Ligation of a DNA or RNA oligo to the 3’ end of long ssRNA using High Conc. T4 RNA Ligase 1 (NEB #M0437)
This protocol is for ligation of a DNA or RNA oligo donor with a 5'P to the 3'OH end of long ssRNA (> 0.3 kb).
| Component | 20 μl reaction |
|---|---|
| RNA acceptor* (>0.3 kb) |
up to 200 ng |
| RNA or DNA oligo donor** | 10 pmol |
| 10X T4 RNA Ligase Reaction Buffer | 2 ul (1X) |
| 100% DMSO (not included) |
2 μl |
| 100 mM ATP | 0.2 μl (1 mM) |
| 50% PEG 8000 | 5-10 μl (final 12.5-25%) |
| Rnase Inhibitor (40 U/ul) | 0.5 ul (1 U/μl) |
| T4 RNA Ligase I (High Concentration) |
1 ul (30 units) |
| Nuclease-free Water | To 20 μl |
| Incubation Time & Temperature | 2 hour at 25°C or 16 hour at 16°C |
*3-OH is required; ** 5-P is required;
Enzyme should be added at the last step.
