Bio-Rad Quantum Prep Plasmid Kits - Manual (Page 5)

5. Add 5 ml of Neutralization Solution. Cap the tube and mix
by inverting the tube 6–8 times. The solution should
become cloudy and develop a flocculant white precipitate.
6. Centrifuge for 10 minutes at 8,000 rpm (7,500 x g).
Carefully pour the supernatant into a new tube. Try not
to transfer any of the precipitate. However, a small
amount of the debris will not affect plasmid purification.
7. Resuspend the Quantum Prep matrix by shaking
vigorously. Add 1.0 ml of Quantum Prep matrix to
the cleared lysate from step 6. Swirl gently for
15–30 seconds to mix. Centrifuge for 2 minutes at
8,000 rpm to pellet the matrix.
8. Carefully pour off the supernatant from the pelleted
matrix.
Add 125 ml of 95% ethanol to the wash buffer
before first use.
9. Add 10 ml of wash buffer to the matrix. Resuspend the
matrix in the wash buffer by shaking.
10. Centrifuge for 2 minutes at 8,000 rpm. Carefully pour
the wash buffer from the pelleted matrix. Add 600
µ l of wash buffer to the matrix and resuspend.
If isolating DNA from an EndA+ strain do an
additional 10 ml wash step at this point.
Additional wash buffer can be ordered using Bio-Rad
catalog number 732-6134. Alternatively the Midiprep
Wash Buffer, composed of 200 mM NaCl, 40 mM
Tris, 4 mM EDTA, pH 7.5 plus an equivalent amount
of 95% ethanol, may be made.
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• To increase DNA concentration, elute with less
than the recommended elution volume or concentrate
the DNA by ethanol precipitation.
• If using EndA+ strains, grow in Luria Broth and
purify DNA from only 20 ml of culture using rec-
ommended volumes and extra wash steps noted.
2.2 Protocol
Note: All steps are performed at room temperature unless
otherwise indicated.
1. Inoculate plasmid-containing bacteria into 50 ml of
liquid media in a 250 ml Erlenmeyer flask. Incubate
overnight (15–18 hr) at 37 °C in a rotary shaker at
300 rpm.
2. Transfer 40 ml of the overnight culture to a 50 ml screw-
cap centrifuge tube. Spin down cells by centrifugation
for 5 minutes at 5,000 rpm (approximately 3,000 x g).
Discard the supernatant.
3. Add 5 ml of Cell Resuspension Solution to the cell
pellet. Vortex the cells to resuspend the pellet. Be
sure that the cell pellet is completely resuspended.
4. Add 5 ml of Cell Lysis Solution. Mix by inverting
the tube 6–8 times. Do not vortex, since this may
cause shearing of the chromosomal DNA, resulting
in contamination of the plasmid DNA. The solution
should become viscous and somewhat clear.
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