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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