Large-scale, endotoxin-free plasmid preparation is critical for downstream applications such as mammalian cell transfection. In this case study, we demonstrate the efficient extraction of Plvx-PGK plasmid DNA from an 80 mL bacterial culture using the Yeasen Endotoxin-Free Plasmid Mega Extraction Kit V2, followed by successful transfection into HEK293T cells.
Materials
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Reagents |
Transfection Reagent: 40802ES Cells: 293T |
Absolute Ethanol / Ethanol (100%) |
Isopropanol |
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Consumables |
50 mL Conical Centrifuge Tubes |
2 mL Microcentrifuge Tubes |
50 mL Centrifuge Tube Racks |
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Equipment |
Thermo Centrifuge with Fixed-Angle Rotor |
Pipettes |
Agarose Gel Electrophoresis System |
Nanodrop spectrophotometer |
Protocol
I. Bacterial Harvest, Lysis, and Clarification
1. Harvest Bacteria: Collect the required bacterial biomass via centrifugation. Aliquot 80 mL of culture into two 50 mL conical tubes.Centrifuge at 6,000 × g for 3 min. Discard the supernatant.
2. Resuspend Bacteria: Add 10 mL of Solution SI. Vortex or shake until no visible bacterial clumps remain.
3. Lysis: Add 10 mL of Solution SII. Invert gently 4–10 times to mix. Incubate until a clear lysate forms (approx. 4 min).
4. Neutralization and Precipitation: Add 8 mL of Solution SIII and 100 µL of RNase A. Invert 10 times to mix. Incubate at room temperature for 10 min. Centrifuge at 10,000 × g for 5 min.
5. Filtration: Filter the supernatant through a plunger filter into a 50 mL conical tube (yielding approx. 25 mL). Add 10 mL of Binding Buffer PB and mix well. Set aside for column loading.
II. Plasmid Purification
6. Column Equilibration: (Recommended to perform while processing step 5).Load 4 mL of Equilibration Buffer BS onto a HiBind Plasmid Mega Column (E2).Incubate for 2 min. Centrifuge at 6,000 × g for 2 min. Discard the flow-through.
7. Binding: Load the mixture from step 5 onto the equilibrated column. Perform multiple centrifugations to bind the plasmid.Load 9 mL per spin, repeating 4 times.Centrifuge at 6,000 × g for 2 min each time.
8. Washing: Wash the column to remove impurities.Add 8 mL of Solution W1 (wash once).Add 8 mL of Wash Solution WS (wash twice).Centrifuge at 6,000 × g for 2 min after each wash.
9. Drying: Centrifuge the empty column at 10,000 × g for 5 min. Let it stand at room temperature for 5 min to completely evaporate residual ethanol.
III. Elution
10. Elution: Add 1.2 mL of Elution Buffer EB directly onto the column matrix.Incubate at room temperature for 5 min.Centrifuge at 10,000 × g for 3 min to collect the eluate.
Practical Notes
1. Binding Buffer Ratio: Maintaining the correct binding buffer volume is critical. Do not reduce the recommended amount; a slight excess is acceptable.
2. Complete Resuspension: Incomplete resuspension of the bacterial pellet may result in inefficient lysis and reduced plasmid yield.
3. Alcohol Mixing: After adding ethanol or isopropanol, mix vigorously to ensure complete homogenization.
Quality Control Results
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Sample |
nanodrop |
A260/A280 |
A260/A230 |
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1 |
700 ng/μL |
1.9 |
2.0 |
The purified plasmid DNA showed excellent purity and met all quality requirements for mammalian cell transfection.
293T Cell Transfection
Figure 1. Efficient HEK293T cell transfection using large-scale Plvx-PGK plasmid DNA.
Conclusion
Using the Yeasen Endotoxin-Free Plasmid Mega Extraction Kit V2, we successfully obtained high-purity Plvx-PGK plasmid DNA from an 80 mL bacterial culture, achieving a final concentration of 700 ng/µL. The plasmid performed reliably in HEK293T cell transfection, demonstrating that this workflow is well-suited for large-scale plasmid preparation for mammalian applications.