There are a large number of options available to researchers that need to perform plasmid preps using the bacterial strain
E.coli. Because I was going to be using my plasmid DNA for transfections, however, I needed to make sure that it was endotoxin free. To do this, I used a kit, made by a California based company called Mo Bio Laboratories, called the UltraClean Endotoxin-Free Plasmid Prep Kit. As its name implies, this kit produces clean, endotoxin-free plasmid DNA that is ideal for transfections for eukaryotic cells, microinjections or even automated sequencing.
The UltraClean kits are available in multiple sizes for use on cultures ranging from 5ml to 500ml. Cells are lysed by what the manufacturer refers to as an improved set of alkaline lysis reagents. Endotoxins are then precipitated into a specialized buffer and the plasmid DNA is left to bind to a silica filter. After this process, endotoxin levels are said to be < 0.1 EU/ mg of supercoiled plasmid DNA.
I decided to use this kit because I needed endotoxin free samples and I wanted an alternative to ethanol precipitation. The kit was easy to use and the resulting plasmid DNA was clean. Unfortunately, having to elute the DNA in 3ml of water left my sample diluted and I had to ethanol precipitate anyway. I had tried to reduce my elution volume, but the size of the silica filter made it difficult to do so. Certain procedures in the protocol were adjusted to fit my requirements, for example, after the lysis solution was added samples were incubated for about 10 minutes. The protocol calls for inverting the tube twice and then adding the neutralization buffer, I find that I do not get complete lysis unless I increase the incubation time prior to adding the neutralization buffer. The white precipitate that is pelleted after centrifugation did not stick to the tube as suggested. I instead used a pipette to remove the supernatant as I turned the tube, allowing some precipitate to stick to the side of the tube.
Overall, I found the Mo Bio endotoxin free Maxi Plasmid Prep kit to be an easy and rapid purification method that produces high quality plasmid DNA. It did produce a lot of plastic waste (necessary for the endotoxin free sample) – this fact and the high elution volume are the down sides to the kit. I have not tried the high nutrient broth recommended by the company, but according to them, using high nutrient broth may be able to increase the yield of my DNA and allow me to work with a culture as small as 3ml. One nice fact is that everything you need to complete this procedure is provided, including endotoxin free water.
Karrie Comatas
Research Technician II
Duke University