Ligation Using T4 DNA Ligase Amrita University

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▶ This video channel is developed by Amrita University's CREATE • http://www.amrita.edu/create • ▶ Subscribe @ • / amritacreate • / amritavlab • ▶ Like us @ • / createatamrita • ▶ For more Information @ • http://vlab.amrita.edu/index.php?sub=... • ▶ Amrita Virtual Lab Project website • http://vlab.amrita.edu • • DNA ligation is the act of joining together DNA strands with covalent bonds with the aim of making new viable DNA or plasmids. • • The enzyme used to ligate DNA fragments is T4 DNA ligase, which originates from the T4 bacteriophage. This enzyme will ligate DNA fragments having overhanging, cohesive ends that are annealed together. • A ligation reaction requires three ingredients in addition to water: • 1. Two or more fragments of DNA that have either blunt or compatible cohesive ( sticky ) ends. • 2. A buffer which contains ATP. The buffer is usually provided or prepared as a 10X concentrate which, after dilution, yields an ATP concentration of roughly 0.25 to 1 mM. Most restriction enzyme buffers will work if supplemented with ATP. • 3. T4 DNA ligase. A typical reaction for inserting a fragment into a plasmid vector (subcloning) would utilize about 0.01 (sticky ends) to 1 (blunt ends) units of ligase.

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