Protocol for Ligation of Fragments with Splinted or Cohesive Ends using Immobilized T4 DNA Ligase (NEB #M0569)

Adaptor ligation can be accomplished using a molar excess of adaptor in a reaction using 1X NEBNext Quick Ligase Reaction Buffer.

  1. Before setting up reaction, mix beads thoroughly by pipetting up and down a minimum of 10 times.

  2. Set up the following reaction on ice (in this order):


    20 µl RXN


    Nuclease-free Water  to 20 µl  

    5X NEBNext Quick Ligase
    Reaction Buffer

    4 µl


    DNA Fragment


    0.06 pmol



    2.5 pmol

    Immobilized T4 DNA Ligase

    2 µl

    10 µg

  3. Gently mix the reaction by pipetting up and down.

  4. Incubate at 25°C for 15 minutes.

  5. Place tube on a magnet for 3 minutes to concentrate the beads and allow easy removal of the supernatant.

  6. Using a clean pipette tip, carefully transfer supernatant to new microfuge tube.

  7. Reaction can be used immediately, stored on ice for a short time (1-2 hours), or stored
    at -20°C indefinitely.