The Basics of DNA Purification

juni 6, 2024

DNA purification is a crucial component of many molecular assays such as PCR, qPCR, and DNA sequencing. It removes proteins that are contaminating salts, proteins, as well as other impurities which hinder the downstream process. It also ensures that the desired DNA is pure and present in order to be used for further analysis. The quality of DNA is measured by spectrophotometry (the ratio of A260 to A280), gel electrophoresis, and other methods.

The first step in the DNA purification process is cell lysis. In this process, the cellular structure is disturbed by reagents or detergents such as SDS to release DNA. To further purify DNA, protein-denatured reagents like sodium dodecylsulfate as well as Ethylene diamine tetraacetic acid (EDTA) are added to denature proteins. They then are removed from the nucleic acid solution using centrifugation and wash steps. If RNA is present in the sample then a ribonuclease treatment may be added to further denature the RNA. The nucleic acids then are concentrated in ice-cold water to separate them from other contaminants.

Ethanol is solvents to eliminate salts or other contaminants from nucleic acids. Researchers can compare results between experiments by using the standard ethanol concentration, which is a good option for workflows with high-throughput. Other solvents such as chloroform and phenol could be used, but these are more harmful and require additional steps to avoid cross-contamination with other cellular debris or proteins. Newer techniques can facilitate the DNA purification process using ethanol with low-ionic strength, which has been shown to be just as effective as traditional organic solvents in purifying DNA [2626. This is especially applicable when used in conjunction with spin column extract kits.

Polymerase chain reaction

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