The Basics of DNA Purification
DNA purification is a vital component of many molecular assays that include PCR or qPCR, as well as DNA sequencing. It removes proteins that are contaminating, salts, and other impurities that interfere with the downstream process. It also ensures that the desired DNA is in good condition and pure to be further analysed. The quality of DNA is measured through spectrophotometry (the ratio of A260 to A280) as well as gel electrophoresis and other methods.
The initial step in the DNA purification process is cell lysis. In this process, the https://mpsciences.com/2021/02/15/science-supplies-for-students/ cell structure is destroyed by reagents or detergents such as SDS to release DNA. To further purify DNA, protein-denatured reagents like sodium dodecyl sulfate and Ethylene diamine tetraacetic acids (EDTA) are added to denature the proteins. Then, they are removed from the nucleic acid solution using centrifugation and wash steps. If there is RNA in the sample, a ribonuclease treatment could be added to further denature RNA. The nucleic acids are concentrated in ice-cold alcohol to separate them from other contaminants.
Ethanol is an everyday solvent that can be used to eliminate salts and other contaminants from nucleic acids samples. Researchers can compare results between tests using a standard ethanol concentration, which is a great choice for high-throughput workflows. Other solvents, such as chloroform or phenol can be used, however, they are more hazardous and require additional steps to avoid cross-contamination. Newer techniques can simplify the DNA purification process using low-ionic-strength ethanol that has been proven to be equally effective as the conventional organic solvents when purifying DNA [26]. This is especially true when combined with spin column extract kits.