DNA Purification Methods
A high-quality DNA specimen is essential for a variety of downstream applications, including PCR and sequencing. DNA purification is a method which removes contaminants from a sample, like proteins or other cellular components. This results in an acid solution that is ready to use. There are a variety of DNA purification methods, each with their particular advantages and disadvantages based on the source material or the application.
The first step in DNA purification is removing proteins from the sample using a proteinase (protein enzyme) or mechanical disruption. After the cellular debris is removed, the DNA may be precipitated by ethanol to form a white, stringy precipitate. The DNA precipitated by ethanol can be resuspended using water or a clean solution. The DNA concentration can be determined by the spectrophotometry method based on the nucleic acid absorbance peak at 260nm.
Another common DNA purification method is salting out, where the cellulose column is employed to capture and hold the DNA. The cellulose matrix is treated with detergents in order to remove contaminants, and then a wash buffer is used to flush out the salts. The DNA binds to the matrix under low salt conditions, and contaminating proteins and RNA can be removed with higher salt solutions. The eluted RNA and DNA are then recovered by ethanol precipitation.
Anion exchange is also a well-known method for cleaning DNA. This method uses resin that attracts positively charged DNA molecules, while an agent that neutralizes negatively charged molecules of DNA is used to clean the column. After the DNA has been extracted it can be concentrated by centrifugation. Then, the DNA can be precipitated out by washing it off with 70 percent ethanol at temperatures that are ice cold.