SpinSmart Plasmid Purification Protocol: High-copy plasmid DNA from E. coli
Start with 1-5 ml E. coli LB culture*, pellet cells in a microcentrifuge for 30 sec at 11,000 x g. Discard the supernatant and remove as much of the liquid as possible.
Add 250 µl PB1 Resuspension Buffer.
Vortex or pipet up and down to resuspend the cell pellet completely.
No cell clumps should be visible.
Add 250 µl PB2 Lysis Buffer.
Invert the tube 6-8 times to mix completely.
Do not vortex!.
Incubate at room temperature for up to 5 min or until lysate appears clear.
Add 300 µl PB3 Neutralization Buffer.
Invert the tube 6-8 times to mix completely.
Do not vortex!.
Centrifuge for 5 min at 11,000 x g at room temperature.
If precipitate is not completely clear from the lysate, centrifuge again for 5 min at 11,000 x g at room temperature.
Place a SpinSmart Plasmid Binding Column in a Collection Tube (2 ml) and load a maximum of 750 µl of the supernatant (from the "Lysate Clarification" section above) onto the column.
Centrifuge for 1 min at 11,000 x g. Discard flow-through and place the SpinSmart Plasmid Binding Column into the Collection Tube (2 ml).
Optional: For host strains containing high levels of nucleases (e.g.
HB101 or strains of the JM series), perform a wash step with 500 µl PB4 Wash Buffer pre-warmed to 50°C.
Centrifuge for 1 min at 11,000 x g before proceeding with Buffer PB5.
Add 600 µl PB5 Wash Buffer (make sure EtOH has been added).
Centrifuge for 1 min at 11,000 x g. Discard flow-through and place the SpinSmart Plasmid Column back into the empty Collection Tube (2 ml).
Centrifuge for 2 min at 11,000 x g and discard the Collection Tube (2 ml).
Place the SpinSmart Plasmid Binding Column in a 1.5 ml microcentrifuge tube (not provided) and add 50 µl PB6 Elution Buffer.
Incubate for 1 min at room temperature.
Centrifuge for 1 min at 11,000 x g.
