Learn about immobilized enzymes on beads and how to use NEB’s Immobilized T4 DNA Ligase.
Immobilized enzymes are enzymes that are bound to a solid support. They are often linked to a bead or a magnetic bead via a covalent bond, and they can be used for a variety of standard molecular biology workflows. Immobilized enzymes can be used in applications that are not compatible with heat inactivation. They are easily removed, and the enzyme can be re-used. This enables higher throughput and reduced sample loss. Here, we will demonstrate the use of Immobilized T4 DNA Ligase, NEB’s exciting new format of the classic enzyme T4 DNA Ligase.
The first step is bead preparation. It is important to ensure the beads are evenly dispersed. To do this, pipet the sample up and down slowly for a minimum of 10 times. Set the volume of your pipette to approximately 50% of the volume in the sample to avoid the formation of air bubbles. Do not vortex. The next step is the reaction set-up.
If the volume of Immobilized T4 DNA Ligase is less than 10% of the total reaction volume, then set-up the reaction in the following order: start by adding the water…then add ligase reaction buffer…then add the DNA substrates…and finally add the immobilized enzyme.
If the volume of immobilized T4 DNA Ligase is more than 10% of the total reaction volume, an additional set of steps is recommended. Pre-treat the enzyme as follows: First, add the immobilized T4 DNA Ligase to a fresh tube. Next, place the tube on a magnetic rack for 3 minutes to pellet the enzyme. In another tube, prepare the substrate in 1X reaction buffer. Then carefully remove the supernatant from the bead pellet and finally, add the substrate mixture to the bead pellet. Do not remove the supernatant until you are ready to assemble the reaction, as the beads should not be allowed to dry out. Be sure to mix the reaction well by pipetting up and down.
Next, incubate for the recommended amount of time and at the recommended temperature. If you choose to incubate the reaction longer than 30 minutes, agitation will be required to ensure the beads remain in suspension. Once incubation is completed, place the sample onto the magnetic rack. The beads will pellet on the side of the tube closest to the magnet. This may take 2 to 3 minutes. You can now remove the supernatant containing your ligated DNA, which will be ligase-free.
We hope these immobilized T4 DNA ligase usage guidelines for bead preparation and reaction set up have been helpful. For more information, visit neb.com/immobilized.