1. Cell lysis and protein denaturation
In the initial stage of plasmid DNA extraction, cells need to be lysed to release the DNA inside. This process is usually achieved by using high concentrations of salts, detergents, or enzymes. In this process, the main role of Tris is to maintain the pH of the solution stable, thus protecting the DNA from the acidic environment generated during the lysis process. In addition, Tris can also help to isolate DNA from lysates by binding to and denaturing proteins.
2. DNA solubilization and stabilization
After cell lysis, plasmid DNA needs to be lysed and isolated from other cellular components. This process is usually achieved by using a high concentration of salt or detergent. In this process, the main role of Tris is to provide a suitable ionic strength and pH environment to promote the solubilization and stabilization of DNA. The hydroxyl and amino groups of Tris can form hydrogen and ionic bonds with DNA molecules, which stabilize its structure and prevent its degradation. In addition, Tris can further protect DNA from degradation by inhibiting the activity of nucleases.
3. DNA purification and recovery
After the plasmid DNA is isolated from the lysate, a series of purification steps are often required to remove residual proteins, RNA, and other impurities. These steps include phenol/chloroform extraction, ethanol precipitation, etc. In this process, the main role of Tris is to maintain the pH of the solution stable, thus protecting the DNA from the acidic or alkaline environment that may arise during purification. In addition, Tris can also help recover pure plasmid DNA from solution by binding to impurities and allowing them to precipitate.
