Protocol for ligation of the 3´ OH of RNA to the 5´ pre-adenylated DNA with T4 RNA Ligase 2 truncated (NEB #M0242)
| Component |
Volume |
| 10X T4 RNA Ligase Reaction Buffer |
2 μl |
| 50% PEG8000* |
4 μl |
| RNA Substrate (10 μM) |
2 μl |
| 5' pre-adenylated DNA linker (20 μM) |
2 μl |
| T4 RNA Ligase 2 truncated |
1 μl (200U) |
| Nuclease-free water |
To 20 μl |
Incubate for 1-2 hour at 25°C.
Stop reaction by adding Proteinase K or quenching with EDTA.
* PEG8000 could be added up to 25% in the reaction for boosting ligation efficiency.
