Cross-Contamination Limits the Use of Recycled Anion Exchange Resins for Preparing Plasmid DNA

DNA of high purity and quality is essential to most molecular biology studies and is easily obtained using silicabased resins or anion-exchange resins. These resins are used in many of the commercial kits available for DNA purification. Qiagen-tips (Qiagen, Valencia, CA, USA) offer one of the most popular anion-exchange resin columns and is one that the manufacturer recommends discarding after a single use (2). Recently, a protocol to recycle these columns was reported, which used a high-salt solution to strip used columns for subsequent purifications (1). It was reported that Qiagen-tips could be reused up to 20 times without an apparent decrease in either their yield or purity. Different plasmids purified from the same column appeared to be free of contamination by restriction endonuclease analysis on agarose gels. To test for the presence of occult contamination, we devised a more stringent method to assess the purity of multiple plasmid DNAs harvested from the same anion exchange column. Three different plasmids were selected, each containing unique inserts of different sizes that could be amplified with unique primer pairs using PCR. They were purified in succession over three days using the recycling protocol. On the first day, the first plasmid was purified using the manufacturer’s maxi protocol. After purification, the column was rinsed with 20 mL of deionized water and stored at room temperature until the next day. On the second day immediately before use, the column was washed 3 times with 20 mL of “renew” buffer (3 M NaCl, 0.15% Triton X-100) and re-equilibrated as normal with the Qiagen equilibration buffer. A second plasmid was then purified according to the manufacturer’s protocol. Again, the column was rinsed with 20 mL of deionized water and left overnight at room temperature. This process was repeated with the third plasmid. A diagnostic restriction enzyme digest was conducted on each purified plasmid and resolved on a gel as the original authors did. No contamination was visible with ethidium staining (data not shown). PCR was conducted on each plasmid preparation using the three unique primer pairs for each of the three plasmid inserts. Unfortunately, we detected contamination from each of the previous DNA preparations in all subsequent preparations (Figure 1). In addition, there was a trend towards decreasing yield with subsequent preparations. We set out to see if our laboratory could apply the recycling protocol to our Qiagen columns in an effort to reduce costs. Because of the importance of clean DNA in molecular biology work, we used PCR to test more stringently the assertions made by the protocol’s authors. Regrettably, contamination was clearly evident in later preparations, thereby limiting its usefulness in applications that require a high degree of purity, such as functional studies. However, the recycling protocol may still be a useful alternative when a low level of contamination is tolerable.