How do I extract DNA from bacteria?
Posted September 30, 2020
Extracting DNA from bacterial cells is relatively easy requiring three simple steps.
- Step 1: Cell lysis – in order to retrieve DNA, the cell membrane must first be disrupted. When working with bacteria, yeast or fungi samples, a chemical lysis buffer composed of detergents, solvents and/or enzymes (e.g. Proteinase K) is commonly used to disrupt the membrane. These chemicals can interact with certain components of the cell wall and dissolve cellular proteins in order to free DNA.
- Step 2: Precipitation – this step is vital as it separates DNA from other cellular debris such as broken-down lipids, proteins or nucleic acids. To do so, first treat the sample with a concentrated saline solution. This not only makes unwanted cellular debris clump together; it also neutralizes the negative charges on DNA molecules making it more stable and less water soluble. Next add alcohol, such as ice-cold ethanol or isopropanol. Since DNA is insoluble in these alcohols it will precipitate out of the aqueous solution and form a pellet upon centrifugation.
- Step 3: Purification – after the DNA has been separated and the supernatant removed, it must be washed with alcohol to remove salts and other impurities, and then re-suspended in an aqueous buffer such as water or 1X TE buffer.
The isolated DNA can be quantified by measuring the absorbance at 260nm and its purity could be analyzed using the A260/A280 ratio.