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  • RNA Circularization using T4 RNA Ligase 1(M0204)

    Introduction

    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.

    Buffers

    T4 RNA Ligase Reaction Buffer

    Protocols

    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.