T4 RNA Ligase II, also known as T4 Rnl2 (gp24.1), catalyses the formation of an internucleotide phosphodiester bond between an oligonucleotide donor molecule with a 5′-terminal phosphate and an oligonucleotide acceptor molecule with a 3′-terminal hydroxyl. 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 nucleic acids. T4 RNA ligase II is expressed and purified from E.coli.
Applications- Ligate a nick in dsRNA- Ligate the 3´ OH of RNA to the 5´ phosphate of DNA in a double stranded structure Reagents Supplied & Storage Condition- T4 RNA Ligase II : 10 unit/㎕, Store at -20°C.- 10x T4 RNA Ligase II Reaction Buffer : Store at 4°C. Reaction Condition- T4 RNA Ligae II in 1X T4 RNA RNA ligase II Reaction Buffer. Incubate at 37°C. 10x Reaction Buffer- 500 mM Tris-HCl (pH 7.5), 20 mM MgCl₂, 10 mM DTT, 4 mM ATP. Storage Buffer- 10 mM Tris-HCl (pH 7.5), 50 mM KCl, 35 mM (NH₄)₂SO₄, 0.1 mM DTT, 0.1 mM EDTA, 50% Glycerol. 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. QC Tests
- Activity, exo and endonuclease activity test, SDS-PAGE purity, performance tests.