Our lab works on the molecular manipulation of unicellular organisms and cell lines. One major aspect of our research is the transfection of plasmid DNA into these cells via injection or electroporation. Usually, large amounts of plasmid DNA (up to 100 ug/sample) are necessary for these procedures. Endotoxins can greatly reduce transfection efficiency and gene expression. Qiagen offers an Endofree Maxi Kit that allows purification of up to 500 ug of endotoxin-free plasmid DNA.
The procedure is essentially made up of five steps: bacterial lysis, lysate filtration, plasmid DNA binding, washing and elution, plasmid DNA precipitation and recovery. All of the necessary buffers and solutions are included, only ethanol needs to be added to one. As in other Qiagen Maxi and Mega plasmid kits, the bacteria are initially lysed and the lysate is pressed through a filter cartridge. The purified lysate is then pored over the column containing the plasmid DNA binding silica-gel. Subsequent washing steps remove endotoxins and other residues. Elution occurs with a low salt buffer and is followed by immediate isopropanol precipitation. After a number of centrifugation and drying steps, the plasmid DNA is re-suspended in endotoxin-free water or buffer TE and is ready to use. The necessary equipment includes a centrifuge capable of obtaining an rpm of over 15,000 x g.
The whole procedure requires 45 min for incubation and an hour for centrifugation. Combine this with column flow rates, drying time and in-between steps, and the purification of endotoxin-free plasmid DNA takes up the better part of a working day. Having said that, it is worth it. The yield can vary and depends on the plasmid you try to purify. Plasmids larger than 10 kb tend to give lower yield, but I regularly get between 300 and 500 ug of plasmid DNA/sample. The manufacturer recommends using 100 ml and 250 ml bacterial cultures for high and low copy plasmids, respectively. However, in my experience, some plasmids give a low yield independent of the culture volume. Also, I recommend having disposable 50 ml Falcon tubes at hand (at least 4/sample).
In contrast to other Qiagen plasmid prep kits, one should prepare a number of things before starting the plasmid prep: pre-cool the centrifuge, prepare endotoxin-treated centrifugation tubes, especially for the final elution and precipitation steps (the Falcon tubes used throughout the plasmid purification are sufficiently clean), obtain sterile 25 ml pipettes, and chill Buffer P3.
After spinning down the cells and re-suspending the pellet in Buffer P1 transfer the soup to a 50 ml Falcon tube. When using the first filter cartridge do not force the cell lysate through the filter. If the volume of the filtered lysate is less then 25 ml, don’t worry, I got plenty of plasmid DNA out of 15 ml lysate. After running and washing the sample of the Endotox column elute the DNA directly into the centrifugation tubes. After addition of isopropanol and the 30 min centrifugation at 15,000 x g, you should see a white pellet. The white is the precipitated salt and most of it will be washed away with ethanol. I successfully use the endotoxin-free DNA for sequencing and to transfect insect cells and malaria parasites.
This kit is rather expensive, however, other kits recommend the removal of endotoxins in a procedure following the actual plasmid prep (e.g. BioVintage). The alternative system I tried, although cheaper, proved to be substantially more costly in terms of time, labor and materials. I also lost more than 50% of my plasmid DNA compared to the Qiagen product. People performing this procedure on a regular basis (more than once a month) will definitely be better off with this kit. In summary, this reliable, although expensive, kit produces ultrapure endotoxin-free plasmid DNA with high yield for small and medium transfection experiments.
Stefan M. Kanzok
Malaria Research Institute
Johns Hopkins Bloomberg School of Public Health
Baltimore, USA