Escherichia Coli

Let’s start the introduction by talking about our main target, Escherichia coli or how it is mostly unknown, E.coli, are a diverse and large group of bacteria found food, the environment, and in the intestinal tracts of humans and animals were it normally inhabits and it can also cause infection in other parts of the body, especially the urinary tract. Its name was given by a German physician called Theodor Escherich and it is the most common member of the genus Escherichia. E.coli is “a Gram-negative, rod-shaped bacterium propelled by long, rapidly rotating flagella” (CredoReferences). It can be also found in water and soil. E.coli is of great use in laboratory research and categorized as one of the most thoroughly studied form of life. It …show more content…
Different methods for purification of plasmid exist; during this laboratory experiment specifically kits were used (Qiagen kit). Kits provide the detailed procedures to follow step-by-step and are simple and quicker to perform. This system serves as a fast and simple plasmid purification method for routine molecular biology laboratory applications (Qiagen). This procedure is based on alkaline lysis method of bacterial cells and the by absorption of DNA onto silica in the presence of high salt. Starting with the preparation and clearing of a bacterial lysate, then absorption of DNA, and finally the wash and elution of plasmid DNA are the three basic steps that conform the procedure. After the preparation and growth of the bacterial cell culture and the following clearing using centrifugation, the next step is purification …show more content…
The procedure uses different buffers to optimize the lysis procedure making sure just DNA is absorbed while undesired products are not retained and in opposite are found in the flow-through. These buffers are: P1 (a resuspension buffer that hydrolyzes not DNA but RNA since it contains the RNase enzyme), P2 (a lysis buffer, it opens, breaks or lyse the cell wall and contains SDS and NaOH), and N3 (a neutralization buffer that contains an acetate buffer which helps in the neutralization of NaOH and guanidine hydrochloride, which protagonist the denaturation of proteins, as well N3 aim in the DNA attachment to the gel membrane in the column due to its high concentration of salt). During the washing and elution processes, Buffer PB is used for a brief wash step and in this way efficiently get ride of the endonucleases and guarantee a no degraded DNA. Buffer PE is also used to wash and efficiently remove salts, highly efficient buffer for DNA cleanup procedures. It is composed of 10 mM Tris-HCl pH 7.5, 80% ethanol. Then Buffer EB or water is used to elute the good plasmid DNA from the column. It is important to mention that the efficiency of elution is dependent on pH. A higher efficiency is met at a pH of around 7.0-8.5. It is crucial to maintain this pH range due to the fact that DNA may possible degrade when no buffering agent to regulate the pH is