The isolation and purification of genomic DNA requires three main processes which include, lysing of cells to release the DNA, purifying the solution by removing unwanted macromolecules, and precipitating the DNA from the solution. The process of isolation and purification requires several reagents. The Nuclei Lysis Solution contains lysozyme and EDTA. The lysozyme is an enzyme that degrades peptidoglycan, which is a rigid exoskeleton cell wall that protect the bacteria (3). Disrupting the peptidoglycan will result the release of the content inside the cell and the death of the cell. In addition to inhibiting DNases, the EDTA in the lysis solution aids the lysozyme to access the peptidoglycan by removing the Mg2+ from the lipopolysaccharide …show more content…
The focus of mini-prep is that plasmid isolation requires the separation from not only the cellular components but also from the genomic DNA. To achieve that, the protocol exploits the size and shape differences of genomic and plasmid DNA. The lysis buffer, Buffer P2, lyse the cells similarly to that of genomic isolation; however, NaOH and SDS are included in Buffer P2 to completely denature the genomic DNA and plasmid DNA. Noted that the two strains of plasmid DNA will remain interlocked in denaturing condition. The pH of the solution can be brought back to neutral with neutralization buffer (N3), which will allow the DNAs to return to their original state. The plasmid DNA will instantly return to circular supercoiled state. Genomic DNA, on the other hand, will become entangled with the precipitated cellular debris due to its long stands. The cellular debris including the genomic DNA can be removed by centrifuged while the plasmid remains in the solution. Instead of using alcohol precipitation, the plasmid DNA is eluted from QIAprep spin column as an alternative way of DNA collection. Mini-prep provides an adequate way of separating plasmid DNA from the genomic DNA. The extracted plasmid will be used as the vector for future DNA