Blunt end ligation typically involves the following steps:
1. Preparation of DNA Fragments: The DNA fragments to be ligated must have blunt ends. This can be achieved using specific restriction enzymes or through the action of DNA polymerases that fill in or chew back overhangs. 2. Addition of Ligation Buffer: A ligation buffer containing ATP is added to facilitate the activity of DNA ligase. 3. Enzyme Addition: T4 DNA Ligase is commonly used to catalyze the formation of a phosphodiester bond between the 5' phosphate and 3' hydroxyl groups of the blunt-ended fragments. 4. Incubation: The reaction mixture is incubated under optimal conditions, typically at 16°C overnight, allowing the ligase to join the DNA fragments.
