New England Biolabs, M0239L, T4 RNA Ligase 2 (dsRNA Ligase)

T4 RNA Ligase 2 ligates nicks in dsRNA and can also be used to ligate the 3´ OH of RNA to the 5´ phosphate of DNA in a double stranded structure. Related Categories RNA Ligation Specification 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. Reaction Conditions 1X T4 Rnl2 Reaction Buffer Incubate at 37°C 1X T4 Rnl2 Reaction Buffer 50 mM Tris-HCl 2 mM MgCl2 1 mM DTT 400 µM ATP (pH 7.5 @ 25°C) Storage Buffer 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 80°C for 5 minutes 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. FAQ Q: How can I achieve optimal ligation of a 5’ phosphorylated ssDNA oligo to the 3’ end of an ssRNA substrate with a ssDNA or ssRNA splint using T4 RNA Ligase 2? A: The reaction buffer included with this enzyme is optimized for nick ligation in dsRNA. To optimize the ligation of nicks in DNA/RNA hybrids, we recommend increasing the magnesium concentration from 2mM to 10mM in the reaction. Addition of 10-15% PEG8000 to the final reaction will further boost ligation efficiency. The current buffer is good for nick ligation in dsRNA, but not for nick ligation in DNA/RNA hybrid. Q: What is the salt tolerance of this RNA ligase? A: NEB # RNA Ligase Salt Tolerance M0204 T4 RNA Ligase 1 (ssRNA Ligase) ≤ 50 mM M0239 T4 RNA Ligase 2 (dsRNA Ligase) ≤ 50 mM M0242 T4 RNA Ligase 2, truncated ≤ 100 mM M0351 T4 RNA Ligase 2, trunc. K277Q ≤ 100 mM M0373 T4 RNA Ligase 2, trunc. KQ ≤ 100 mM M0375 SplintR Ligase ≤ 50 mM E2610 5' Adenylation Kit (Mth Ligase) ≤ 300 mM M0319 Thermostable 5' App DNA/RNA Ligase ≤ 50 mM