Animal Tissue Cell Total DNA Extraction Kit

• Animal Tissue Cell Total DNA Extraction Kit uses a centrifugal adsorption column that can specifically bind DNA and a unique buffer system for rapid extraction of genomic DNA from animal tissues and cells. The unique binding solution/proteinase K rapidly lyses cells and inactivates intracellular nucleases, and genomic DNA is selectively adsorbed on the silicon matrix membrane in the centrifuge column. Then a series of rapid rinse-centrifugation steps are used to remove cell metabolites, proteins and other impurities. Southern-blot and various enzyme digestion reactions.
• The kit is fast and simple, and a single sample can be generally completed within 30 minutes, and the purity of genomic DNA after extraction is high.

* Before the first use, 60 mL ethanol (96%-100%) should be added to Wash Buffer.

• Column equilibration: The adsorption column should be pretreated with balanced buffer before the experiment to improve the nucleic acid binding ability. Take the required amount of adsorption column (adsorption column into the collection tube), add 100 µL of balanced buffer, centrifuged at 13,000×g for 60 seconds, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube for later use.

1. Cultured cells

• Collect about 10⁶-10⁷ suspended cells into a 1.5 mL centrifuge tube (no more than 1×10⁷ cells); For adherent cells, the cells were first digested with trypsin and blown down for collection.
• Centrifuged at 13,000×g for 60 seconds to remove the supernatant and leave the cells to settle.
• The cells were resuspended in 200 µL of 1×PBS, centrifuged at 13,000×g for 60 seconds, the supernatant was discarded, and the cell precipitate was resuspended in 180 µL of 1×PBS.
• Add 20 µL proteinase K (20 mg/mL) solution, mix thoroughly, then add 200 µL binding buffer, vortex and shake thoroughly at once, and place at 70°C for 10 minutes.

2. Animal organization

• Take fresh or thawed tissue (≤25 mg), add 180 µL of lysate buffer, quickly grind and mixon ice to make a homogenate.
• Add 20 µL proteinase K solution (20 mg/mL) and mix thoroughly immediately by vortexing and shaking.
• Place the lysates in a water bath at 56°C for 1-3 hours or until tissue digestion is complete during which time gently shake several times to help lysis.
• Add 200 µL binding buffer, immediately vortex and shake to mix thoroughly, and place at 70℃ for 10 minutes.
• Connecting operation step 3.
• After cooling, add 100 µL isopropanol, immediately vortex and shake to mix thoroughly.
• Add the mixture (including the flocculent precipitate) to the adsorption column (the adsorption

3. After cooling, add 100 µL isopropanol, immediately vortex and shake to mix thoroughly.
4. Add the mixture (including the flocculent precipitate) to the adsorption column (the adsorption column is put into the collection tube), centrifuged at 13,000×g for 60 seconds, and discard the waste liquid.
5. Add 500 µL of inhibitor removal solution, centrifuge at 12,000×g for 60 seconds, and discard the waste solution.
6. Add 600 µL of wash buffer (check whether absolute ethanol has been added to the reagent bottle), centrifuge at 12,000×g for 60 seconds, discard the waste solution, and repeat this step once.
7. Put the adsorption column back into the collection tube, centrifuged at 13,000×g for 2 minutes, and removed the rinse solution as far as possible to avoid the residual ethanol in the rinse solution inhibiting the downstream reaction.
8. Put the adsorption column in a new 1.5 mL centrifuge tube, add 100 µL elution buffer to the center of the membrane, and place it at room temperature for 3-5 minutes.
9. Genomic DNA was eluted by centrifugation at 12,000×g for 60 seconds and stored at -20°C.

• Store at 2℃~30℃ for 24 months. Protease K was stored in readymade glycerol buffer at 2℃~8℃ for 12 months, Store at -25℃~-15℃ for 2 years.

• Transported on room temperature.