New England Biolabs, M0242L, T4 RNA Ligase 2, truncated

Related Categories RNA Ligation Applications RNA Cloning Specification 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. FAQ Q: How can I increase ligation efficiency? A: In general, increased reaction time, lowered reaction temperature, and molecular crowding all yield more complete ligation reactions. Reaction times can be increased as long as 24 hours or more. For ligations longer than 2 hours, we routinely incubate below 16°C. Some scientists report ligation on ice (0°C) for 24 hours. We recommend the use of PEG 4000 or 8000 up to 15% (w/v). Increasing PEG concentrations further did not further increase ligation efficiency. Q: Is PEG 8000 available for purchase? A: PEG 8000 is included as a component of the T4 RNA Ligase Reaction Buffer (NEB #B0216). 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