Plasmid Extraction Mini Kit

• Plasmid Extraction Mini Kit is a silica gel column-based plasmid extraction product. It uses a unique new material in the centrifugal adsorption column to efficiently and specifically adsorb plasmid DNA, and high-quality plasmid DNA can be obtained within 25 minutes. The kit can extract plasmid DNA less than 15 kb from bacterial broth. Each adsorption column could bind up to 60 μg of plasmid DNA. The plasmid extracted using this kit can be used for downstream experiments, such as restriction enzyme digestion, transformation, PCR and sequencing. The yield and purity of plasmid extraction are related to the species of host bacteria, culture conditions, degree of cell lysis, stability of plasmid, types of antibiotics added and other factors.

* Add 80 mL ethanol (96%~100%) to Wash Buffer before first use;
* Transfer all of RNase A to a P1 Buffer bottle before first use, refer to Precaution 2.

• 1. 1~3 mL of the bacterial solution from overnight cultures was transferred to a centrifuge tube, centrifuged at 11,000×g for 1 minute, and the supernatant was discarded.
• 2. Add 200 μL P1 Buffer, and use a pipette to gently blow to completely suspend the bacterialprecipitate.
• 3. Add 200 μL P2 Buffer, turn the centrifuge tube gently up and down for 5 to 10 times, stand at room temperature for 2 to 5 minutes to lye the cells until the solution is clear, and the lye time should not exceed 5 minutes.
• 4. Add 300 μL P3 Buffer, and immediately turn it gently up and down for 5 to 10 times, and mix thoroughly to avoid too much white precipitate adhered to the tube wall.
• 5. Centrifuge at maximum speed (≤18,000×g) for 5 minutes.
• 6. Place the Spin Column on the Collection Tube and suck the supernatant obtained in step 5 into the Spin Column. After centrifugation at 11,000×g for 30 seconds, the filtrate was discarded and the Spin Column was returned to the Collection Tube.
• 7. Add 400 μL W1 Buffer to the Spin Column, centrifuge at 11,000×g for 30 seconds, discard the filtrate, and put the Spin Column back into the Collection Tube.
• 8. Add 700 μL Wash Buffer to Spin Column, centrifuge at 11,000×g for 30 seconds, discard the filtrate, and put the Spin Column back into the Collection Tube. Note: Ensure that 80mL ethanol (96%-100%) has been added to Wash Buffer.
• 9. Centrifuge at maximum speed (≤18,000×g) for 3 minutes to dry the Spin Column and thoroughly remove residual ethanol.
• 10. Put the Spin Column in a new 1.5 mL centrifuge tube, add 50 μL~100 μL Elution Buffer or ddH₂O to the center of the Spin Column membrane, and let it stand for 1 minute.
• 11. The plasmid DNA was eluted by centrifugation at maximum speed (≤18,000×g) for 1 minute. The eluted plasmid DNA was stored at -20°C.

• a. Read the instructions carefully before using.
• b. Before the first use, 0.5 mL P1 Buffer was added to the RNase A tube, vortexed and mixed. After a short centrifugation, all the mixed solution was transferred to the P1 Buffer bottle, mixed and stored at 4°C.
• c. Before the first use, 80mL ethanol (96%-100%) should be added to Wash Buffer.
• d. If P2 Buffer is found to be cloudy, it can be restored to clarity by heating in a 37℃ water bath.
• e. The centrifuge operation of this experiment can be completed at 11,000×g~18,000×g.
• f. This product is limited to the scientific research of professionals. For your safety and health, please wear a laboratory coat and wear disposable gloves to operate.

• RNase A is stored at -25℃~-15℃.
• P1 Buffer after the addition of RNase A was stored at 2℃~8℃.
• Other components were stored at room temperature (2℃~30℃) for 12 months.

• Transport on blue ice.