Protocol for ligation of the 3´ OH of RNA to the 5´ pre-adenylated DNA with T4 RNA Ligase 2 truncated KQ (NEB #M0373)
|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 KQ|| 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;