Home Protocols Protocol for one-step adenylation and ligation of 5´p DNA and 3’OH RNA using Mth RNA Ligase (NEB #M2611) and Thermostable 5’ App DNA/RNA Ligase (NEB #M0319)

Protocol for one-step adenylation and ligation of 5´p DNA and 3’OH RNA using Mth RNA Ligase (NEB #M2611) and Thermostable 5’ App DNA/RNA Ligase (NEB #M0319)

  1. Set up the reaction as follows:

    Components Volume
     10X NEBuffer 1 2 μl
    50% PEG 8000 8 μl
    RNA Substrate (10 μM) 1 μl
    5′P-DNA linker (20 μM) 2 μl
    ATP (1 mM) 2 μl
     Thermostable 5′App DNA/RNA Ligase (20 μM) 1 μl
     Mth RNA Ligase (50 μM)  0.5 μl
     Nuclease-free H2O (NEB #B1500S) to 20 μl
  2. Incubate for 2 hours at 65°C in a PCR machine.
  3. Stop the reaction by adding 1 µl Proteinase K (NEB #P8107), incubated at 37°C for 15 minutes or quenching with EDTA (10 mM final concentration). Proteinase K treatment is recommended to release RNA bound by the ligase.

Note: 50% PEG 8000 is not included but can be purchased separately as part of T4 RNA Ligase Reaction Buffer pack (NEB #B0216L).