T4 RNA Ligase 2 (dsRNA Ligase)

Description

T4 RNA Ligase 2, also known as T4 Rnl2 (gp24.1), has both intermolecular and intramolecular RNA strand joining activity (1-3). Unlike T4 RNA Ligase 1 (NEB #M0204), T4 RNA Ligase 2 is much more active joining nicks on double stranded RNA than on joining the ends of single stranded RNA. The enzyme requires an adjacent 5´ phosphate and 3´ OH for ligation. The enzyme can also ligate the 3´ OH of RNA to the 5´ phosphate of DNA in a double stranded structure (4).

Highlights

  • Best choice for ligating nicks in dsRNA
  • Suitable for ligating 3' OH of RNA to 5' phosphate of DNA in a DNA/RNA hybrid
  • Isolated from a recombinant source
  • Supplied with 10X Reaction Buffer

Product Source

An E. coli strain that carries the T4 RNA Ligase 2 gene (I. Schildkraut).

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
T4 Rnl2 Reaction Buffer10X

Advantages and Features

Applications

  • Ligate a nick in dsRNA
  • Ligate the 3´ OH of RNA to the 5´ phosphate of DNA in a double stranded structure

Properties and Usage

Unit Definition

One unit is defined as the amount of enzyme required to ligate 0.4 µg of an equimolar mix of a 23-mer and 17-mer RNAs in a total reaction volume of 20 µl in 30 minutes at 37°C.

Concentration:
10,000 units/ml

Reaction Conditions:
1X T4 Rnl2 Reaction Buffer

1X T4 Rnl2 Reaction Buffer:
50 mM Tris-HCl
2 mM MgCl2
1 mM DTT
400 µM ATP
pH 7.5 @ 25°C

Reaction Conditions

1X T4 Rnl2 Reaction Buffer
Incubate at 37°C

Storage Temperature

-20°C

Storage Conditions

10 mM Tris-HCl
50 mM KCl
35 mM (NH4)2SO4
0.1 mM DTT
0.1 mM EDTA
50% Glycerol
pH 7.5 @ 25°C

Heat Inactivation

No

Specific Activity

40,000 units/mg

Unit Assay Conditions

1X T4 Rnl2 Reaction Buffer and 0.4 μg of an equimolar mix of the 23-mer and 17-mer RNAs. After incubation at 37°C for 30 minutes, the ligated product is detected on a 15% polyacrylamide gel.

References

  1. Ho, C.K. and Shuman, S. (202). Proc. Natl. Acad. Sci. USA. 99, 12709-12714.
  2. Ho, C.K. et al. (2004). Structure. 12, 327-339.
  3. Nandakumar, J. et al. (2004). J. Biol. Chem.. 279, 31337-31347.
  4. Nandakuman, J. and Shuman, S. (2004). Mol. Cell. 16, 211-221.

FAQs

  1. What products does NEB offer for functional genomics and reverse genetics?

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

Selection Charts

Interactive Tools

Quality Control

Quality Control Assays

The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page. Further information regarding NEB product quality can be found here.
  • Endonuclease Activity (Nicking):
    The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
  • Exonuclease Activity (Radioactivity Release):
    The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
  • Phosphatase Activity (PNPP):
    The product is tested in a reaction containing a p-nitrophenyl phosphate (PNPP), a chromogenic substrate for most phosphatases. After incubation the percent degradation is determined by spectrophotometric analysis of released p-nitrophenol at 405 nm.
  • Protein Purity (SDS-PAGE):
    The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.
  • RNase Activity (2 Hour Digestion):
    The product is tested in a reaction containing a RNA substrate.  After incubation for 2 hour there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.

Safety Data Sheet

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

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.