The process of end to end ligation involves several steps:
Preparation of DNA Fragments: The DNA fragments to be ligated are first generated through restriction enzyme digestion or PCR amplification. Purification: The fragments are purified to remove any contaminants that might interfere with the ligation process. Setting Up the Ligation Reaction: The purified DNA fragments are mixed with a ligase enzyme (commonly T4 DNA ligase) and an appropriate buffer to facilitate the reaction. Incubation: The reaction mixture is incubated at a specific temperature to allow the ligase enzyme to catalyze the formation of phosphodiester bonds. Analysis: The ligated product is then analyzed using techniques such as gel electrophoresis or sequencing to confirm the success of the ligation.
