What factors are most important to assure complete digestion of an agarose gel slice?
First, be sure to use the right amount of GELase according to the gel buffer and the protocol you choose. Beyond that, the most critical factor for complete digestion of a gel slice is to be sure that all of the gel is completely melted prior to adding GELase and that it remains molten throughout the digestion. During the process of gelling, the agarose polysaccharides wrap around each other to form helices and then these helices wrap around each other to form bundles of helices. Thus, in its gelled state, many of the chemical bonds of agarose which would be cleaved by GELase are buried inside these helical bundles and are inaccessible to the enzyme. This is why it is so important to obtain complete melting before beginning the digestion and to prevent re-gelling during it. How can I assure that the gel is completely melted? First, the time needed for melting at 70C depends on the tube or container used. In general, glass conducts heat much better than plastic, and tubes with thinner w
