Plasmid Midiprep Kit

The kit adopts the classical alkaline lysis method and the purification method of efficient and specific binding of plasmids by fiberglass membranes. It is suitable for purifying high purity plasmids from bacteria cultured with 20~50 mL. According to the copy number of plasmids, the yield is about 100~400 μg. 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 is 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 4°C for 6 months.

3. Please add 24 mL anhydrous ethanol to Buffer PD before use.

4. Please add 64 mL anhydrous ethanol to Buffer PW before use.

5. 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.

6. Please pre-cool Buffer P3 at 4°C before use.

1. Column balance: Add 2 mL Buffer BL to HiBind DNA Midi Column, centrifuge at 4,500×g for 2 min at room temperature, discard the filtrate, and put the HiBind DNA Midi Column back into the Collection Tube (the treated column is best used immediately).

2. Harvest bacterial culture from 20~50 mL overnight cultured bacterial fluid by centrifugation at 8,000×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 that the amount of bacteria should be increased appropriately, and the dosage of Buffer P1, Buffer P2 and Buffer P3 should be increased in equal proportion.

3. Add the 2.5 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 2.5 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 to avoid genomic DNA shear breaks 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, and consideration should be given to reducing the amount of bacteria.

5. Add 2.5 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 8,000×g for 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 15 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 Midi Column (no more than 4 mL each time).

7. Centrifuge at 4,500×g for 3 min at room temperature, discard the filtrate in the Collection Tube. Put the HiBind DNA Midi Column back into the Collection Tube (the solution in step 6 can be passed through the column many times).

8. Add 3.5 mL Buffer PD (confirm whether to add anhydrous ethanol) to the HiBind DNA Midi Column, centrifuge at 4,500×g for 3 min at room temperature, discard the filtrate. Put the HiBind DNA Midi Column back into the Collection Tube.

9. Add 3.5 mL Buffer PW (confirm whether or not to add anhydrous ethanol) to HiBind DNA Midi Column, centrifuge at 4,500×g for 3 min at room temperature, discard the filtrate in the Collection Tube. Put the HiBind Midi DNA Column back into the Collection Tube.

10. Repeat step 9.

11. Centrifuge at 4,500×g for 10 min at room temperature, place the HiBind DNA Midi Column in a new Nuclease-Free 15 mL Centrifuge Tube. Open the lid and place 10 min at room temperature to volatilize the ethanol thoroughly.

12. Add 500~1000 μL Buffer TE or Nuclease-Free Water to the membrane center of the HiBind DNA Midi Column and place it at room temperature for 5 min. Centrifuge at 4,500×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 4,500×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. When the copy number of the extracted plasmid is low or the plasmid fragment is larger than 10kb, the amount of bacteria collection should be increased, 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!