Set up the labeling reaction at room temperature until the ligase is added to avoid DMSO precipitation. The addition of DMSO is essential.
- Set up a 30 μl reaction as follows:
1 X Reaction Buffer (50 mM Tris-HCl, pH 7.8, 10 mM MgCl2, 10 mM DTT)
1 μg RNA
10 % (v/v) DMSO
1 μM [32P]pCp
1 μl (20 units) T4 RNA Ligase
- Incubate overnight at 16°C