Nmbhat:3-way ligation

Materials

 * 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

Method

 * 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&deg;C (room temperature) for 30 min.
 * 4) Place the tube at 65&deg;C for 10 min to denature the ligase.
 * 5) Store at -20&deg;C