Wizard® Plus Midipreps DNA Purification System Promega

For research requiring large amounts of plasmid DNA, kits such as the Wizard® Plus Midiprep DNA Purification System are available. The Wizard® Plus Midiprep kit is designed to extract plasmids from 10-100 ml of bacterial culture. The kit works best with plasmids that are less than 20 kb in size, and can yield from 10-200 µg of DNA depending on the volume of culture, bacterial strain and the plasmid. The kit contains Cell Resuspension Solution, Cell Lysis Solution, Neutralization Solution, DNA Purification Resin, Column Wash Solution, and enough Midicolumns for 25 preps. All solutions can be stored at room temperature, and are contained in a box with sliding drawers for easy access. It is important to point out to first time users that a vacuum step is required to wash and purify the DNA. Vacuum or aspirator strength must be 15-18 inches of mercury (Hg). In addition to a vacuum pump or aspirator, you will also need a vacuum manifold. Manifolds for 2 or 20 samples are available from Promega (Vac-Man® Jr. and Vac-Man®), which allow multiple samples to be processed at one time.

The plasmid isolation procedure begins with centrifugation of the bacterial culture to collect a cell pellet, which is resuspended in Cell Resuspension Solution. Cells are then lysed with Cell Lysis Solution and the lysate subsequently neutralized. A second centrifugation step removes cell debris by generating a tight pellet and clear supernatant containing the DNA. I have found that using a pipette to carefully remove the supernatant works well; I prefer using a pipette to filtering the sample, which is the alternative. A DNA Purification Resin is then added to the supernatant to bind the plasmid DNA – care must be taken to mix the resin well before use as it can settle over time. The resin-supernatant mix is then added to the column, and drawn down into a reservoir by applying a vacuum. The DNA bound to the resin is then washed twice by vacuum. Each wash step requires 15 ml of Column Wash solution to be pulled through the tightly packed resin, a process which can take up to 45 minutes. During this time it is essential to watch the column to prevent it emptying completely and allowing the resin to dry out. The reservoir is then detached from the column and the DNA eluted with nuclease free water by centrifugation and elution into an eppendorf tube.

I have used the Wizard® Plus Midiprep kit on plasmids from 4 to 13 kilobases in size and found that in all cases it yields high concentrations of pure, non-nicked DNA, which I have used successfully in transfections, in vitro transcription, restriction digests and sequencing with no further purification. This system gave superior results over similar kits for purification of larger plasmids (~13 kb). Although the manual indicates that volumes of 10 ml can be purified, I suggest purifying from larger culture volumes in order to get the best value from the kit. Although the procedure takes slightly longer, on average, to complete than the 90 minutes suggested by the manufacturer, it is still reasonably quick and quite simple to use.