QIAfilter Plasmid Midi Kit (100)

From QIAGEN, this plasmid purification kit relies on anion-exchange technology to isolate plasmid DNA from bacterial lysates that have been clarified by filtration. According to supplier documentation, the kit can yield up to 10 mg of DNA of a purity comparable to that obtained through two rounds of CsCl gradient centrifugation, suitable for transfection-grade applications.

The Quick-Start Protocol from QIAGEN indicates that the kit components can be stored at room temperature (15–25°C) for at least 2 years. For high-copy plasmids, the recommended LB culture volume is 25 ml, while low-copy plasmids require 50–100 ml. Bacterial cultures are harvested after 12–16 h of growth by centrifugation at 6000 × g for 15 min at 4°C, then resuspended in Buffer P1 (with RNase A added), lysed with Buffer P2, and neutralized with prechilled Buffer P3. LyseBlue reagent can be added optionally to Buffer P1 at a 1:1000 ratio to visualize lysis; the lysate turns blue after Buffer P2 and becomes colorless once Buffer P3 is mixed thoroughly.

After clarification through the QIAfilter Cartridge, the lysate is applied to an equilibrated QIAGEN-tip, washed with Buffer QC, and eluted with Buffer QF. For constructs ≥45 kb, prewarming the elution buffer to 65°C may help increase yield. DNA is precipitated with room-temperature isopropanol, washed with 70% ethanol, and air-dried for 5–10 min before redissolution in a suitable buffer such as TE (pH 8.0) or 10 mM Tris·Cl (pH 8.5). Isopropanol and 70% ethanol must be supplied by the user.