Nmbhat:3-way ligation

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


10.5μL ligation mix

  • 1.0 µL T4 ligase buffer
  • 3.0 µL insert A
  • 3.0 µL insert B
  • 3.0 µL vector
  • 0.5 µL T4 DNA ligase


  1. Vortex each item before adding it to ensure that it is uniformly mixed.
  2. Add them to the tube in the order given above. When pipetting the ligase, be sure not to get too much glycerol on the sides of the pipet tip.
  3. Let sit at 22.5°C (room temperature) for 30 min.
  4. Place the tube at 65°C for 10 min to denature the ligase.
  5. Store at -20°C