Bio-rad Ligation and Transformation Module Manuel d'utilisateur télécharger pdf (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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Bio-rad Ligation and Transformation Module Manuel d'utilisateur Page 17