If your laboratory uses bacteria to amplify plasmid DNA, you are already familiar with at least one DNA miniprep system. This kit from QIAGEN allows you to go from bacterial culture to reasonably pure plasmid in about 20 minutes using a simple lysis, followed by elution from a supplied bind-and-release spin column. The only pieces of equipment you need are standard 1.7mL microcentrifuge tubes, pipettors and a benchtop microcentrifuge. Alternatively, the protocol can be done using a standard vacuum manifold. QIAGEN offers packages of 50 and 250 preparations, with the price working out to just over $1/miniprep.
The protocol calls for a bacterial pellet from 2mL of overnight culture. The pellet is then resuspended in a buffered solution (P1), and the cells lysed in a second solution (P2). The solution is then neutralized with another buffer (N3) and spun down. Next, the supernatant is spun through the supplied column. Two subsequent wash steps with buffers PB and PE follow, and finally elution with warm water or aqueous buffer typically yields about 15μg of purified plasmid DNA (The binding capacity of the membrane is theoretically 20μg). QIAGEN's protocol is extremely easy to learn. We have had high school interns in our lab who quickly mastered these minipreps. The protocol is also easily adapted for several parallel minipreps, and so is good for working with mutiple clones or plasmid variants. Sequencing of these plasmids shows good purity levels, and is often successful for 600-700 base pairs past the sequencing primer.
All of the solutions can be stored conveniently under normal room conditions, with the exception of buffer P1, which must be refrigerated after the addition of the included RNase. As a minor improvement, QIAGEN kits now include a new LyseBlue reagent, a pH dependent indicator, which is added to Buffer P1. When Buffer P2 is added, the solution turns blue, rendering clumps of unlysed cells more visible. Once these clumps are dissolved, Buffer N3 is added, and the solution again becomes colorless.
The somewhat enigmatic naming scheme for the solutions is quickly overcome with a little experience. One minor irritation of this kit is that the solutions are supplied out of ratio so that you will always run out certain reagents faster than others. More than once I've had to go to other labs to borrow their buffer PB or buffer PE. Overall, however, this is an excellent kit which provides sure results every time. Our lab uses it exclusively.
Chu H. Choi, Ph.D.
Postdoctoral Fellow
Beth Israel Deaconess Medical Center