New England Biolabs, M0319S, Thermostable 5´ App DNA/RNA Ligase

Thermostable 5´ App DNA/RNA Ligase ligates a 5´ pre-adenylated 3´-blocked DNA linker to the 3´-OH of RNA and ssDNA. Related Categories RNA Ligation,, DNA Ligases Applications Non-Cloning Ligation Specification Reaction Conditions 1X NEBuffer™ 1 Incubate at 65°C 1X NEBuffer™ 1 10 mM Bis-Tris-Propane-HCl 10 mM MgCl2 1 mM DTT (pH 7 @ 25°C) Storage Buffer 50 mM KCl 10 mM Tris-HCl 0.1 mM EDTA 1 mM DTT 50% Glycerol pH 7.5 @ 25°C Heat Inactivation No FAQ Q: How can you synthesize an adenylated DNA linker? A: An efficient method is to use the 5’ DNA adenylation kit (NEB# E2610) to convert pDNA to AppDNA. Q: Can I use the wild type Mth RNA ligase included in the 5’ DNA adenylation kit instead of the Thermostable 5’AppDNA/RNA ligase for library construction? A: No. Wildtype Mth is very efficient at adenylation, but not ligation. Q: How much Thermostable 5’AppDNA/RNA ligase do I need to add to a reaction? A: The ligase is a dimer and has very low turnover so add at least a two molar excess of the enzyme to substrate. Q: Why do you recommend adding MnCl2 to the DNA ligation but not the RNA reaction? A: The MnCl2 is not included in the RNA ligation to reduce metal mediated breakdown of the RNA. Q: Does the Thermostable 5’ AppDNA/RNA ligase ligate dsRNA or dsDNA? A: No, the enzyme only uses single stranded substrates. Q: Will PEG or increased time enhance the amount of ligated product? A: PEG does not enhance ligation. There may be an increase in ligated product when the incubation time is increased. Q: Why is the RNA running as a smear or stuck in the well? A: The enzyme binds tightly to the RNA which may result in it running as a smear or stuck in the well. Heating at 90°C for 3 minutes or treatment with Proteinase K releases the RNA; for some samples Proteinase K treatment is required. 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