Plasmid DNA Midiprep Kit
No:DMDPD020& DMDPD050
For research use only
Sample: up to 100 ml bacterial cells
Yield: up to 250μg of plasmid
Introduction
The BioDiamond Plasmid DNA Midiprep Kit provides a fast, simple, and cost-effective plasmid midiprep method for isolation of plasmid DNA from cultured bacterial cells. The BioDiamond Plasmid DNA Midiprep Kit 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 a high amount of salt. Phenol extraction and ethanol precipitation are not required, and high-quality plasmid DNA is eluted with a small volume of Tris buffer (included in each kit) or water (pH is between 7.0 and 8.5). Plasmid DNA purified with BioDiamond Plasmid DNA Midiprep 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 40-50 minutes.
Kit Contents
No:DMDPD020& DMDPD050
For research use only
Sample: up to 100 ml bacterial cells
Yield: up to 250μg of plasmid
Introduction
The BioDiamond Plasmid DNA Midiprep Kit provides a fast, simple, and cost-effective plasmid midiprep method for isolation of plasmid DNA from cultured bacterial cells. The BioDiamond Plasmid DNA Midiprep Kit 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 a high amount of salt. Phenol extraction and ethanol precipitation are not required, and high-quality plasmid DNA is eluted with a small volume of Tris buffer (included in each kit) or water (pH is between 7.0 and 8.5). Plasmid DNA purified with BioDiamond Plasmid DNA Midiprep 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 40-50 minutes.
Kit Contents
NOTE
★ Add the provided RNase A solution to Buffer M1, mix, and store at 2–8°C.
★ Add ethanol (96–100%) to Buffer W2 before use (see bottle label for volume).
★ Check Buffers before use for salt precipitation. Redissolve any precipitate by warming to 37°C.
★ Buffers M2, M3, and W1 contain irritants. Wear gloves when handling these buffers.
Quality Control
In accordance with BioDiamond’s ISO-certified Total Quality Management System, the quality of the BioDiamond Plasmid DNA Midiprep Kit is tested on a lot-to-lot basis to ensure consistent product quality.
Additional requirements
*Ethanol (96~100%)
*50 ml centrifuge tubes
Protocol
Step 1 Bacterial Cells Harvesting
- Transfer 50~100 ml bacterial culture to a centrifuge tube.
- Centrifuge at 6,000 x g for 5 minute and discard the supernatant.
Step 2 Resuspend
- Resuspend pelleted bacterial cells in 4 ml Buffer M1 (RNase A added)
- Add 4 ml Buffer M2 and mix thoroughly by inverting the tube 10 times (Do not vortex) and then stand at room temperature for 2 minutes or until the lysate is homologous.
- Add 6 ml Buffer M3 and mix immediately and thoroughly by inverting the tube 10 times (Do not vortex). Centrifuge at 6,000 x g for 10 minutes.
- Place a MP Column in a 50 ml centrifuge tube. Apply the supernatant (from step 4) to the MP column by decanting or pipetting.
- Centrifuge at 6,000 x g for 3 minutes. Discard the flow-through and place the MP column back into the same 50 ml centrifuge tube.
- Add 8 ml of Buffer W1 into the MP Column. Centrifuge at 6,000 x g for 3 minutes. Discard the flow-through and place the MP column back into the same 50 ml centrifuge tube.
- Add 12 ml of Buffer W2 (Ethanol added) into the MP Column. Centrifuge at 6,000 x g for 3 minutes. Discard the flow-through and place the MP column back into the same 50 ml centrifuge tube.
- Centrifuge at 6,000 x g again for 3 minutes to remove residual Buffer W2.
- To elute DNA, place the MP column in a new 50 ml centrifuge tube.
- Add 2 ml Buffer EL or water (pH is between 7.0 and 8.5) to the center of each MP column, let stand for 2 minutes, and centrifuge at 6,000 x g for 3 minutes.
Troubleshooting
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