DNA purification

Online Free life science study material. Hi DNA Purification: There are three types of DNA Total cell DNA, Plasmid DNA and Phage DNA. For purifying Total Cell DNA: Brief summary: First we culture cells, the culture is then centrifuged to get pellet of cells. From the pellet cell extract is removed from which pure DNA is separated which is then concentrated for later use. DNA purification: Lysozyme enzyme is added to cell extract which digest polymeric compound that give rigidity to cell wall. For removal of lipid SDS is added. For removal of protein,protease is added for protein digestion and then phenol and chloroform in 1:1 ratio or only phenol is adde to precipitate protein. Ribonuclease is used to degrade RNA. Concentration of DNA: Organic extraction gives concentrated DNA, rest procedures give dilute DNA so to concentrate it following procedure is used. Ethanol is layered over DNA solution and DNA molecule is precipitated at interface. A glass rod is used to pull out long fibre. Alternatively ethanol is mixed with dilute DNA solution, the precipitate can be collected by centrifugation. This method leaves short chain of nucleic acid and RNA is lost at this stage. Measurement of DNA concentration: UV absorbance spectrophotometer are used to accurately measure DNA concentration. Amount of UV absorbed is directly proportional to amount of DNA A260(Absorbance at 260nm)of 1.0= 50 mg of double stranded DNA/ml. We can also check purity of DNA sample by UV spectrophotometer. If A260/A280=1.8 it means pure sample of DNA. if A260/A280 is less than 1.8 it means impurity of phenol or protein. Free life science studymaterial.