Plasmid Maxiprep Kit

By using the classical alkaline lysis method and the reversible binding of silica gel membrane to nucleic acid, the kit can extract 500 μg~1500 μg plasmid DNA from Escherichia coli cultured overnight with 100 mL. The extracted plasmid can be directly used in various molecular biology experiments, such as restriction enzyme digestion reaction, ligation reaction, PCR amplification, sequencing, transformation, etc.

RNase A is shipped with wet ice and stored at -20°C. Other reagents are shipped and stored at room temperature; valid for 12 months.

1. Anhydrous ethanol and 50 mL Nuclease-free centrifuge tube are required but not supplied in this kit.

2. Add all the RNase A provided in the kit to Buffer P1 before use, and it can be stored at 2~8°C for 6 months.

3. Please add 75 mL anhydrous ethanol to Buffer PD and 163 mL anhydrous ethanol to Buffer PW before use.

4. If Buffer P2 precipitates, please heat it in a water bath at 37°C for a few minutes to restore clarification. After using Buffer P2, the lid should be closed immediately to avoid long-term contact with air.

5. Please pre-cool Buffer P3 at 4°C or ice bath before use.

1. Column balance: Add 2.5 mL Buffer BL to HiBind DNA Column, centrifuge at 10,000×g for 2 min, discard the filtrate, and put the HiBind DNA Column back into the Collection Tube (the treated columns are best used on the same day).

2. Harvest bacterial culture from 100 mL fresh bacterial fluid by centrifugation at 10,000×g for 1 min at room temperature. Collect the culture in the 50 mL centrifuge tube (remove the supernatant as much as possible). Plasmids with low copy or more than 10 kb suggest collecting bacteria cultured overnight with 200mL, and the dosage of Buffer P1, Buffer P2 and Buffer P3 should be increased in equal proportion.

3. Add the 8 mL Buffer P1 (please check whether or not to add the RNase A first), use a pipette or vortex oscillator to thoroughly suspend the bacteria (be sure to disperse the bacteria thoroughly, otherwise it will affect the lysis, resulting in low quality and purity of the extracted plasmid).

4. Add 8 mL Buffer P2, immediately and gently turn it upside down for 8~10 times to make the bacteria fully lysed (this step should not be violently shaken, and should be done within 5 min). At this point, the solution should become clear and sticky, if it does not become clear, it may be that there are too many bacteria and the lysis is not complete, it can be reversed gently 5~8 times again until the solution is completely clear.

5. Add 8 mL Buffer P3 (pre-cool at 4°C in advance), immediately and gently upside down 10~12 times, the solution appears compact agglomerate, centrifuge at 10,000×g for 10~15 min at room temperature, slowly pour the supernatant into the Column Filter, push the handle to filter, and the filtrate is collected in Nuclease-Free 50 mL centrifuge tube (self-provided).

6. Add anhydrous ethanol 0.5 times the volume of the above solution, mix upside down and transfer to HiBind DNA Column (no more than 10 mL each time).

7. Centrifuge at 10,000×g for 2 min at room temperature, discard the filtrate. Put the HiBind DNA Column back into the Collection Tube (the solution in step 6 can be passed through the column many times).

8. Add 10 mL Buffer PD (confirm whether to add anhydrous ethanol) to the HiBind DNA Column, centrifuge at 10,000×g for 2 min at room temperature, discard the filtrate. Put the HiBind DNA Column back into the Collection Tube.

9. Add 10 mL Buffer PW (confirm whether or not to add anhydrous ethanol) to HiBind DNA Column, centrifuge at 10,000×g for 2 min at room temperature, discard the filtrate. Put the HiBind DNA Column back into the Collection Tube.

10. Repeat step 9.

11. Centrifuge at 10,000×g for 5 min at room temperature, place the HiBind DNA Column in a new Nuclease-Free 50 mL Centrifuge Tube. Open the lid and place 10 min at room temperature to volatilize the ethanol thoroughly.

12. Add 1~1.5 mL Buffer TE or Nuclease-Free Water to the center of the membrane and place it at room temperature for 5 min. Centrifuge at 10,000×g for 5 min. If you need to increase the efficiency of plasmid recovery, the obtained solution can be re-added to HiBind DNA Column, place at room temperature for 5 min and centrifuge at 10,000×g for 5 min.

13. The obtained plasmid DNA can be preserved for a long time at -20°C.

1. Please read the Product Manual carefully before use.

2. If the copy number of plasmid is low or the plasmid DNA is larger than 10kb, it is recommended to collect the overnight culture of 200 mL, and the dosage of Buffer P1, Buffer P2 and Buffer P3 should be increased in equal proportion.

3. Buffer TE can be preheated at 60~65°C and the elution incubation time can be prolonged to improve the elution efficiency.

4. Ethanol should be completely volatilized before eluting the plasmid to avoid the influence of residual ethanol on downstream experiments.

5. For your safety and health, please wear a lab coat and disposable gloves.

For Research Use Only!