Plasmid Purification Protocol
All centrifugation steps are performed at maximum speed (12-14,000 x g).
- Transfer an overnight culture(1-2 ml) of plasmid-containing cells to a microcentrifuge tube. Pellet the cells by centrifugation for 30 seconds. Remove all of the supernatant by aspirating or pipeting.
- Add 200 ml of the Cell Resuspension Solution and vortex or pipet up and down until the cell pellet is completely resuspended.
- Add 250 ml of the Cell Lysis Solution and mix by gently inverting the capped tube about 10 times(do not vortex). The solution should become viscous and slightly clear if cell lysis has occurred.
- Add 250 ml of the Neutralization Solution and mix by gently inverting the capped tube about 10 times (do not vortex). A visible precipitate should form.
- Pellet the cell debris for 5 minutes in microcentrifuge. A compact white debris pellet will form along the side or at the bottom of the tube. The supernatant (cleared lysate) at this step contains the plasmid DNA.
- While waiting for the centrifugation step at step 5, insert a Spin Filter into one of the 2 ml wash tubes supplied with the kit. Mix the Quantum Prep matrix by repeated shaking and inversion of the bottle to insure that it is completely suspended.
- Transfer the cleared lysate (supernatant) from step 5 to a Spin Filter, add 200 ml of thoroughly suspended matrix, then pipet up and down to mix. If you have multiple samples, transfer the lysates first, then add matrix and mix. When matrix has been added to all samples and mixed, centrifuge for 30 seconds. The correct final formation of the Wash Buffer is 50% ethanol, added by the user. Add one volume, 63ml of 100% ethanol to the Wash Buffer before first use of the Quantum Prep Kit.
- Remove the Spin Filter from the 2 ml tube, discard the filtrate at the bottom of the tube, and replace the filter in the same tube. Add 500 ml of Wash Buffer and wash the matrix by centrifugation for 30 seconds.
- Remove the Spin Filter from the 2 ml tube, discard the filtrate at the bottom of the tube and replace the filter in the same tube. Add 500 ul of Wash Buffer and wash the matrix by centrifugation for a full 2 minutes to remove residual traces of ethanol.
1.Remove the Spin Filter and discard the microcentrifuge tube. Place the filter in one of the 1.5 ml collection tubes supplied with the kit or any other standard 1.5 ml microcentrifuge tube which will accommodate the Spin Filter. Add 100 ml of deionized H 2O or TE. Elute the DNA by centrifugation for 1 minute at top speed.
1.Discard the Spin Filter and store the eluted DNA at -20°C.
- Molecular Biology