Increasing the concentration length of incubation, and adding PEG can increase the number of intramolecular ligations. Blocking one end of the molecule with a dideoxy terminator will prevent the molecule from forming a circle.
T4 RNA Ligase Reaction Buffer
- Set up a 20 μl reaction as follows:
1 X Reaction Buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM DTT)
RNA (example: 10 μM 5´-[32P]rA20, 10 μM in 5´ termini)
1 μl (10 units) T4 RNA Ligase
0.5 μl Rnase inhibitor (M0314 , 40 μ/ul)
20-50 μM ATP
- Incubate at 25°C for 1-2 hours.
For longer oligos, overnight incubation at 16°C may improve yield.
- Boil for 2 minutes to terminate the reaction.