Team:WashU/Protocols/GelPurification

Gel Extraction from an Agarose Gel

1. Excise the DNA fragment of interest from the agarose gel with a clean, sharp scalpel or razor blade. Trim away excess gel to minimize the amount of agarose.
2. Weigh the gel slice in a tared colorless tube.
3. Add 3 gel volumes of the Gel Solubilization Solution to the gel slice. In other words, for every 100 mg of agarose gel, add 300 microliters of Gel Solubilization Solution. Incubate the gel mixture at 50-60 degrees Celsius for 10 minutes, or until the gel slice is completely dissolved. Vortex briefly every 2-3 minutes during incubation to help dissolve the gel.(NOTE:To adequately dissolve a gel with an agarose concentration greater than 2%, it is necessary to increase the ratio of the GEl Solubilization Solution volume to the gel weight to 6:1.)
4. Prepare the binding column. Preparation of the binding column can be completed while the agarose is being solubilized in step 3. Place the GenElute Binding Column G into one of the provided 2 ml collection tubes. Add 500 microliters of the Column Preparation Solution to each binding column. Centrifuge for one minute. Discard flow-through liquid.
   
5. Check the color of the mixture. Make sure the mixture is yellow (similar to fresh Gel Solubilization Solution without gel slice) prior to proceeding to the following step. If the color of the mixture is red, add 10 microliters of the 3 M Sodium Acetate Buffer, pH 5.2, and mix. The color should now be yellow. If not, add the 3 M Sodium Acetate Buffer, pH 5.2, in 10 microliter increments until the mixture is yellow.
6. Add 1 gel volume of 100% isopropanol and mix until homogenous. For a gel with agarose concentration greater than 2%, use 2 gel volumes of isopropanol.
7. Load the solubilized gel solution mixture from 6 into the binding column that is assembled in a 2 ml collection tube. It is normal to see an occasional color change from yellow to red once the sample is applied to the binding colun. If the volume of the gel mixture is greater than 700 microliters, load the sample onto the column in 700 microliter portions. Centrifuge for 1 minute after loading the column each time. Discard the flow-through liquid. (NOTE: Do not be alarmed if the flow-through has changed color.)
8. Add 700 microliters of the Wash Solution (diluted from Wash Solution Concentrate G as described under Preparation Instructions) to the binding column. Centrifuge for one minute. Remove the binding column from the collection tube and discard the flow-through liquid. Place the binding column back into the collection tube and centrifuge again for one minute without any additional wash solution to remove excess ethanol. Residual Wash Solution will not be completely removed unless the flow-through is discarded before the final centrifugation.
9. Transfer the binding column to a fresh collection tube. Add 50 microliters of Elution Solution to the center of the membrane and incubate for 1 minute. Centrifuge for one minute. For efficient recovery of intact plasmid DNA, preheat the elution solution to 65 degrees Celsius prior to adding it to the membrane. Eluting at 65 degrees Celsius improves plasmid DNA recoveries by 2 to 3-fold. Yields of large linear DNA fragments (> 3 Kb) can also be increased by up to 20% by preheating the elution solution to 65 degrees Celsius.