A Brief Introduction to DNA
Deoxyribonucleic acid is one of the most important life-based molecules that carries genetic instructions to guide biological inheritance, biological development, and vital functioning. The structure of DNA in eukaryotic cells is called a double helix that is formed by antiparallel coiling of two polynucleotide chains. DNA extraction is a critical and basic step involved in molecular experiments. In many laboratories, alkaline lysis method is used to extract DNA. For a successful DNA extraction, there are several measurement criteria, it is required to ensure integrity of the primary structure of nucleic acid, A260/A280=1.8~2.0, the low concentration of salt ions, and the minimal pollution of other biomacromolecules such as proteins, polysaccharides and lipid molecules. High-quality and purified DNA is very important for the downstream research, such as genomic research and epigenomic research.
DNA Extraction from Cells Cultured in vitro
1. Collect cells. (The cells are directly centrifuged to obtain cell pellet, and the adherent cells need to be digested with trypsin and collected by centrifugation.)
2. Resuspend the cells and rinse once with pre-cooled PBS.
3. Repeat step 2.
4. Add 2 mL of DNA extraction buffer (10m mol/L Tris-cl, 0.1 mol/L EDTA, 0.5% SDS) and mix.
5. Add proteinase K to a final concentration of 100 μL/mL and a water-bath is performed at 50 °C for 3 hours.
6. Add an equal volume of phenol, centrifuge at 2,500 rpm for 15 minutes,and collect the aqueous phase.
7. Add an equal volume of phenol chloroform isoamyl alcohol mixture,centrifuge at 2,500 rpm for 15 minutes, and collect the aqueous phase.
8. Add 0.1 volume of 3M sodium acetate and 3 volumes of pre-cooled absolute ethanol at -20 °C overnight.
9. Centrifuge at 12,000 rpm for 10 minutes under 4 °C centrifuge.
10. Wash with 80% of the pre-cooled ethanol twice.
11. Resuspend the white pellet in an appropriate amount of TE buffer or ddH2O.The resulting solution is the cellular DNA.
DNA Purification and Concentration
1. Place the DNA solution in a centrifuge tube and add 0.1 volume of 3 M sodium acetate and 3 volumes of pre-cooled absolute ethanol at -20 °C overnight.
2. Centrifuge at 14,000 rpm for 10 minutes at 4 °C and discard the supernatant.
3. Wash with 80% of the pre-cooled ethanol twice.
4. Resuspend the white pellet in an appropriate amount of TE buffer or ddH2O. The resulting solution is the cellular DNA.
Sodium acetate provides salt ions for the precipitation of DNA, which can be added a little more and finally needs to be removed. Part of the DNA is lost during the process of DNA purification and concentration, and the recovery of DNA is approximately 80%.
0.7 times by the volume of isopropanol at room temperature is added (for example, 0.7 mL of isopropanol is added to 1 mL of the plasmid to be concentrated). After mixing, the mixture is centrifuged at 14,000 rpm for 10 minutes at 4 °C, and the supernatant is carefully aspirated to avoid contact with the precipitate. Note that the isopropanol-precipitated DNA is a glassy, nearly transparent, granular precipitate that is difficult to see as compared to salt-containing precipitates produced by ethanol precipitation. Centrifuge the centrifuge tube as gently as possible to avoid loose sediment or some particles suspended into the solution. Do not pour off the supernatant directly. Use a pipette to gently absorb the supernatant and take care to avoid aspiration.