ligated product?
  7. Why is the RNA running as a smear or stuck in the well?
  8. What is the salt tolerance of this RNA ligase?

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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