QIAGEN Plasmid Maxi Kit (100)

QIAGEN's Plasmid Maxi Kit uses gravity-flow anion-exchange tips for purification of transfection-grade plasmid DNA. Bacterial lysates are cleared by centrifugation, and plasmid DNA is concentrated and desalted by isopropanol precipitation. The kit delivers up to <500 µg of high-copy plasmid DNA per preparation, with culture volumes of 100–500 ml suitable for high-copy, low-copy, or cosmid DNA. Processing is manual (gravity flow), handling 1 sample per run with a time per run of 160 min. The system uses prepacked anion-exchange tips that operate by gravity flow and never run dry, and it avoids toxic substances such as phenol, chloroform, ethidium bromide, and CsCl. Applications include transfection, cloning, sequencing, capillary sequencing, and in vitro transcription.

Recommended LB culture volumes are 100 ml for high-copy plasmid and 500 ml for low-copy plasmid. The bacterial pellet is resuspended in 10 ml Buffer P1, 10 ml Buffer P2 is added with vigorous inversion (4–6 times), and the mixture is incubated at room temperature (15–25°C) for 5 min. Prechilled Buffer P3 (10 ml) is added, and the lysate is incubated on ice for 20 min before centrifugation at ≥20,000 x g for 30 min at 4°C, with re-centrifugation of the supernatant at ≥20,000 x g for 15 min at 4°C. A QIAGEN-tip 500 is equilibrated with 10 ml Buffer QBT, the supernatant is loaded by gravity flow, and the tip is washed with 2 x 30 ml Buffer QC. DNA is eluted with 15 ml Buffer QF into a 50 ml vessel; prewarming the elution buffer to 65°C may increase yield for constructs larger than 45 kb. DNA is precipitated with 10.5 ml (0.7 volumes) of room-temperature isopropanol and centrifuged at ≥15,000 x g for 30 min at 4°C. The pellet is washed with 5 ml of room-temperature 70% ethanol at ≥15,000 x g for 10 min, air-dried for 5–10 min, and redissolved in a suitable buffer (e.g., TE buffer pH 8.0, or 10 mM Tris·Cl, pH 8.5).

Add RNase A solution to Buffer P1 and store at 2–8°C. Optionally add LyseBlue reagent to Buffer P1 at a ratio of 1:1000. Prechill Buffer P3 at 4°C and check Buffer P2 for SDS precipitation. Isopropanol and 70% ethanol are required. Kits can be stored at room temperature (15–25°C) for up to 2 years if not otherwise stated on the label.