HiSpeed Plasmid Maxi Kit (10)

This plasmid purification kit from QIAGEN provides a workflow for isolating plasmid DNA from bacterial cultures. The manufacturer's Quick-Start Protocol states that the kit can be stored at room temperature (15–25°C) for at least 2 years, and it is used together with the buffers P1, P2, P3, QBT, QC and QF, a QIAfilter Cartridge, a HiSpeed Tip and a QIAprecipitator Module.

The protocol begins by pelleting an overnight LB culture — 150 ml for a high-copy plasmid or 250 ml for a low-copy plasmid — at 6000 x g for 15 min at 4°C. The bacterial pellet is resuspended in 10 ml Buffer P1 (to which RNase A solution is added and stored at 2–8°C), then lysed by adding 10 ml Buffer P2 and incubating at room temperature (15–25°C) for 5 min. After neutralisation with 10 ml prechilled Buffer P3, the lysate is filtered through the QIAfilter Cartridge. The cleared lysate is loaded onto a HiSpeed Tip pre-equilibrated with 10 ml Buffer QBT, washed with 60 ml Buffer QC, and plasmid DNA is eluted with 15 ml Buffer QF.

DNA is precipitated by adding 10.5 ml isopropanol to the eluate, then captured on the QIAprecipitator Module. The membrane is washed with 2 ml of 70% ethanol and dried by pressing air through forcefully. The DNA is finally eluted into a 1.5 ml collection tube using 1 ml Buffer TE, with a second elution step performed into the same tube. An optional LyseBlue reagent may be added to Buffer P1 at a ratio of 1:1000 so that lysis turns the solution blue and complete neutralisation returns it to colourless.