Home Protocols RNA Circularization using T4 RNA Ligase 1

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)
    Single-stranded RNA with 5´P and 3´OH ends (200 ng-1 µg) 
    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.