Plasmid midiPREP Kit

The Plasmid midiPREP Kit provides a fast, simple, and cost-effective plasmid midiprep method for isolating the plasmid DNA from the cultured bacterial cells. The Plasmid midiPREP Kit is based on the alkaline lysis of bacterial cells, followed by binding DNA onto the glass fiber matrix of the spin column in the presence of high salt. Phenol extraction and ethanol precipitation are not required, and the high-quality plasmid DNA is eluted in a small volume of the Tris buffer (included in each kit) or water (pH is between 7.0 and 8.5). The plasmid DNA purified with the Plasmid midiPREP Kit is suitable for a variety of routine applications, including the restriction enzyme digestion, sequencing, library screening, in vitro translation, transfection of robust cells, ligation, and transformation. The entire procedure can be completed within 40 minutes.

• Sample: Up to 100 ml bacterial cells.
• Yield: Up to 250 μg of plasmid.
• Endotoxin value: <0.003 EU/μg.

Kit Contents

• Safe: Eliminates the use of phenol, chloroform, ethidium bromide, and cesium chloride, thus minimizing the exposure to and disposal of hazardous materials.
• Time saving: Complete the process in less than 40 minutes.

• Add the provided RNase A solution to the Buffer M1, mix, and store at 2-8°C.
• Add 100 ml of ethanol (96-100%) to the Buffer W2 before use.

The purified plasmid DNA can be immediately used in any downstream molecular biology application.

Step 1 Bacterial Cells Harvesting

1. Transfer 50 ml of the bacterial culture to a 50 ml centrifuge tube.
2. Centrifuge at 6,000 x g for 5 minute and discard the supernatant.

Step 2 Resuspend

Resuspend pelleted bacterial cells in 4 ml of the Buffer M1 (RNase A added)

Step 3 Lysis

Add 4 ml of the Buffer M2 and mix thoroughly by inverting the tube 10 times (Do not vortex) and then stand at the room temperature for 2 minutes or until the lysate is homologous.

Step 4 Neutralization

1. Add 6 ml of the Buffer M3 and mix immediately and thoroughly by inverting the tube 10 times (Do not vortex).
2. Centrifuge at 6,000 x g for 10 minutes.

Step 5 Binding

1. Place a MD Column in a 50 ml centrifuge tube.
2. Apply the supernatant (from step 4) to the MD column by decanting or pipetting.
3. Centrifuge at 6,000 x g for 3 minutes.
4. Discard the flow-through and place the MD column back into the same 50 ml centrifuge tube.

Step 6 Wash

1. Add 8 ml of the Buffer W1 into the MD Column.
2. Centrifuge at 6,000 x g for 3 minutes.
3. Discard the flow-through and place the MD column back into the same 50 ml centrifuge tube.
4. Add 12 ml of the Buffer W2 (Ethanol added) into the MD Column.
5. Centrifuge at 6,000 x g for 3 minutes.
6. Discard the flow-through and place the MD column back into the same 50 ml centrifuge tube.
7. Centrifuge at 6,000 x g again for 3 minutes to remove residual Buffer W2.

Step 7 Elution

1. To elute DNA, place the MD column in a new 50 ml centrifuge tube.
2. Add 2 ml of the Buffer E or water (pH is between 7.0 and 8.5) to the center of each MD column, let it stand for 2 minutes, and centrifuge at 6,000 x g for 3 minutes.

• Check buffers before use for salt precipitation. Re-dissolve any precipitate by warming up to 37°C.
• Add the provided RNase A solution to the Buffer M1, mix, and store at 2-8°C.
• Add 100 ml of ethanol (96-100%) to the Buffer W2 before use.
• Check buffers before use for salt precipitation. Re-dissolve any precipitate by warming to 37°C.
• Buffers M2, M3, and W1 contain irritants. Wear gloves when handling these buffers.
• During operation, always wear a lab coat, disposable gloves, and protective equipment.
• Research Use Only. Not intended for any animal or human therapeutic or diagnostic uses.