Endo-Free Plasmid Midiprep Kit

The kit uses a glass fiber membrane with specific adsorption to plasmid and a specialbuffer combination, which can effectively extract various endotoxin-free plasmid (endotoxin content≤0.1 EU/μg) from cultured bacteria. According to the different copy number of plasmid, the yield of plasmid is generally 100~400 μg. The extracted high purity plasmid can be used in many biological experiments, such as restriction enzyme digestion, ligation reaction, PCR amplification, sequencing, transformation, transfection, etc.

RNase Aisshipped with wet ice and stored at -20°C. Other reagentsare shipped and stored at room temperature; valid for12 months.

1. Anhydrous ethanolis required but not supplied in this kit.

2. Prepare 42℃ water bath or heating module.

3. 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.

4. Please add64 mL anhydrous ethanol to Buffer PW before use.

5. If BufferP2 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 BufferP3 at 4°C before use.

1. Column balance: Add 2 mL Buffer BL to HiBind DNA Midi Columns,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 freshbacterial fluid by centrifugation at8,000×g for1 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 the2.5mL BufferP1 (please check whether or not toadd the RNase A first), use a pipette orvortex 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. Add2.5mL BufferP2, immediately and gently turn it upside down for 8~10 times to make the bacteria fullylysed (this step should not be violently shaken, and should be done within 5 min). At thispoint, 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. Add2.5 mLBufferP3 (pre-cool at 4°C in advance), immediately and gently upside down 10~12 times, the solution appears compact agglomerate, centrifuge at8,000×g for15 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 750 μLBuffer ER, place 10 min on the ice after mixing upside down (mix upside down 3~5 timesduring the period), the solution is transparent blue.

7. Incubate 5 min at 42°C, the solution recovers turbidity.centrifuge at4,500×g for5 min at room temperature, the solution is divided into upper and lower layers, the upper layer is clear water phase and the lower layer is blue. Carefully collect the upper water phase into the new Nuclease-Free 15 mL centrifuge tube (self-provided).

8. 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).

9. Centrifuge at4,500×g for3 min at room temperature, discard the filtrate. Put the HiBind DNA Midi Column back into the Collection Tube (the solution in step 8 can be passed through the column many times).

10. Add3.5 mL Buffer ED to the HiBind DNAMidiColumn, 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.

11. Add3.5 mL Buffer PW (Confirm whether or not to add anhydrous ethanol) to HiBind DNAMidiColumn, centrifuge at 4,500×g for 3 min at room temperature, discard the filtrate. Put the HiBind Midi DNA Column back into the Collection Tube.

12. Repeat step11.

13. Centrifuge at4,500×g for10 min at room temperature, place theHiBind 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.

14. Add 500~1000μL Buffer TE or Endo-Free Water to the center of the membrane and place it at room temperature for 5 min.Centrifuge at4,500×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 at4,500×g for 5 min.

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

1. Please read the Product Manual carefully before use.

2. Avoid direct contact with Buffer P2 and P3. Close the lid immediately after using Buffer ED.

3. 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.

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

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

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

For Research Use Only!