• The temperature optimum for T4 DNA ligase mediated ligation in vitro is 16°C. However ligation is also achieved by incubation at 4°C by incubating over night or at room temperature condition by incubating for 30 minutes.
  
  
• Adenylate and DNA-adenylate are the important intermediates of the phosphodiester bond forming pathway.

Fig 2-4.1.1: The mechanism of DNA joining by DNA ligase.