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DEVELOPMENT OF NEW METHODS FOR PURE PLASMID DNA ISOLATION WITHOUT USING COMMERCIAL KITS

Avsar, A.R. (2023) I am responsible for conducting experiments in the laboratory. Additionally, I contribute to report writing and literature review. Cakıroglu, A. (2023). Alp Cakıroglu, my team member, is responsible for literature review and report writing


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The Project Summary: Although the purpose of already available plasmid isolation methods is to provide high-purity plasmids, some of them can not achieve the desired goal. For this reason, commercial kits that provide high-quality isolation are used. However, these kits are expensive and difficult to obtain from abroad, especially when pandemic conditions are under question. Unlike ready-made isolation kits, manual plasmid isolation is inexpensive, and the degree of purity obtained varies according to the design of the study. The most common manual method for pDNA purification after isolation is phenol-chloroform-isoamyl alcohol extraction. Since the chemicals used in the method are toxic and harmful to the environment, their use should be limited. In this project, bacteria were multiplied, and reusable isolation columns filled with hydrophobic materials were created. These columns are similar to those used in the commercial kits, but cheaper and economical. Also, they are less harmful to the environment as compared to the phenol-chloroform-isoamyl alcohol purification. The efficiency of the columns in the isolation and purification of plasmids was evaluated both qualitatively by agarose gel electrophoresis and quantitatively by spectrophotometrical measurements. When pDNA isolation was performed with a fiberglass column, it was observed that the amount of obtained plasmid increased with the increase of column material. The increse in the amount of pDNA showed logarithmic correlation with the amount of column material. On the other hand the amount of pDNA obtained with silica columns showed an exponential increase with the increase in the amount of silica. It was observed that the amount of pDNA obtained with both silica columns and fiberglass columns was higher than other purification methods. The fiberglass filters provided low yield and the test results were highly variable. Therefore, no correlation could be established for fiberglass filters.

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