The Basics of DNA Purification

DNA purification is a vital component of many molecular assays that include PCR as well as qPCR and DNA sequencing. It removes contaminants, such as proteins, salts and other impurities that can interfere with downstream processes. It also ensures the desired DNA is completely clean and available in a way that it can be used for further analyses. The quality of DNA is assessed using spectrophotometry (the ratio of A260 to A280), gel electrophoresis, and a variety of other methods.

In the initial stage of a DNA purification procedure the cellular structure will be disrupted by detergents or reagents such as SDS to release DNA. To further purify the DNA, protein-denatured reagents such as sodium dodecylsulfate and Ethylene diamine tetraacetic acid (EDTA) are added to denature proteins. They are then removed from the nucleic acids solution through centrifugation and wash steps. If RNA is present in the sample and is not removed, it can be denatured by adding ribonuclease. The nucleic acid is then concentrated in ice-cold water to separate them from other contaminants.

Ethanol is an everyday solvent that can be used to eliminate salts and other contaminants from nucleic acid samples. Researchers can compare results between tests using an ethanol concentration that is standard, which is a good choice for high-throughput workflows. Other solvents, such as chloroform or phenol, can be used, however, they are more toxic and require additional steps to avoid cross-contamination. Newer methods can make it easier to complete the process of DNA purification by using ethanol with a low-ionic strength that has been proven to be as effective as the conventional organic solvents when purifying DNA [2626. This is particularly relevant when used in conjunction with spin column-based extract kits.

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