DNA purification is a vital step in many molecular assays that include PCR as well as qPCR and DNA sequencing. It removes contaminating proteins as well as salts and other impurities that can hinder the downstream process. It also ensures the desired DNA is pure and available to be used for further analyses. The quality of DNA is determined through spectrophotometry (the ratio of A260 to A280) as well as gel electrophoresis and other methods.
The first step in a DNA purification procedure is cell lysis, where the cellular structure is broken with detergents or reagents like SDS to release DNA. To further purify the DNA, reagents with protein denature such as sodium dodecyl sulfate and Ethylene diamine tetraacetic acid (EDTA) are used to denature proteins. Then, they are removed from the nucleic acid solution with centrifugation and washing steps. If there is RNA in the sample then a ribonuclease treatment may be added to further denature RNA. The nucleic acids are then concentrated in ice-cold alcohol to separate them from other contaminants.
Ethanol is a popular solvent that can be used to eliminate salts and other contaminants from nucleic acids samples. A standard concentration of ethanol permits researchers to compare results across studies, making it a suitable choice for workflows that require high-throughput. Other solvents, like chloroform or phenol, could be utilized, however they are more toxic and require additional steps to avoid cross-contamination. The purification of DNA is made easier by using ethanol with a low ionic strength. This has been shown to work just as conventional organic solvents at making DNA purer. This is particularly relevant when used in conjunction with a spin column extraction kit.