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):
   
   

   COMPONENTS	20 µl RXN	FINAL CONCENTRATION OR AMOUNT
   Nuclease-free Water	to 20 µl
   5X NEBNext Quick Ligase Reaction Buffer	4 µl	1X
   DNA Fragment		0.06 pmol
   Adaptor		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.