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.