A quality DNA sample is vital for a variety of downstream applications, including PCR and sequencing. DNA purification is a procedure that removes contaminants from the sample, for example proteins or other cell components. This results in an nucleic acid solution that is ready to use. There are a variety of DNA purification methods, each with their own advantages and drawbacks depending on the starting material or the application.
The initial step in DNA purification is to remove proteins from the sample by using a protease (proteinase enzyme) or mechanical disruption. After the cellular debris has been eliminated, DNA can be precipitated by the aid of ethanol to create a stringy, white precipitate. The DNA that is precipitated can be resuspended, either in water or a sterilized solution. The amount of DNA in the buffer can be determined by spectrophotometry, based on the nucleic acid absorbance peak at 260nm.
Salting out is a common method for DNA purification. A cellulose column is used for this procedure to capture and bind DNA. The cellulose matrix is cleaned with detergents to remove contaminants, and then the wash buffer is used to wash away the salts. The DNA binds to the matrix when the salt levels are low, and contaminating proteins and RNA can be removed with higher salt solutions. The DNA that is eluted is recovered through precipitation with ethanol.
Anion exchange is a popular technique for DNA purification. This method employs a cation exchange resin to attract positively-charged DNA molecules, while neutralizing resins allow negatively charged RNA to be ejected of the column. After the DNA has why not look here been eluted from the anion exchange column it can be concentrated by centrifugation, and then washed using ice-cold 70% ethanol to precipitate out the DNA.