Consider the three major classes of biologically important molecules: proteins, lipids, and nucleic acids. Which steps of the miniprep procedure act on proteins? On lipids? On nucleic acids?

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enginr eNotes educator| Certified Educator

A miniprep procedure is used to isolate plasmid DNA from bacterial cells. The cells are first lysed and then the DNA is adsorbed onto a silica membrane. The membrane is then washed before the DNA is removed by elution. The procedure is relatively simply and involves a number of prepared buffer solutions that come with the kit. These buffer solutions are normally referred to as P1, PE, P2, N3 and PB. 

The bacterial pellet containing the plasmid of interest is initially re-suspended in buffer P1, which contains RNase A, glucose, Tris and EDTA. The EDTA prevents DNases from breaking down the plasmid. The RNase breaks down RNA following cell lysis.

Buffer P2 is then added. P2 contains SDS (a strong surfactant) and a base. P2 is the lysis buffer which breaks down the cell wall and membrane, denatures proteins, and separates double stranded DNA into single stranded DNA. 

The neutralization buffer, N3, is added. The small plasmids will recombine to double stranded DNA. However, the large genomic DNA gets tangled and precipitates out of solution with the other cell components. The plasmids remain in solution. The precipitate is removed by centrifugation.

The supernatent containing the plasmids is then added to the miniprep tubes which contain the silica membranes. The plasmid DNA binds to the membranes. The membranes are then washed with buffer PB (optional) and PE. 

Finally, buffer EB is added to the miniprep tubes. The plasmid DNA detaches from the silica membrane and is collected in the resulting elutant.

To summarize, lipids and proteins are primarily removed during the lysis procedure. With respect to nucleic acids, the RNA is also removed early in the procedure along with the genomic DNA, while the plasmid DNA is retained until the very end.