QIAfilter Plasmid Maxi Kit (10)

From QIAGEN, the QIAfilter Plasmid Maxi Kit is designed for maxi-scale purification of plasmid DNA from bacterial cultures and is classified under the Secondary Sample Technology consumables portfolio.

According to the Quick-Start Protocol, kit components can be stored at room temperature (15–25°C) for at least 2 years. Recommended LB culture volumes are 100 ml for high-copy plasmids and 250 ml for low-copy plasmids. Before beginning, RNase A solution is added to Buffer P1, and LyseBlue® reagent may optionally be added to Buffer P1 at a 1:1000 ratio. Buffer P3 is prechilled to 4°C, and isopropanol plus 70% ethanol are required.

The workflow combines a QIAfilter Cartridge with a QIAGEN-tip. Bacterial cells are harvested by centrifugation at 6000 x g for 15 min at 4°C, resuspended in 10 ml Buffer P1, lysed with 10 ml Buffer P2, and neutralized with 10 ml prechilled Buffer P3. The lysate is filtered through the QIAfilter Cartridge and loaded onto a QIAGEN-tip equilibrated with 10 ml Buffer QBT, washed with 2 x 30 ml Buffer QC, and DNA is eluted with 15 ml Buffer QF. For constructs ≥45 kb, prewarming the elution buffer to 65°C may help increase yield. DNA is precipitated with 10.5 ml room-temperature isopropanol (centrifugation at 15,000 x g for 30 min at 4°C), the pellet is washed with 5 ml room-temperature 70% ethanol, and the DNA is redissolved in a suitable slightly alkaline buffer such as TE buffer, pH 8.0, or 10 mM Tris·Cl, pH 8.5.