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.
- Before setting up reaction, mix beads thoroughly by pipetting up and down a minimum of 10 times.
- Set up the following reaction on ice (in this order):
20 µl RXN
FINAL CONCENTRATION OR AMOUNT
Nuclease-free Water to 20 µl
5X NEBNext Quick Ligase
Immobilized T4 DNA Ligase
- Gently mix the reaction by pipetting up and down.
- Incubate at 25°C for 15 minutes.
- Place tube on a magnet for 3 minutes to concentrate the beads and allow easy removal of the supernatant.
- Using a clean pipette tip, carefully transfer supernatant to new microfuge tube.
- Reaction can be used immediately, stored on ice for a short time (1-2 hours), or stored
at -20°C indefinitely.