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  • RNA Circularization using T4 RNA Ligase 1(M0204)


    Increasing the concentration length of incubation, and adding PEG can increase the number of intramolecular ligations. Blocking one end of the molecule with a dideoxy terminator will prevent the molecule from forming a circle.


    T4 RNA Ligase Reaction Buffer


    1. Set up a 20 μl reaction as follows:
      1 X Reaction Buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM DTT)
      RNA (example: 10 μM 5´-[32P]rA20, 10 μM in 5´ termini)
      1 μl (10 units) T4 RNA Ligase
      0.5 μl Rnase inhibitor (M0314 , 40 μ/ul)
      10% PEG8000
      20-50 μM ATP
    2. Incubate at 25°C for 1-2 hours.
      For longer oligos, overnight incubation at 16°C may improve yield.
    3. Boil for 2 minutes to terminate the reaction.