A high-quality DNA sample is required for a variety of downstream applications, including PCR and sequence. DNA purification is the process of removing contaminants like proteins and other components of the cell, from a specimen to produce a nucleic acid-free solution that is ready to use. There are a myriad of DNA purification techniques each with its particular advantages and disadvantages based on the source material or the application.
The first step of DNA purification is to eliminate protein from the sample by using a protease (proteinase enzyme) or mechanical disruption. After the cellular debris has been removed, the DNA can be precipitated using the aid of ethanol to create a white, stringy precipitate. The DNA that is precipitated is resuspended in water or a sterile buffer. The DNA’s concentration can be measured by an spectrophotometry method, based upon the peak of absorbance for nucleic acids at 260 nm.
Salting out is another common method for DNA purification. A column of cellulose is utilized in this click this link now method to capture and attach DNA. The cellulose matrix needs to be treated with detergents to remove any contaminants. A wash buffer is then used to get rid of the salts. DNA binds with the matrix under low salt conditions. Proteins and RNA that are contaminating are then removed using solutions with higher salt levels. The eluted DNA and RNA are then recovered using the ethanol precipitation.
Anion exchange is also a popular method for purifying DNA. This method employs a cation exchange resin to attract positively-charged DNA molecules, while a neutralizing resin allows negatively-charged DNA to be flushed out of the column. After the DNA has been extracted and then dissolved, it can be concentrated using centrifugation. The DNA is removed by washing with 70% ethanol at ice-cold temperatures.
Leave a Reply