There are so many miniprep kits available today that it difficult to choose which one would be the best for your application. I have used several different techniques and a variety of kits for isolating plasmid DNA in my career and I have found the ChargeSwitch® kit to be the best fort my needs. The isolated plasmids worked well for standard cloning operations and restriction digests. Since the plasmids had reduced endotoxin content (<50 EU/ìg DNA), they were ideal for transfection into mammalian cells as well.
The basic protocol is quite simple and it can be completed in approximately 15-20 minutes. The protocol calls for 1-5 ml of overnight bacterial culture to be centrifuged in 1.5 ml microcentrifuge tubes. The pellet is resuspended in the Resuspension buffer and then Lysis buffer is added, followed by Precipitation buffer. Once the precipitate is removed, the endotoxin removal reagent (ETRR) is added to the tube along with the magnetic beads. The samples are then placed onto the MagnaRack™ (also available from Invitrogen) for 1 minute to pull the beads against the back wall of the tube. The samples are washed twice (each step taking 1 minute) and eluted (an additional minute incubation). The magnetic beads work by changing their surface charges from positive to negative (and vise versa) depending on the pH of the surrounding buffer. In a low pH environment, the beads are positively charged and therefore, are able to bind to the negatively-charged nucleic acid backbone. The advertised yield for the kit is 20 ìg of DNA per 1.5 ml of culture. I found the advertised yield to be fairly accurate.
I regularly need more than 20 ìg of DNA so I process several tubes at once (the rack can magnetize 12 tubes at a time). I also found that I can concentrate the DNA by eluting 1 tube at a time and using the eluate to elute the next tube and so on. To ensure that I have fully eluted the DNA from the beads, I use fresh elution buffer and perform a second elution, which I then add to the first elution. The first time I used this kit, I tested the efficacy of this practice and found it to work quite well. I routinely generated several hundred micrograms of plasmid. I don’t have any major complaints about this kit. If you are looking for a miniprep kit that can produce high quality DNA in less than 30 minutes, then Invitrogen’s ChargeSwitch® ER Mini kit is something you should try.