Spin Column Nucleic Acid Purification
The principle behind spin-column extraction builds on the fact that nucleic acids bind to silica. The process of spin column-based nucleic acid purification involves four main steps, which are: cell lysis, binding, washing up and elution.
To begin, cultured cells are lysed by using a lysis buffer that contains various detergents, salts, and inhibitors. Next, binding solution is added to the lysate and transferred to the column. The lysis buffer works to remove cellular membranes which enclose the DNA, exposing it to the chaotropic agents within the binding solution. These agents work to break the hydrogen bonds within the DNA molecule, providing the conditions necessary for silica to bind to nucleic acids.
The column is then centrifuged, forcing the nucleic acids (with the binding buffer) to pass through the column which contains the silica, and the flow through the solution is then discarded. So, in principle, the nucleic acids stay within the silica membrane in the column, and the extra liquid (like binding buffer) pass through. Afterward, the washing up process is performed. Wash buffer is added to the column and centrifuged to remove impurities. Finally, the elution buffer is added, and the column is centrifuged, allowing for nucleic acids to be collected for further research.
Beads Nucleic Acid Purification
Beads are another popular option which enables scientists to isolate nucleic acid in a fast and easy way. Silica is a common material for beads, but with an added magnetic coat.
The basic principle of using magnetic beads is similar to the working principle of spin columns. The cultured cells are lysed and a binding buffer which contains chaotropic agents are added to the lysate. The addition of binding buffer to the lysate leads to binding of nucleic acids to magnetic beads. Afterward, a magnetic force is applied, and all the magnetic beads (binding with targeted molecules) are aggregated together, and the unbound molecules are left within the solution. The supernatant which contains unbound molecules are removed and the magnetic beads are resuspended with elution buffer to release the bonded molecules. Finally, magnetic force is applied to the system again to facilitate the collection of the nucleic acid.