Protocol Ligation of an oligo to the 3’end of RNA using T4 RNA Ligase 1(M0204)



  1. Set up a 20 μl reaction as follows: 
    1 X T4 RNA Ligase Reaction Buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM DTT) 
    15-25% (wt/vol) PEG 8000
    10% DMSO (optional)
    0.5 μl RNase Inhibitor, Murine (M0314 - 40U/ul, optional)
    1 μl (10 units) T4 RNA Ligase 1
    1 mM ATP
    20 pmol RNA
    40-200 pmol DNA or RNA oligo

  2. Incubate at 25°C for 2 hours or at 16°C for 16 hours

  3. Stop the reaction by column cleanup with Monarch® RNA Cleanup Kit (10 μg) (T2030)

1) The ssRNA acceptor should have a 3’-OH group
2) The DNA or RNA donor should have a 5’-PO4 group and blocked at the 3’ end
3) The reaction can also be stopped by adding an equal amount of 25 mM EDTA (pH8.0)