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Cat. No.: T4RL2-1k (for 1KU)

Cat. No.: T4RL2-5k (for 5KU)

Description

T4 RNA Ligase 2 is an ATP-dependent double-stranded RNA ligase (dsRNA Ligase). It is also referred to as T4 Rnl2 (gp24.1) and is used for intramolecular circularization and intermolecular linear ligation of double-stranded RNA. T4 RNA Ligase 2 differs from T4 RNA Ligase 1 in that its activity for ligating nicks in double-stranded RNA is significantly higher than its activity for ligating single-stranded RNA ends. T4 RNA Ligase 2 can also be used for the ligation of 3' hydroxyl groups of RNA chains and 5' phosphate groups of DNA chains within or between double-stranded nucleic acids (double-stranded RNA, RNA/DNA hybrids, and/or DNA double-strands). The ligation reaction of T4 RNA Ligase 2 requires the presence of a 5' phosphate and a 3' hydroxyl group, which can occur between the 5' phosphate of an RNA or DNA chain and the 3' hydroxyl group of an RNA chain.

The reaction process of T4 RNA Ligase 2 for the ligation of dsRNA sticky ends is as follows: First, T4 RNA Ligase 2 directly consumes ATP, forming an intermediate product called T4 RNA Ligase 2-AMP and releasing pyrophosphate. Then, T4 RNA Ligase 2-AMP binds to the nick site of the dsRNA and transfers the AMP from the intermediate product T4 RNA Ligase 2-AMP to the 5' phosphate end of the nick site of the dsRNA, forming an adenylated nicked dsRNA intermediate. Finally, under the catalysis of T4 RNA Ligase 2, the 3' hydroxyl group of the nick site in the dsRNA attacks the 5' phosphate group at the same site, forming a 3'-5' phosphodiester bond and releasing AMP.

Application

T4 RNA Ligase 2 is primarily used for ligating nicks in double-stranded RNA (i.e., ligation of sticky ends in double-stranded RNA). It can also be used for the ligation of 3' hydroxyl groups of RNA and 5' phosphate groups of DNA within double-stranded structures.

Source

Recombinant protein expressed in Escherichia coli, with the expressing gene derived from T4 bacteriophage T4 RNA ligase 2.

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.

Assay Conditions

50 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 1 mM DTT, 400 µM ATP, incubation at 37°C for 30 minutes.

Purity

Free of DNA endonucleases and exonucleases, RNases, and phosphatases.

Enzyme Storage Solution

10 mM Tris-HCl (pH 7.5), 50 mM KCl, 35 mM (NH4)2SO4, 0.1 mM EDTA, 0.1 mM DTT, 50% (v/v) Glycerol.

10X Reaction Buffer

500 mM Tris-HCl (pH 7.5 at 25℃), 20 mM MgCl2, 10 mM DTT, 4 mM ATP.

Inactivation or Inhibition

T4 RNA Ligase 2 can be inactivated by heating at 85°C for 5 minutes, or its activity can be inhibited by adding proteinase K or EDTA.

Precautions

• If the substrate for ligation is ssRNA, we recommend using products such as T4 RNA Ligase 1.
• Select the appropriate operational method based on your specific application. Additional reagents may be required, such as RNase inhibitor, DEPC water, etc.
• The provided T4 Rnl2 Reaction Buffer (10X) is suitable for ligation reactions involving nicks in double-stranded RNA. If you are performing ligation reactions involving the connection of RNA 3' hydroxyl groups and DNA 5' phosphate groups in RNA/DNA hybrid chains, it is necessary to increase the final concentration of MgCl2 to 10 mM and add PEG8000 to a final concentration of 10-15% in the reaction system. This will significantly enhance enzyme activity without affecting reaction characteristics.
• This product is intended for scientific research purposes by professionals only. It should not be used for clinical diagnosis or treatment, food or drug purposes, and should not be stored in a residential setting.
• For your safety and health, please wear appropriate lab attire and disposable gloves when handling the product.

Storage

The minimum shelf life is 1 year at -20°C.

Related:

• T4 RNA Ligase
• T4 RNA Ligase 1 (ssRNA Ligase)
• T4 RNA Ligase 2 (dsRNA Ligase)
• T4 RNA Ligase 2, truncated
• T4 RNA Ligase 2, truncated KQ

SBS Genetech is recognized as one of the global major leading industry players in Ligase Market by third-party market researchers. For more details, please visit Ligase Market - A Global and Regional Analysis Focus on Product, Source, Application, End User, and Country - Analysis and Forecast, 2022-2032