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

 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.