Product Class: Other

T4 RNA Ligase 2, truncated
NEBU cloned at NEB recombinant 25 65 Heat

Product Introduction

  • T4 RNA Ligase 2, truncated (T4 Rnl2tr) specifically ligates the pre-adenylated 5´ end of DNA or RNA to the 3´ end of RNA.
  • The enzyme does not require ATP for ligation but does need the pre-adenylated substrate.
  • T4 Rnl2tr is expressed from a plasmid in E. coli, which encodes the first 249 amino acids of the full length T4 RNA Ligase 2.
  • Unlike the full length ligase, T4 Rnl2tr cannot ligate the phosphorylated 5´ end of RNA or DNA to the 3´ end of RNA.
  • This enzyme, also known as Rnl2 (1–249) has been used for optimized linker ligation for the cloning of microRNAs.
  • This enzyme reduces background ligation because it can only use pre-adenylated linkers.
Catalog # Size Concentration
M0242S 2000.0 units 200000 units/ml
M0242L 10000.0 units 200000 units/ml

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Product Information

Description

T4 RNA Ligase 2, truncated (T4 Rnl2 truncated) specifically ligates the pre-adenylated 5´ end of DNA or RNA to the 3´ end of RNA. The enzyme does not require ATP for ligation but does need the pre-adenylated substrate. T4 Rnl2 truncated is expressed from a plasmid in E. coli which encodes the first 249 amino acids of the full length T4 RNA Ligase 2. Unlike the full length ligase, T4 Rnl2 truncated is unable to adenylate the 5´ end of the substrate, and as a result it cannot ligate the phosphorylated 5´ end of RNA or DNA to the 3´ end of RNA (1-3). This enzyme, also known as Rnl2 (1-249) has been used for optimized linker ligation for the cloning of microRNAs. This enzyme reduces background ligation because it can only use adenylated primers (4-5).

Product Source

An E. coli strain that carries the cloned truncated T4 RNA Ligase 2 gene.
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

Properties & Usage

Unit Definition

200 units is defined as the amount of enzyme required to give 80% ligation of a 31-mer RNA to the pre-adenylated end of a 17-mer DNA Universal miRNA Cloning Linker (#S1315) in a total reaction volume of 20 µl in 1 hour at 25°C. 

5´-FAM-rArGrUrCrGrUrArGrCrCrUrUrUrArUrCrCrGrArGrArUrUrCrArGrCrArArUrA-3´ 

5´-rAppCTGTAGGCACCATCAAT–NH2-3´ 

Molarity = 14 μM

Reaction Conditions

1X T4 RNA Ligase Reaction Buffer 
Incubate at 25°C

1X T4 RNA Ligase Reaction Buffer 
50 mM Tris-HCl
10 mM MgCl2
1 mM DTT
(pH 7.5 @ 25°C)

Storage Buffer

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

Heat Inactivation

65°C for 20 minutes

Unit Assay Conditions

1X T4 RNA Ligase Reaction Buffer supplemented to 10% (w/v) PEG MW 8000, 20 pmol of 5´-FAM labeled RNA, and 40 pmol preadenylated DNA linker. After incubation at 25°C for 1 hour, the ligated product is detected on a 15% denaturing polyacrylamide gel.

Application Features

  • Ligate a pre-adenylated DNA or RNA sequence tag to any RNA 3´-end
  • Join a single stranded adenylated primer to small RNAs for cDNA library creation
  • Join a single stranded adenylated primer to RNA for strand-specific cDNA library construction

References

  1. Ho, C.K. et al. (2004). Structure. 12, 327-339.
  2. Ho, C.K. and Shuman, S. (2002). Proc. Natl.Acad.Sci. USA. 99, 12709-12714.
  3. Nandakumar, J. et al. (2004). J. Biol. Chem. 279, 31337-31347.
  4. Aravin, A. and Tusch, T. (2005). FEBS Letters. 579, 5830-5840.
  5. Pfeffer, S. et al. (2005). Nat. Meth.. 2, 269-276.

Protocols, Manuals & Usage

Protocols

  1. Protocol for ligation of the 3´ OH of RNA to the 5´ pre-adenylated DNA with T4 RNA Ligase 2 truncated (NEB #M0242)

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. How can I increase ligation efficiency?
  2. Is PEG 8000 available for purchase?

Tech Tips

Since pre-adenylated oligos are used in the reaction, the ligation efficiency is very high. Usually 1-2 hr at 25C is sufficient to achieve desirable results.

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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