Protocol for ligation of the 3´ OH of RNA to the 5´ pre-adenylated DNA with T4 RNA Ligase 2 truncated (NEB #M0242)
| 10X T4 RNA Ligase Reaction Buffer
| 50% PEG8000*
| RNA Substrate (10 μM)
| 5' pre-adenylated DNA linker (20 μM)
| 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.