Home Protocols RNA Circularization using T4 RNA Ligase 1(NEBM#0204)

RNA Circularization using T4 RNA Ligase 1(NEBM#0204)

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. Incubate at 65°C for 15min to terminate the reaction.