Protocol for ligation of the 3´ OH of RNA to the 5´ pre-adenylated DNA with T4 RNA Ligase 2 truncated K227Q (NEB #M0351)
| 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 K227Q |
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.
