It is crucial to have a high-quality DNA that is free of contaminants like protein, debris and RNA prior to carrying out a PCR as well as cloning or DNA sequencing. Purifying DNA is also referred as DNA Isolation, and is an essential step in molecular biology. In this article, you will be taught the fundamentals of DNA purification as well as how to improve your DNA extraction processes for more efficient results.
The first step in the DNA purification procedure is to prepare a solution containing an amalgamation of alkaline buffer and water. This buffer makes DNA soluble, and it can be easily separated from other components in the sample. After the DNA is placed in an alkaline solution and a water solution, it is then treated with detergents and chaotropics salts to break up cell membranes and nuclei. This releases the DNA. RNase can also be added to eliminate any contamination RNA from the sample.
The DNA is then separated by organic solvents like chloroform and phenol from other cellular components such as proteins and fats. After the DNA is separated from lipids or proteins, it is then precipitated using alcohol or ruby alcohol.
Gel electrophoresis and spectrophotometers can be used to determine the quality of DNA. A good quality sample of DNA should have an absorbance ratio between 250 nm and 280nm. 1.8. A low ratio could indicate problems with the protein binding steps or salt carryover from wash or buffers for binding.