The QIAquick nucleotide removal kit uses silica-gel membrane technology to purify DNA molecules from reaction samples. The kit contains columns with silica-gel membranes (QIAquick columns) as well as buffers for binding, washing and elution. In this system, the purification of the DNA is dependent on the salt concentration and pH of the buffers - oligonucleotides and double-stranded DNA can be bound to the membrane in a high-salt buffer and eluted in a low-salt buffer. The binding buffer is optimized for selective adsorption of DNA molecules (> 17 nucleotides) to the silica-gel membrane, while the wash buffer is optimized to remove unincorporated nucleotides, enzymes and other impurities.
The purification procedure is simple and fast. Briefly, the reaction sample is mixed with 10 volumes of binding buffer and applied to the QIAquick column followed by centrifugation for 1 min at 6000 rpm. The labeled DNA is bound to the membrane of the column. Then the column is washed with 500ml of wash buffer and centrifuged for 1 min at 6000 rpm. I usually wash the column twice. After the second wash, the column is placed into an empty tube and spun for an additional 1 min at 13000 rpm to remove the residual wash buffer. This step is important because, according to Qiagen, the wash buffer may interfere with downstream applications. To elute the DNA, elution buffer is added to the column and the column is centrifuged for 1 min at 13000 rpm. If the elution volume is not critical for the downstream applications, I usually elute the column twice with 100ml of elution buffer in each elution. If the elution volume is critical, I generally add 30~50ml elution buffer to the center of the membrane, let the column stand for 1 min, and then centrifuge. The recovery of the labeled DNA is comparable in either way.
I routinely use this kit to remove unincorporated nucleotides and enzymes from a variety of enzymatic reactions such as random-primed labeling, PCR labeling and 5’, 3’ end-labeling. In my experience, most of the unincorporated nucleotides in the reaction are removed (> 99%). In addition, the recovery of labeled oligonucleotides and DNA fragments is good, I consistently get a 70~90% recovery. The quality of purified DNA is excellent - I have used the labeled DNA for various downstream applications such as library screening, Northern /Southern blotting and primer extension experiments. The purified DNA works well in all of these applications, I rarely have any problems with background.
In my hands, this kit has worked extremely well. The purification procedure is simple (no phenol/chloroform extraction or ethanol precipitation) and the only equipment needed for this protocol is a microcentrifuge, which keeps radioactive contamination to a minimum. And, in addition, because the whole procedure only takes about 10 minutes, your exposure time to radioactive material is limited.
Chengshi Hwang, PhD
Postdoctoral Researcher
University of Massachusetts