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