1. Mix each reaction described in the materials.

Assuming a 3:1 insert-to-vector ratio, the amount of insert to add can be calculated as 3 x (amount of vector) x (length of insert) / (length of vector).

Scale the reaction if DNA concentrations are low.

Add the ligase last.

Avoid repeated freeze/thaw cycles of the ligase buffer.

2. Incubate following the ligase manufacturer's instructions.

3. Denature the ligase by incubating at 65°C for 10 min.

4. Immediately proceed with transformation or store at -20°C.

If many colonies result from the negative (vector + ligase) control, the vector was not completely digested and/or phosphatase treatment did not work.

Additional controls can also be used to troubleshoot failed ligations.