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
Component |
Volume |
---|---|
H2O |
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) |
Total |
20 µl |
- Incubate for 1 hour at 25°C.
- 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.