Potassium acetate DNA extraction protocol

Material preparation
1. ** Reagents **: Potassium Acetate, Glacial Acetic Acid, Distilled Water, Ethanol, Isopropanol, Tris-EDTA (TE) buffer, etc.
2. ** Equipment **: Centrifuge, Pipette, Pipette Tips, Centrifuge Tubes, Water Bath, Electrophoresis Apparatus, Gel Imaging System, etc.

Operation steps
1. ** Cell lysis **
- Transfer the sample of DNA to be extracted (e.g. cell suspension) to a suitable centrifuge tube, centrifuge at an appropriate rotational speed (e.g. 5000 rpm) for a few minutes, discard the supernatant and collect the cell pellet.
- Add an appropriate amount of lysis solution (e.g. lysis buffer containing protease K) to the pellet and mix well to allow the cell to lyse. Incubate the centrifuge tube in a 37 ° C water bath for a period of time (e.g. 30 minutes) to promote digestion of the protein.
2. ** Potassium acetate treatment **
- Prepare a solution of potassium acetate, usually in a certain proportion (such as the ratio of 5M potassium acetate: glacial acetic acid: water = 60:11.5:28.5).
- Add an appropriate amount of prepared potassium acetate solution to the lysed solution, and mix it gently upside down. At this time, a white precipitate will be observed in the solution. This is because potassium acetate combines with impurities such as proteins and polysaccharides to form a precipitate, while the DNA remains in the supernatant.
- Leave the centrifuge tube on ice for several minutes (such as 10 minutes) to promote the formation of the precipitate. Then centrifuge at a higher speed (e.g. 12000 rpm) for 10 - 15 minutes.
3. ** Supernatant transfer with nucleic acid pellet **
- Carefully aspirate the supernatant after centrifugation and transfer to a new centrifuge tube, taking care not to absorb the pellet.
- Add an appropriate amount of isopropanol (generally 0.6 - 1 times the volume of the supernatant) to the supernatant and mix gently upside down. At this time, white filamentous DNA can be seen to precipitate.
- Leave the centrifuge tube at room temperature for a few minutes (e.g. 5 - 10 minutes), then centrifuge at 12,000 rpm for 5 - 10 minutes to allow the DNA to settle at the bottom of the tube.
4. ** Wash & Dry **
- Discard the supernatant, add an appropriate amount of 70% ethanol to the centrifuge tube containing the DNA pellet, and gently wash the DNA pellet to remove residual impurities and salts.
- Centrifuge again at 12,000 rpm for 2 - 3 minutes, discard the ethanol, and turn the centrifuge tube upside down on a clean absorbent paper to allow the residual ethanol to evaporate naturally, allowing the DNA pellet to dry. Be careful not to over-dry, so as not to make it difficult for the DNA to dissolve.
5. ** DNA dissolution **
- After the DNA pellet has dried, add an appropriate amount of TE buffer (or distilled water) to the centrifuge tube and gently blow or shake to fully dissolve the DNA. The centrifuge tube can be incubated in a 37 ° C water bath for several minutes to accelerate the dissolution.
6. ** Quality test **
- Take an appropriate amount of dissolved DNA solution and detect it by agarose gel electrophoresis (Agarose Gel Electrophoresis). Prepare an appropriate concentration of agarose gel (e.g. 1% - 2%), add a nucleic acid dye (e.g. EB or SYBR Green), mix the DNA sample with the loading buffer and load it.
- Carry out electrophoresis at an appropriate voltage. After the electrophoresis is completed, observe the DNA bands through the gel imaging system to judge the integrity and purity of the DNA. At the same time, the absorbance of DNA solution at 260 nm and 280 nm can be determined by UV spectrophotometer, the ratio of A260/A280 is calculated, and the purity of DNA is evaluated. The ideal ratio should be between 1.8 - 2.0.

Precautions
1. The operation should be as gentle as possible to avoid violent oscillation and prevent DNA breakage.
2. All reagents should be kept pure to avoid contamination. Pipette gun heads should be changed each time to prevent cross-contamination.
3. When drying DNA precipitation after ethanol washing, pay attention to control the drying time to avoid DNA being too dry and difficult to dissolve.
4. When performing electrophoresis testing, pay attention to setting the appropriate voltage and time to obtain clear DNA bands.