1. Harvest 1-2 mL of fresh bacterial culture by centrifugation for 1 minute at 10,000 rpm. Pour off the supernatant and blot the inverted tube on a paper towel to remove residue medium. Remove the residue medium completely.
2. Add 200 µL Buffer F1 and completely resuspend bacterial pellet by vortexing or pipetting (Complete resuspension is critical for optimal yields).
3. Add 200 µL Buffer F2, mix by inverting 10 times (do not vortex) and incubate at room temperature for 2 minutes until the solution becomes clear.
4. Add 200 µL Buffer F3 to the sample from step 3, mix completely by inverting the vial for 10 times. Incubate at room temperature for 2-3 min. Transfer the whole lysate to a lysate clearance column.
5. Centrifuge at 12,000 rpm for 30 seconds. Note: If the lysate still remains in the column, spin for another 30 seconds.
6. Discard the lysate clearance column and add 200 µL 100% ethanol to the flow through in the collection tube, mix well by pipetting and transfer the sample to a DNA mini column.
7. Spin at 12,000 rm for 30 seconds. Discard the flow through and use use the collection tube
8. Optional: Add 300 µL Buffer KB and centrifuge at 12,000 rpm for 30s. Discard the flow-through and put the column back to the collection tube. Note: This step is NOT necessary if the plasmid is being purified from endA- strain such as DH5a and Top 10. Buffer KB wash is necessary for endA+ strains such as HB101, JM110, JM 101 and their derived strains.
9. Add 600 µL DNA Wash Buffer (Add ethanol to DNA wash buffer before use) into the spin column, centrifuge at 12,000 rpm (14,000 - 18,000 x g) for 30 seconds at RT. Remove the spin column from the tube and discard the flow-through.
10. Reinsert the spin column into the collection tube and centrifuge for 1 minute at 13,000 rpm. Note: Residual ethanol will be removed more effectively with the column lid open.
11. Carefully transfer the spin column into an Elution tube (Provided) and add 50-100 µL Elution Buffer into the column and let it stand for 1 minute. Elute the DNA by centrifugation at 12,000 rpm (14,000-18,000 x g) for 30 seconds to elute DNA.
Note: The first elution normally yields around 70% of the plasmid DNA bound to the column. Add the eluted DNA back to the column for another elution yields 20-30% of the DNA bound to the column.