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  • 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.