Bio-Rad Ligation and Transformation Module - Manual (Page 17)

DNA Ligation
Ligation is the process of joining two pieces of linear DNA into a single
piece through the use of an enzyme called DNA ligase. DNA ligase catalyzes
the formation of a phosphodiester bond between the 3'-hydroxyl on one
piece of DNA and the 5'-phosphate on a second piece of DNA.
The most commonly used DNA ligase is T4 DNA ligase (named because it
originated in a bacteriophage named T4). There are several ways that the
efficiency of DNA ligation can be optimized. First, like any enzyme, there
are conditions that are optimal for ligase activity:
• T4 DNA ligase requires ATP and magnesium ions for activity
• The concentration of vector and insert DNA in solution must be high for
efficient ligation
• The molar ratio of insert to vector DNA should be approximately equal,
although the optimal ratio may not be 1:1
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