Extraction of pure DNA from agarose is essential for many downstream assays including cloning and radioisotopic or fluorescent DNA sequencing. We use Qiagen’s QIAquick Gel Extraction Kit for this purpose. The kit is suitable to extract and purify DNA of 70 bp to 10 kb from standard or low melt agarose gels in TAE or TBE. We generally purify PCR products from 1% agarose gels, but you can purify from up to 2% agarose, even more than 2% agarose by making small modifications to the procedure. The kit is available in 50 and 250 reaction sizes. This kit can also be used for DNA clean-up from enzymatic reactions, but we have not used the kit for this purpose yet.
The principle of this kit depends on binding properties of the silica-gel membrane. It is reported by the manufacturer that each silica membrane column can bind up to 10 µg DNA. While using this kit, the important point that one should not forget is that adsorption of DNA to silica depends on pH. pH should be less than 7.5 for efficient adsorption; one of the buffers is specially optimized as a pH indicator.
While a microcentrifuge can be used, we have only used the vacuum manifold method. Since our procedure is vacuum-driven, we first make sure to have a vacuum manifold and prepare it before beginning the extraction process. The procedure begins with excising the DNA fragment from the agarose gel with a sharp scalpel. Although the procedure is robust, you should be careful about minimizing the size of the gel slice by removing extra agarose. Then, place the gel slice in a clean tube and weigh it. Add three volumes of Buffer QG to the sample. Buffer QG is the most critical agent because as it solubilizes the agarose gel, it indicates the pH by color change. After incubating the sample on a heat block at 50ºC for 10 minutes, the color of the mixture should be yellow; this means that the mixture is at the optimal pH. If the color of the sample is violet or orange you should add 3 M sodium acetate, pH 5.0. You should observe the color of the mixture turn to yellow. I think this is a safe and easy method for pH optimization.
During the 10 minute incubation step, you should insert the columns into the luer connectors of the vacuum manifold. You can use the QIAvac manifolds or any other manifolds that have a luer connector.
When you are satisfied that the gel has completely dissolved, add one gel volume of isopropanol to the sample and mix. To bind the DNA, load the sample mix onto the column and apply vacuum. Wait until the sample passes through the column completely. To be sure about removing all traces of agarose, you may apply Buffer QG onto the membrane and apply vacuum again. This step is optional but I recommend performing this step. Then, the washing step comes. Buffer PE is for removal of excess salt from the membrane. After addition of Buffer PE, you again apply vacuum. After removing salts, you transfer the column to a provided collection tube and centrifuge. Buffer PE contains ethanol. In the centrifugation step, you should be sure to remove residual ethanol from the membrane. The last step is elution, as expected. You can add different amounts of elution buffer for the desired concentration. You can use distilled sterile water or Buffer EB, and obtain eluate containing pure DNA by centrifuging for one minute. Elution efficiency is dependent on pH. The maximum efficiency is achieved between pH 7.0 and 8.5. So, I would recommend using Buffer EB or measuring the pH if you will use water.
It generally took little time to complete the process: 20 or 25 minutes. The process is easy. Although it depends on multiple factors, the yield is satisfactory, changing from 70 to 90%.
We generally extract DNA from agarose gels for sequencing and transformation assays. Qiagen’s QIAquick Gel Extraction Kit may be a convenient candidate if you want to extract pure DNA from agarose gel. The procedure is rapid, easy to apply, user-friendly and efficient.