- Add appropriate amount of deionized H2O to sterile 0.6 mL tube
- Add 1 μL ligation buffer to the tube.
Vortex buffer before pipetting to ensure that it is well-mixed.
Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation.
- Add appropriate amount of insert to the tube.
- Add appropriate amount of vector to the tube.
- Add 0.5 μL ligase.
Vortex ligase before pipetting to ensure that it is well-mixed.
Also, the ligase, like most enzymes, is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 1 μL, just touch your tip to the surface of the liquid when pipetting.
- Let the 10 μL solution sit at 22.5°C for 30 mins
- Denature the ligase at 65°C for 10min
- Dialyze for 20 minutes if electroporating
- Use disks shiny side up
- Store at -20°C