Assemble reaction mix into 10 µL volume in a microfuge tube. Reaction may be scaled up to 20 µL if DNA concentrations are low.
Add reagents in following order: water, buffer, insert, vector, T4 ligase.
Gently mix by stirring gently with pipette tip.
Typical Incubation time and temperature is 15°C for at least 4 hours. Incubation at 4°C for overnight is commonly used.
Prepare a control that excludes the insert. This allows for detection of re-annealing due to incomplete vector digestion (If vector is not completely digested the colonies generated by the vector reaction will greatly outnumber that of the vector + insert reaction).
Ligation Reaction can be inactivate d by incubation at 65°C for 20 minutes. This step is optional.