Plasmid DNA extraction is a commonly used technique in molecular biology laboratories for the purification of plasmid DNA from bacterial cells. In this laboratory report, we will describe the procedure for extracting plasmid DNA from Escherichia coli (E. coli) cells using a commercial kit.
The first step in the plasmid DNA extraction process is to prepare the bacterial culture. This involves growing the E. coli cells in a liquid growth medium, such as Luria-Bertani (LB) broth, until they reach the desired density. The cells are then harvested by centrifugation and resuspended in a lysis buffer, which contains enzymes that break down the cell wall and release the plasmid DNA.
Next, the lysed cells are treated with a proteinase K enzyme, which breaks down proteins and other cellular contaminants. The mixture is then incubated at a high temperature to denature the proteins and facilitate the binding of the plasmid DNA to a spin column.
After the incubation period, the mixture is transferred to a spin column and centrifuged, which allows the plasmid DNA to be selectively retained on the column while the contaminants pass through. The spin column is then washed with a series of buffers to remove any remaining contaminants, and the purified plasmid DNA is eluted from the column by adding a solution of low-salt buffer.
Finally, the eluted plasmid DNA is concentrated and purified using a spin filter, and the concentration is determined using a spectrophotometer. The purified plasmid DNA can then be used for various downstream applications, such as cloning, sequencing, or expression studies.
In conclusion, plasmid DNA extraction from E. coli cells is a straightforward and efficient method for purifying large quantities of high-quality plasmid DNA. The use of commercial kits simplifies the procedure and reduces the risk of contamination, making it a widely used technique in molecular biology laboratories.
