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Reagents

• T4 DNA ligase
• 10x T4 DNA Ligase Buffer
• Deionized, sterile H2O
• Purified, linearized vector (likely in H2O or EB)
• Purified, linearized insert (likely in H2O or EB)

Method

1. Add appropriate amount of deionized H2O to sterile 0.6 mL tube
2. Add 1 μL ligation buffer to the tube.
3. Vortex buffer before pipetting to ensure that it is well-mixed.
4. Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation.
5. Add appropriate amount of insert to the tube.
6. Add appropriate amount of vector to the tube.
7. Add 0.5 μL ligase.
8. Vortex ligase before pipetting to ensure that it is well-mixed.
9. 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 0.5 μL, just touch your tip to the surface of the liquid when pipetting.
10. Let the 10 μL solution sit at 22.5°C for 30 mins
11. Denature the ligase at 65°C for 10min
12. Dialyze for 20 minutes if electroporating
13. Use disks shiny side up
14. Store at -20°C