To isolate plasmid DNA from bacterial cells, researchers use the alkaline lysis method, which relies on a precise sequence of chemical treatments. The process begins by centrifuging the bacterial culture to concentrate the cells into a pellet and discarding the supernatant liquid. Next, the cell pellet is resuspended in a glucose-Tris-EDTA buffer, which destabilizes the cell membrane without lysing the cells. Subsequently, an alkaline lysis solution containing sodium dodecyl sulfate (SDS) and sodium hydroxide is added. The SDS solubilizes the cell membrane lipids, while the sodium hydroxide denatures both the plasmid DNA and the larger chromosomal DNA. Crucially, before the mixture can degrade the DNA entirely, a potassium acetate neutralizing buffer is introduced. This acidic buffer restores neutral pH, causing the small circular plasmid DNA to rapidly renature and remain in solution, while the large chromosomal DNA and proteins precipitate out as a tangled white mass. After centrifuging this mixture to pellet the precipitate, the supernatant containing the dissolved plasmid DNA is transferred to a fresh tube. Finally, isopropanol is added to precipitate the plasmid DNA from the supernatant, which is then collected by a final centrifugation step.
Based on the passage, if a researcher transfers the liquid to a fresh tube prior to centrifuging the neutralized mixture, which of the following is the most likely consequence for the final isolated product?
- The final plasmid DNA product will be contaminated with chromosomal DNA and proteins.Cevap
- BThe cell membrane lipids will remain bound to the plasmid DNA in the final solution.
- CThe plasmid DNA will fail to renature and will remain permanently denatured.
- DThe sodium hydroxide will degrade the circular plasmid DNA before it can be collected.