Elute the released products
What is Elution?
Elution is a term used in analytical chemistry to describe the emergence of chemicals from the column of a chromatograph. As they elute, the chemicals typically flow into a detector. Predicting and controlling the order of elution is a key aspect of column chromatographic methods.
- Remove the DNA fragments from the agarose gel with a clean scalpel. Try to get as small a piece of gel as possible, without touching the DNA.
- Weigh the gel slice in a clorless tube. Add 3 times the volume of Buffer QG to 1 volume of gel.
- Incubate at 50°C for 10 minutes, or until all the gel has dissolved. Mix the tube using the vortex mixer every 2 or 3 minutes to make sure it dissolves.
- Check if the mixture is yellow in color. If it is, it means that its pH is around 7.5. This is the pH at which the adsorbtion (stickiness)of the QIAquick membrane is most efficient. If it is orange or violet, it is too acidic. Add 10µl of 3 M sodium acetate (pH 5.0) and mix, if that is the case.
- Add isopropanol of a volume equal to the volume of the gel to the sample and mix. The isopropanol precipitates the DNA.(?)
- Place QIAquick spin column in a 2ml collection tube.
- To collect the DNA, pour the sample into the QIAquick column, and centrifuge for 1 min.
- Discard flow-through and place QIAquick column back in the same collection tube.
Collection tubes are re-used to reduce plastic waste.
- Add 0.75 ml of Buffer PE to QIAquick column, let it stand for 2 to 5 minutes and then centrifuge for 1 min at 13,000 rpm.
- Discard the flow-through and centrifuge the QIAquick column for another minute at 13,000 rpm.
- The QIAquick column should then be placed in a fresh eppendorff tube.
- To elute (extract) the DNA, add 50 μl of Buffer EB (10 mM Tris·Cl, pH 8.5) or H2O to the center of the QIAquick membrane (a small disc of silica gel) and centrifuge the column for 1 min.
For stronger concentration of DNA, add 30 μl elution buffer to the center of the QIAquick membrane, let it stand for 1 minute, and then centrifuge for 1 minute.