Every molecular biologist is concerned about the purity of their DNA samples. Whether performing RT-PCR, nested PCR, sequencing or fragment analysis, a critical step for a successful outcome is the purity of the PCR products. There are many different purification systems available today based on slightly different purification schemes. The Qiagen QIAquick PCR Purification Kit is one of them and it is based on a silica-gel membrane system that selectively binds DNA.
The kit is made up of binding buffer, wash buffer and small columns composed of selectively binding membranes in standard 2 ml tubes. The PCR samples are mixed with binding buffer, loaded into the columns and briefly centrifuged. The buffer provides the correct salt concentration and pH for DNA to optimally adsorb to the silica-gel membrane, enabling fragments of at least 100 base pairs to be retained in the column, while contaminants and smaller oligomers flow through. A subsequent washing with an alcohol buffer removes residual salts, leaving highly purified DNA bound to the membrane. At this point, double or single stranded DNA molecules can easily be eluted with a low salt concentration buffer or pure water. We found out that using warm water at this point increased our yield, probably because the higher temperature relaxes the membrane fibres, assisting DNA unbinding.
I have been using QIAquick for a couple of years now and I think it is one of the best PCR purification systems I have ever worked with. It is based on a very simple principle; thus it is easy to follow each step of the procedure. In addition, the efficiency is incredible as substantiated by the bands I obtained following agarose-gel electrophoresis. This is very important when you have to perform many reactions with the purified DNA and product yield is a critical issue. Moreover, sample quality is good; most low weight oligomers are removed, while longer ones are rare and are usually due to PCR problems (non-specific amplification, too short elongation time, etc.) which cannot be fixed by purification alone. Finally, the feature I like most is the speed. The QIAquick is incredibly fast, I can usually purify 24 samples in less than 30 minutes.
I find the QIAquick system to be quite simple to use and handy, in spite of utilizing plain tubes. On the other hand, this is a relatively expensive kit, since a single purification can cost up to three times more than competitors, depending on the technique used. This could be an issue for laboratories performing a high number of purifications, but there are other options available for those circumstances. Overall, QIAquick is a superb system, capable of providing sharp results even with poor samples thanks to its high efficiency.
Dr. Andrea Galli
University of Milan, Italy