Protocol for ligation of 3′OH of RNA and 5′PO4 of DNA in ds structure with T4 RNA Ligase 2 (NEB #M0239)

Step 1: Preparation of Nicked DNA/RNA hybrid substrates

The DNA/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 DNA/RNA hybrid




9.4 µl 

10X Reaction Buffer (NEB #B0239S)

2 µl

MgCl2 (100mM)

1.6 µl

50% PEG8000

4 µl

Nicked DNA/RNA 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.

Note: If using T4 RNA Ligase reaction buffer (NEB #B0216S) instead, no extra MgCl2 is needed in the reaction.