Since molecular cloning has become routine laboratory technique, manufacturers offer countless sources of enzymes to generate and manipulate nucleic acids. Thus, selecting the appropriate enzyme for a specific task may seem difficult to the novice. This review aims at providing the readers with some cues for understanding the function and specificities of the different sources of polymerases, ligases, nucleases, phosphatases, methylases, and topoisomerases used for molecular cloning. We provide a description of the most commonly used enzymes of each group, and explain their properties and mechanism of action. By pointing out key requirements for each enzymatic activity and clarifying their limitations, we aim at guiding the reader in selecting appropriate enzymatic source and optimal experimental conditions for molecular cloning experiments.