Protocol for ligation of Nicks in dsRNA with T4 RNA Ligase 2 (NEB #M0239)

Step 1: Preparation of Nicked dsRNA substrates

The RNA mixture (at equal molar ratio) was heated at 65°C for 3 min, followed by putting on ice for 2 min. The stock can be stored at −20°C.

Step 2: Nick Ligation in dsRNA




15 µl 

10X Reaction Buffer (NEB #B0239S)

2 µl

Nicked dsRNA Substrate (10 µM) 

2 µl

T4 RNA Ligase 2

1 µl (10U)


20 µl

  1. Incubate for 1 hour at 25°C.

  2. Stop reaction by adding Proteinase K or quenching with EDTA.