Protocol for Ligation of Fragments with Cohesive Ends using Immobilized T4 DNA Ligase (NEB #M0569)
Fragment Joining: Cohesive Ends
- Before setting up reaction, briefly spin down Immobilized T4 DNA Ligase stock tube.
- Mix beads thoroughly by slowly pipetting up and down a minimum of 10 times. We recommend setting the volume of the pipette to 50% of the volume in the Immobilized T4 DNA Ligase stock tube, to avoid frothing or accidental aspiration of beads into the pipette during mixing.
- Set up the following reaction on ice (in this order):
*Recommendations are based on a standard ligation reaction in a cloning workflow. Molar ratios of the two DNA fragments being ligated (vector and insert) can be adjusted based on the relative size differential. In the example shown above, a 1:3 ratio has been used.COMPONENTS
20 µl RXN
FINAL CONCENTRATION OR AMOUNT
Nuclease-free Water
to 20 µl
10X T4 DNA Ligase Reaction Buffer
2 µl
1X
Vector DNA (4 kb)
50 ng
0.020 pmol*
Insert DNA (1 kb)
37.5 ng
0.060 pmol*
Immobilized T4 DNA Ligase**
1 µl
10 µg
** If IM T4 DNA Ligase is more than 10% of the total reaction volume, we recommend setting up the reaction as follows: Add IM T4 DNA Ligase into a fresh tube. Put the tube on a magnet for 3 minutes to pellet the beads. In another tube, prepare the substrate in 1X reaction buffer. Carefully remove the supernatant from the bead pellet. 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.)
- Gently mix the reaction by pipetting up and down. Incubate at 25°C for 30 minutes, or at 16°C overnight (if the incubation time is more than 30 minutes, agitation may be necessary to keep the ligase in suspension).
- 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. The supernatant contains ligated DNA which will be ligase-free.
