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)
- 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
- Incubate for 2 hours at 65°C in a PCR machine.
- 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).