Use 1–5 mL of a saturated E.coli LB culture, pellet cells in a standard benchtop microcentrifuge for 30 s at 11,000 x g. Discard the supernatant and remove as much of the liquid as possible.
Add 250 μL Buffer A1. Resuspend the cell pellet completely by vortexing or pipetting up and down. Make sure no cell clumps remain before addition of Buffer A2!
Add 250 μL Buffer A2. Mix gently by inverting the tube 6–8 times. Do not vortex to avoid shearing of genomic DNA. Incubate at room temperature for up to 5 min or until lysate appears clear.
Add 300 μL Buffer A3. Mix thoroughly by inverting the tube 6–8 times. Do not vortex to avoid shearing of genomic DNA!
Centrifuge for 5 min at 11,000 x g at room temperature. Repeat this step in case the supernatant is not clear!
Place a NucleoSpin® Plasmid/Plasmid (NoLid) Column in a Collection Tube (2 mL) and decant the supernatant from step 3 or pipette a maximum of 750 μL of the supernatant onto the column. Centrifuge for 1 min at 11,000 x g. Discard flow-through and place the NucleoSpin® Plasmid/Plasmid (NoLid) Column back into the collection tube. Repeat this step to load the remaining lysate.
Recommended: If plasmid DNA is prepared from host strains containing high levels of nucleases (e.g., HB101 or strains of the JM series), it is strongly recommended performing an additional washing step with 500 μL Buffer AW, optionally preheated to 50 °C, and centrifuge for 1 min at 11,000 x g before proceeding with Buffer A4. Additional washing with Buffer AW will also increase the reading length of DNA sequencing reactions and improve the performance of critical enzymatic reactions.
Add 600 μL Buffer A4 (supplemented with ethanol, see section 3). Centrifuge for 1 min at 11,000 x g. Discard flow-through and place the NucleoSpin® Plasmid/Plasmid (NoLid) Column back into the empty collection tube.
Centrifuge for 2 min at 11,000 x g and discard the collection tube.
Place the NucleoSpin® Plasmid/Plasmid (NoLid) Column in a 1.5 mL microcentrifuge tube (not provided) and add 50 μL Buffer AE. Incubate for 1 min at room temperature. Centrifuge for 1 min at 11,000 x g.
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