Plasmid miniPREP Kit

The Plasmid miniPREP Kit provides a fast, simple, and cost-effective method for isolation of plasmid DNA from cultured Gram positive and Gram negative bacterial cells. The design is based on alkaline lysis of bacterial cells followed by binding of DNA onto the glass fiber matrix of the spin column in the presence of highly concentrated salts. Plasmid DNA purified with this kit is suitable for a variety of routine applications including restriction enzyme digestion, sequencing, library screening, in vitro translation, transfection of robust cells, ligation and transformation. The entire procedure can be completed within 15-20 minutes.

• Fastest Procedure: Completed within 15-20 mins.
• Sample: up to 4 ml bacterial cells.
• Yield: up to 30 μg of plasmid.

Kit Contents

• Add the provided RNase A solution to the Buffer S1, mix, and store at 2-8°C.
• Add 60 ml of ethanol (96-100%) to the Buffer W2 before use.

Step 1 Bacterial Cells Harvesting

1. Transfer 1.5 ml bacterial culture to a microcentrifuge tube.
2. Centrifuge at 14,000 x g for 1 minute and discard the supernatant.

Step 2 Resuspend

Resuspend pelleted bacterial cells in 200 μl of the Buffer S1 (RNase A added).

Step 3 Lysis

Add 200 μl of the Buffer S2 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 300 μl of the Buffer S3 and mix immediately and thoroughly by inverting the tube 10 times (Do not vortex).
2. Centrifuge at 14,000 x g for 3 minutes.

Step 5 Binding

1. Place a PM column in a Collection Tube. Apply the supernatant (from step 4) to the PM column by decanting or pipetting.
2. Centrifuge at 14,000 x g for 30 seconds, then discard the flow-through, and place the PM column back into the same collection tube.

Step 6 Wash

1. Add 400 μl of the Buffer W1 into the PM column.
2. Centrifuge at 14,000 x g for 30 seconds.
3. Discard the flow-through and place the PM column back into the same collection tube.
4. Add 600 μl of the Buffer W2 (Ethanol added) into the PM column.
5. Centrifuge at 14,000 x g for 30 seconds.
6. Discard the flow-through and place the PM column back into the same collection tube.
7. Centrifuge at 14,000 x g again for 2 minutes to remove the residual Buffer W2.

Step 7 Elution

1. To elute DNA, place the PM column in a clean 1.5 ml microcentrifuge tube.
2. Add 50-200 μl of the Buffer E or H₂O (pH between 7.0 and 8.5) to the center of each PM column, let it stand for 2 minutes, and centrifuge at 14,000 x g for 2 minutes.

＊Check the buffers before use for salt precipitation. Redissolve any precipitate by warming to 37°C.

• Check buffers before use for salt precipitation. Redissolve any precipitate by warming up to 37°C.
• Buffers S2, S3 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.