Frozen buffers for enzyme reaction must be mixed well because

 Almost all buffers used for enzyme reaction assay are basically stored at -20-30℃, which usually contain salts and viscous liquids. The thawed buffers from freezers don't have uniform density of materials because the water (H2O) is frozen with excluding salts. Moreover, all components in a buffer are separated according to their weight during freezing. Glycerol and sucrose go down to the bottom of the container. Therefore, the thawed buffers are not the same one we made before, unless they are mixed well. This is a very simple, but important matter. Even when we think a buffer mixed well, it is not sometimes enough. If you add 1ul dye into 500ul H2O and mix the liquid by tapping, you can know how much labor we need to get a uniformly mixed solution.