3. Centrifuge at maximum speed (10000-13000 rpm) for 10 min at room temperature. Transfer the supernatant promptly to a fresh tube and recentrifuge for 10 min at maximum speed. Transfer the supernatant to a fresh tube. After centrifugation the supernatant should be clear. Suspended or particulate material (causing the sample to be turbid) will clog the column and reduce or eliminate flow. Alternatively, the centrifugation process may be replaced by use of a QIAfilterTM Midi Cartridge.
4. Precipitate the DNA in the supernatant with 0.7 volumes room-temperture isopropanol. Centrifuge at maximum speed for 30 min at room temperature ande carefully decant the supernatant.
5. Redissolve the DNA pellet in a small volume of TE, pH 7.0, and add Buffer QBT to obtain a final volume of at least 1 ml. Apply the sample to a previously equilibrated QIAGEN-tip 20.
Note: To compare the amount of plasmid in the culture with amount obtained after purification, remove a comparable aliquot (11%) of the final purified sample and quantify the amount of plasmid each contains by comparison to standards on an agarose gel. A recovery of 80-% is typical.
6. Continue with the standard purification protocol at step 7 (page 11).
Note: An extra 5-10% increase in recovery of cosmid DNA may be obtained by heating Buffer QF to 65℃ before elution (step 8).
Purification of BACs, PACs, PIs, and other large plasmids
BACs, PACs, PIs, and other large plasmid constructs can be purified on QIAGEN-tip 20 with a few modifications to the standard protocol. Recommendations are give below. Cultures should be grown overnight in 5 ml LB medium.
1. Resuspend bacterial cells in 0.6 ml Buffer P1, and also increase volumes of lysis buffers P2 and P3 to 0.6 ml.
2. Elute DNA using 2×0.4 ml pre-warmed (65℃) Buffer QF.
3. After isopropanol precipitation, redissolve DNA in 20 µl 10 mM Tris-Cl, pH 8.5, incubating overnight at 4℃ to redissolve completely.
（此步可用TE缓冲液（pH 8）溶解DNA替代）。
