MiniElute® PCR Purification Kit from Qiagen

Isolation of DNA fragments from PCR reactions, agarose gels, or enzymatic reactions is often performed in the daily laboratory routine. The goal is to remove all enzymes, independent of size and secondary structure, together with salts, oligomers, and nucleotides. For a lot of downstream applications, an extremely small elution volume, allowing for high end-concentrations is required.

The MiniElute® PCR Purfication Kit from Qiagen is available in two different sizes, for 50 or 250 purifications, to isolate DNA sizes ranging from 70 bp- 4 kb. The kit comes with spin columns with silica membranes, collection tubes, buffers and pH indicator. A detailed protocol for performing the isolation using a microcentrifuge or alternatively a vacuum manifold, is included. Some reagents need to be supplied by the user, namely ethanol as well as 3 M sodium acetate at pH 5.0, which might be necessary for PCR purifications to adjust the reaction to the optimal pH.

I used this kit on a regular basis for either purification or subsequent concentration of DNA after PCR in preparation for in vitro transcription reactions, or for cleaning up DNA fragments after performing whole genome amplification using a DNA amplification kit (GenoMatrix Whole Genome Amplification Kit from Active Motif).

The MiniElute system uses a simple bind-wash-elute procedure. This makes the protocol easy, fast, and reproducible. One major advantage I really enjoyed was the pH indicator dye which gets added to the DNA binding buffer. DNA adsorption requires a pH ≤7.5, and the pH indicator will appear yellow in this range. If the pH is more in the basic range, the mixture will turn towards a more orange or violet color. In this case, the addition of a small volume of 3M sodium acetate, pH 5.0, is indicated. Using this kit, it happened several times that the pH indicator showed a non-optimized pH which I always corrected by adding the suggested amount (10 ul) of the 3M sodium acetate, pH 5.0.

Following the very detailed protocol of the kit is very easy. The pH indicator is added to the binding (PB) buffer and is used as described above. Binding buffer is directly added to the PCR sample or other enzymatic reaction without needing to remove any mineral oils which might have been used to cover the PCR reaction. The mixture gets applied to the spin column and the DNA is adsorbed to the silica membrane by centrifugation. Impurities are washed away and pure DNA is eluted with a small volume of low salt buffer or water, ready to use in all subsequent applications.

Altogether, this kit performed very well in my hands, especially the convenient pH indicator came in very handy.