For some downstream assays, such as ligation and transformation, restriction enzyme digestion,
in vitro transcription, hybridization and radioactive and fluorescent sequencing, a researcher has to clean up PCR products from the amplification mixture. During this process, all enzymes should be removed, independent of size and secondary structure. There are several suitable kits for this purpose. We use Qiagen’s MinElute PCR purification kit to obtain pure PCR product, generally for sequencing. The kit is suitable for direct purification of double-stranded PCR products that are between 70 bp and 4 kb. Thus, common 20-40mer oligonucleotides are removed. Starting materials may be
PCR reactions, labeled, modified or digested DNA (for example, products from restriction digests, kinase or enzymatic labeling reactions). The kit can be purchased in 2 sizes: 50 or 250 reactions. While using this kit, you have two choices: A vacuum manifold or a microcentrifuge. In our experience, yield is about the same with both techniques.
The system uses spin-column technology with a silica-gel membrane which has selective binding properties. DNA adsorbs to the silica-membrane in the presence of high salt concentrations, while contaminants pass through the column. The second factor that influences the binding of DNA to the column is pH. pH should be less than 7.5 for the efficient adsorption. Appropriate salt concentration and pH are supplied by the buffers provided in the kit.
The MinElute System uses a simple bind-wash-elute procedure. Five volumes of Buffer PE, binding buffer, is added directly to the PCR sample or other enzymatic reaction. The good thing about this step is you do not need to remove the mineral oil or kerosene. Then, you transfer this mixture to the MinElute spin column. The binding buffer contains a pH indicator. pH indicator dye in the solubilization and binding buffer allows easy visual determination of optimal pH for DNA adsorption. Nucleic acids adsorb to the silica membrane in the high-salt conditions provided by the buffer. You can centrifuge the tube containing the column or you can use a vacuum to pull the liquid through the column. Impurities are then washed away by Buffer PE. After discarding the flow through, you should centrifuge an additional 1 minute in order to dry the membrane. The last step is to elute the DNA from the column. It is achieved by applying a small volume of low-salt buffer (provided) or water just in the middle of the column. Elution efficiency is dependent on pH. The maximum efficiency is achieved between pH 7.0 and 8.5. So, I would recommend using Buffer PE or measuring the pH if you will use water.
The column with specialized assembly allows concentration of DNA fragments small volumes, as little as 10 µl. With using this kit, you can recover 70-80% of the DNA from the reaction.
The procedure is very simple and fast. We consistently obtain reproducible recoveries. The purification generally takes 10 minutes or less. Our lab generally uses this kit in order to purify PCR products for sequencing. The Qiagen MinElute PCR Purification Kit is a valuable tool for purifying PCR products efficiently and quickly for many downstream assays.
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Sevgi Bagislar
Research Assistant
Department of Molecular Biology and Genetics
Bilkent University
Turkey
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