I use the GenElute™ Gel DNA Extraction Kit from Sigma when I want to carry out extraction of plasmid DNA from agarose gels. Most of my plasmid DNA extractions vary between 200bp (for an insert released from a plasmid digested with a restriction enzyme) and 5000bp, but the kit can definitely be used for larger or smaller fragments as well. I have mostly purified DNA from a 0.8% agarose gel. DNA from a 2.0% gel can also be purified. The kit I use contains reagents sufficient for 70 reactions, however different sizes are available from Sigma, and it is a good practice to purchase smaller kits to first test it out.
The kit includes several solutions that are required for the extraction process: 1) Column Preparation Solution, 2) Gel Solubilization Solution (for melting the agarose), 3) Wash Solution Concentrate (to be diluted with ethanol), and 4) Elution Buffer. Additionally, DNA binding columns and collection tubes are provided with the kit. Sigma claims that the column has a binding capacity of 10ug, but I have never tested that limit.
After I cut the gel piece containing the DNA of interest, I weigh it and add the Gel Solubilization Buffer [3 parts (volume) buffer to 1 part (weight) gel]. I then heat the gel piece to about 60ºC for 3-5 minutes. I prefer longer durations of heating for smaller DNA fragments as I use 1.5% agarose for smaller DNA fragments and that requires a few more minutes of heating compared to the 0.8% agarose for larger DNA. However, the duration of heating varies depending on the amount of gel cut-out for melting. The color of the solution should remain yellow after this step. If it does not, then small amounts of sodium acetate (pH 5.2) have to be added until the color returns to yellow (this was never required in my experiments). One gel volume of isopropanol is then added to the mixture. In my experience, this addition can be skipped with no change in the yield or purity of DNA.
In the meantime, the column should be prepared for DNA binding by rinsing it with the Column Preparation Solution. I then load the gel solution on to the column and spin it at high speed (12000 rpm in any fixed angle rotor.). For smaller DNA molecules (200 to 600 bp), I prefer spinning at slightly lower speeds (6000 rpm), which gives the DNA more time to bind to the column rather than being pulled into the flowthrough by the centrifugal force. Once the spin is complete, I use the Wash Buffer to clean the column and then I elute the DNA with the Elution Buffer. The DNA obtained is generally salt-free.
The total time required for the extraction is about 20 minutes. This is very rapid compared to some of the gel extraction kits I used to use. All the steps in this kit are performed at room temperature. I also get a good recovery of DNA: about 75-85% depending on the size.
I have also tried DNA extraction from polyacrylamide gels using protocols I have obtained from elsewhere. The kit does work well for acrylamide gels; however, the yields are not as good as those obtained from agarose gels. Also, a gel diffusion buffer has to be used, and this is not provided in this kit.
A detailed protocol is provided with the kit; however, I encourage people to experiment with different steps and use a combination of different solutions to optimize their yields.