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 .
