1. Please prepare yourself: anhydrous ethanol, PBS and 1.5mL centrifuge tubes.
2. Take out the separated liquid and washing liquid and perform the following operations:
A) precipitation: adding 25.5 ml of anhydrous ethanol into 4.5 ml; Add 5 1 ml anhydrous ethanol to 9 ml.
B) Washing solution: 9 ml is added with 2 1 ml anhydrous ethanol; 18 ml, add 42 ml of absolute ethanol.
C) If there is any precipitate, the prepared precipitate can be dissolved at 37℃ and shaken well before use.
3. Cell treatment: a) culturing monolayer adherent cells on a culture plate, discarding the culture medium, washing with PBS once, and discarding PBS;
B) The adherent cells cultured in the culture bottle are digested with trypsin, treated as cell suspension, and the cell quantity is preferably 105- 106, and centrifuged at 1000 rpm for 5 minutes, then the supernatant is completely discarded, and 100μL PBS is added for shaking until the cells are suspended;
C) The cultured cells are suspended cells, and centrifuge at 1 000 rpm for 5 minutes, completely discard the supernatant, and add 100μL PBS for shaking until the cells are suspended.
4. Add 200μL of lysate and 20μL of digestive juice A, shake well and let stand at room temperature for 5 minutes.
5. Add 20 μL of digestive juice B, shake well, and take a water bath at 56℃ 10 minute until the cells are completely lysed.
6. Add 500μL of precipitate, and gently invert and mix. If there is translucent suspended matter, it will not affect DNA extraction and subsequent experiments.
7. Put the adsorption column into the collection tube, transfer the above solution into the adsorption column, let it stand for 2 minutes, centrifuge at 12000 rpm 1 min, and discard the waste liquid in the collection tube.
8. Put the adsorption column back into the collection tube, add 500μL of washing liquid to the adsorption column, centrifuge at 65438±02000 rpm for 65438 0 minutes at 4℃, and discard the waste liquid in the collection tube.
9. Put the adsorption column back into the collection tube and centrifuge at 4℃ 12000 rpm for 2 minutes, leaving residual washing liquid.
10. Take out the adsorption column, put it into a new 1.5 mL centrifuge tube, add 50-200 μL eluent, stand for 3 minutes, centrifuge at 12000 rpm for 2 minutes, and collect DNA solution. The extracted DNA can be used in the next experiment or stored at -20℃.