Today, it seems like every company on the market sells a plasmid miniprep kit, thus, researchers have several to choose from. Our lab is currently using Qiagen’s QIAprep miniprep kit as our miniprep kit of choice. Personally, this is the only kit I have used in my research career. We use this kit primarily to isolate DNA from 5ml overnight cultures of bacteria following transformation. DNA isolated using this kit has worked well in many applications, including restriction digests, sequencing and as template DNA for PCR.
The basic protocol is probably similar to other miniprep kits. Depending on the number of samples, the procedure usually requires less than one hour of hands on time. The protocol uses 2ml of an overnight bacterial culture. I have found that 2ml epptubes work best for this part of the procedure. The bacterial culture is spun down and the pellet is resuspended in buffer. The bacteria is then lysed for 5min at room temperature and neutralized. The resulting solution is centrifuged and the cleared lysate is loaded onto a spin column. This kit can be used with either a centrifuge or a vacuum manifold. I usually use a centrifuge, but it would probably be a little faster if a vacuum manifold was used. The lysate is spun through the column; the membrane is then washed once and, following two spins, the DNA is ready to be eluted. The DNA can be eluted with either the supplied elution buffer or with water. I usually elute with 50ul of water and DNA isolated using this kit has performed well in subsequent restriction digestions and sequencing. I usually use between 5-10ul of the sample for each digestion and for sequencing. The DNA also performs well as a template for PCR.
If you are looking for increased yield from your Qiagen QIAquick spin minipreps, I have a couple of suggestions that might help. First, you can use 4ml of an overnight culture instead of just 2ml. I simply spin down 2ml of culture, remove the supernatant, and spin down another 2ml in the same tube. It should be noted that this is not required to obtain high yields using this kit. Another way to increase your yield when eluting with water (and not in a hurry) is to allow the column to incubate 30-60 minutes at room temperature before the final spin (after adding the water). This incubation will increase your DNA yield significantly. I am not sure if this incubation will increase your yield if eluting with the elution buffer, since I primarily elute with water.
I cannot think of any major disadvantages of this miniprep kit. Personally, I have not experimented with miniprep kits from different companies because I have never had a reason to change from Qiagen. I have found this product extremely reliable with reproducible yields and purity every time. I know exactly how much DNA I will get from my minipreps and how much DNA to use in restriction digestions and to send for sequencing. If you are looking for a high quality miniprep kit that is reliable, I suggest trying Qiagen’s QIAprep spin miniprep kit - it has worked well for us.
Aaron Tooley
Graduate Student
Dept. of Pathology
University of California, San Francisco