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Plasmids are small double-stranded DNA pieces of DNA that exist outside the chromosome of the cell in which they are located. Almost all plasmids are circular. They contain a small number of genes, which can include genes that make cells resistant to antibiotics. Plasmids are used extensively in cloning DNA. You insert some foreign DNA into the plasmid DNA, and then infect cells with the DNA in a process called transformation.
There are several ways to purify plasmids. These methods are often called “minipreps” because you are purifying DNA from a small volume of cells. Minipreps involve lysing the cells and purifying the DNA via centrifugation and/or membrane binding. The method we will do uses a silica-gel membrane to bind the DNA, which has been developed by the company Qiagen. You will purify the plasmid DNA and analyze it.
We lyse the cells using a modified alkaline lysis procedure. This technique was invented by Birnboim and Doly. Other types of minipreps may use a different procedure for lysing the cells. In general, in the alkaline lysis method, the cells are brought to a high pH to not only lyse the cells, but also to denature the DNA. The DNA solution is then neutralized. Since plasmid DNA is circular and supercoiled, when the pH is brought back down to neutral, the plasmid DNA snaps back to being double-stranded. By contrast, genomic DNA is so large that it is broken into linear pieces no matter what we do. The linear DNA denatures in alkali and forms precipitates when the pH is lowered. The precipitate is removed via centrifugation. The supernatant is then applied to the silica-gel membrane to further purify the plasmid DNA. Under the right conditions double-strand plasmid DNA sticks to the membrane while single-strand DNA and RNA do not. The membrane does not require the use of organic solvents and lets us easily wash the DNA to remove contaminants. We then elute the DNA from the membrane by using a low-ionic strength buffer. The plasmid DNA that we obtain is very pure, and can be restricted or used in DNA sequencing.

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