Gel-purification is a standard procedure performed to recover desired DNA fragments from agarose gels after electrophoretic separation. After dissolving the gel fragment and running it through a specialized filter, this procedure yields DNA freed from impurities such as salts, free nucleotides and enzymes, suitable for downstream applications.
The first step in the gel purification procedure involves casting the agarose gel and performing electrophoresis of the DNA samples. Once the gel-run is complete, desired DNA fragments are visualized against UV light and fragments are selected after comparing against a molecular weight standard. If the gel is unstained, the band location can be approximately determined based on a comparison to the DNA ladder. While cutting the gel with a razor blade, one must take care to recover as much DNA as possible with as little agarose as possible. When handling ethidium bromide stained gels and working in front of UV light, gloves and protective eyewear should be used. After cutting the desired DNA from the gel, dispose of the gel and running buffer properly, in compliance with institutional safety protocols. Once isolated, the piece of gel is placed in a microfuge tube and weighed on a balance. Using the approximation that 100 mg of gel occupies 100 l, a volume of solubilization buffer that is 4X the gel weight is added to the gel piece. After being placed in buffer, the gel piece is incubated at around 50°C to melt the agarose. Once melted, the solubilized gel is added into a spin column and the solution is centrifuged, which will cause all of the DNA and other particulates to stick to the filter. Next, the bound DNA is washed by adding 70% ethanol to the filter, followed by centrifugation, which will remove residual impurities from the filter. Flow through is discarded, and this washing step is then generally repeated up to three times. The empty filter is spun again to remove residual ethanol, and the silica filter is allowed to dry at room temperature. Water or elution buffer is added to the filter, and with another round of centrifugation, purified DNA is collected in the bottom of the tube.
Prepared by
Aqief Afzal
Research Assistant, ASRBC
