Complete Guide to Plasmid Maxi Preparation: A Step-by-Step Workflow

1. Bacterial Harvesting: Collect the bacterial culture in a 50 mL centrifuge tube and centrifuge at 10,000 rpm for 2 minutes. Discard the supernatant.

                                                                                                                     After bacterial collection

2. Bacterial Resuspension: Add the appropriate reagent and thoroughly resuspend the bacterial pellet using a vortex mixer.

                                                                                                                          After resuspension

3. Cell Lysis: Gently mix the lysate until it becomes clear. For samples with a high bacterial density, allow the lysate to stand briefly if necessary.

After adding the lysis solution 

                                                                        After gentle mixing during lysis, the solution becomes clear and transparent.

4. Neutralization: Add the neutralization solution and gently mix until a uniform flocculent precipitate forms. After thorough mixing, centrifuge the sample.

After neutralization

                                                                                                              After mixing during neutralization

5. Filtration: Transfer the clarified supernatant into a filter unit, filter the solution, and collect the filtrate.

Filtered supernatant

6. Magnetic Bead Binding: Add magnetic beads to the supernatant and mix thoroughly on a shaker. After mixing, place the centrifuge tube on a magnetic rack and gently discard the supernatant.

7. Washing: Add protein removal solution to the centrifuge tube, centrifuge, and discard the supernatant.

8. Purification: Add purification solution to the centrifuge tube, centrifuge, and discard the supernatant.

9. Elution: Add 1,000 μL elution buffer or ultrapure water to the 50 mL centrifuge tube containing magnetic beads. For low-copy plasmids, reducing the elution volume is recommended to obtain a higher plasmid concentration. Fully resuspend the magnetic beads, centrifuge, and collect the plasmid-containing eluate in a new centrifuge tube while avoiding transferring the magnetic beads.