The QIAquick Gel Extraction Kit is intended for extraction of DNA fragments ranging in size from 70 bp to10 kb from agarose gels in TAE or TBE buffer. The kit uses silica-gel-membrane technology to bind DNA in a column while removing contaminants and agarose from the sample, followed by elution of the clean DNA.
Once an agarose gel has been run, the DNA band can be sliced from the gel with a clean, sharp scalpel and placed into an eppendorf tube. A pH-sensitive binding buffer is added to solubilize the agarose in a heating block at 50ºC for 10 minutes. The optimal pH is 7.5 or less, in order for the DNA to adsorb to the silica column. The buffer’s color will indicate if your sample is in the right pH range; if not, there is a way to correct the pH by adding 3M sodium acetate, pH 5.0. After solubilization of the gel, the solution is applied to a spin column for DNA adsorption. After adsorption of the DNA, the sample is washed, to remove any unwanted impurities, such as salt, enzymes, dyes, etc.; these will wash away in the flow through.
The DNA is then eluted under conditions of basic pH (ideally between pH 7.0 and 8.5) and low salt concentration. You can use the supplied elution buffer or water; just make sure it is within this pH range. If you elute in water, be sure to store your DNA at –20ºC, as DNA may degrade without the aid of a buffering reagent. After the procedure, it is wise to run an agarose gel to observe the final concentration of the clean DNA, as some of the DNA will invariably be lost during the procedure.
The entire gel extraction procedure takes about 45 minutes to complete, once the agarose gel is run. We have had good success with this kit: The instructions are very easy to follow and all the necessary reagents are supplied, except 100% isopropanol. The only special equipment required is a small tabletop centrifuge or vacuum manifold and a heating block.
Graduate Student
Department of Physiology
Emory University