Before doing an PCR reaction or cloning experiment, or even DNA sequencing, it is essential to have a high-quality DNA that is free of contaminants, such as debris, protein and RNA. Purifying DNA is also referred as DNA isolation and is an essential step in molecular biology. This article will help you understand the fundamentals of DNA extraction and how to optimize it for better results.
The first step in the DNA purification process is to make a solution that consists of the mixture of alkaline buffer and water. This buffer makes the DNA more soluble so that it can easily be separated from the other components of the sample. After the DNA is placed in an alkaline solution and a water solution, it’s treated with detergents and chaotropics salts to break up cells’ membranes and nuclei. This allows the DNA to be released. RNase can be added to the sample to eliminate any DNA that is contaminating.
DNA is separated from other cellular components like proteins and lipids using organic solvents such as phenol and chloroform. After the DNA is removed from lipids or proteins, it is then precipitated using alcohol or rubbing alcohol.
Spectrophotometry and electrophoresis may be used to determine the quality of DNA. A high-quality DNA sample should have an absorbance of 260 nm up to at least 280 nm. 1.8. A low ratio could signal a problem in the protein binding steps, or salt carryover from the wash or binding buffers.
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