Note
All centrifugation speeds are given in units of g. Please refer to Table 1 for information on vonverting g-force to rpm. If centrifuges/rotors for the required g-forces are not available, use the maximum g-force possible and increase the spin time proportionally. Spin until all liquid passes through the column.
All steps are carried out at room temperature.
Harvest Cells
Pellet 1-5 ml of an overnight recombinant E. coli culture by centrifugation. The optimal volume of culture to use depends upon the plasmid and culture density. For best yields, follow the instructions in the note below. Transfer the appropriate volume of the recombinant E. coli culture to a microcentrifuge tube and pellet cells at ≥12000 × g for 1 minute. Discard the supernatant.
Note: for best results with recombinant E. coli grown in LB (Luria Broth), use 1-3 ml of culture for high copy plasmids or 1-5 ml of culture for low copy plasmids. With recombinant E. coli grown in rich media such as TB (Terrific Broth) or 2 × YT, use only 1 ml of culture. Higher volumes can cause a reduction in yield.
1. Resuspend cells
Completely resuspend the bacterial pellet with 200 µl of the Resuspension Soulation. Vortex or pipette up and down to thoroughly resuspend the cells until homogeneous. Incomplete resuspension will result in poor recovery.
Another rapid way to resuspend the cell pellets is to scrape the bottoms of the microcentrifuge tube back and forth 5 times across the surface of a polypropylene microcentrifuge tube storage rack with 5 × 16 holes.
2. Lyse cells
Lyse the resuspended cells by adding 200 µl of the Lysis Solution. Immediately mix the contents by gentle inversion (6-8 times) until the mixture becomes clear and viscous. Do not vortex. Harsh mixing wil shear genomic DNA, resulting in chromosomal DNA contamination in the final revovered plasmid DNA. Do not allow the lysis reaction to exceed 5 minutes. Prolonged alkaline lysis may permanently denature supercoiled plasmid DNA that may render it unsuitable for most downstream applications.
3. Neutralize
Precipitate the cell debris by adding 350 µl of the Neutralization/Binding Solution. Gently invert the tube 4-6 times. Pellet the cell debris by centrifuging at ≥12000 × g or maximum speed for 10 minutes. Cell debris, proteins, lipids, SDS, and chromosomal DNA should fall out of solution as a cloudy, viscous precipitate. If the supernatant contains a large amount of floating particulates after cengtrifugation, recentrifuge the suspernatant before proceeding to step 6.
4. Prepare Column
Insert a GenElute Miniprep Binding Column into a provided microcentrifuge tube, if not already assembled. Add 500 µl of the Column Preparation Solution to each miniprep column and centrifuge at 12000 × g for 30 seconds to 1 minute. Discard the flow-through liquid.
Note: The Column Preparation Solution maximizes binding of DNA to the membrane resulting in more consistent yields.
