Ligation of a DNA or RNA oligo to single stranded RNA using T4 RNA Ligase 1

This protocol is intended to ligate a DNA or RNA oligo donor with 5'P to the 3'OH end of a single-stranded RNA acceptor.

Component  20 μl reaction 
 ssRNA acceptor*  1-20 pmol
RNA or DNA oligo donor**  5-40 pmol 
10X T4 RNA ligase buffer  2 ul (1X) 
10 mM ATP  2 ul (1 mM) 
50% PEG8000  5-10 ul (final 15-25%) 
Rnase inhibitor (40 u/ul)  1 ul (2 u/ul) 
T4 RNA ligase 1 Enzyme   1 ul (10 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.