With the widespread adoption of molecular cloning as a routine laboratory technique, there is now a vast array of enzyme sources available from manufacturers for the generation and manipulation of nucleic acids. However, for those new to the field, the task of selecting the most suitable enzyme for a specific task can be daunting. This review aims to provide readers with valuable insights into the function and specificities of various sources of polymerases, ligases, nucleases, phosphatases, methylases, and topoisomerases commonly used in molecular cloning. We offer a comprehensive description of the most frequently utilized enzymes within each group, along with an explanation of their properties and mechanisms of action. By highlighting the key requirements for each enzymatic activity and addressing their limitations, our objective is to assist readers in making informed decisions when selecting the appropriate enzymatic source and determining the optimal experimental conditions for their molecular cloning experiments.