Introduction
There are a number of reasons why a restriction enzyme may not cut efficiently. The enzyme may have lost its activity due to denaturing under poor storage conditions. Contaminants present in the digest may have interfered with the operation of the enzyme. Improper use of a micropipetter may have resulted in little or no enzyme being present in the reaction mixture.
Supercoiled plasmids
In order to visualize the results of the experiment, we will perform agarose electrophoresis on our digested samples. Examination of the gel can allow us to detect failed plasmid digests because differences in the physical shape of cut and uncut plasmids cause them to migrate at different rates through the gel even when they are the same length. Uncut plasmids can be in two forms: relaxed and supercoiled (or superhelical). These two forms can be understood by comparing them with a rubber band. A normal rubber band is like the relaxed form. If you grasp one side of the band between your thumb and forefinger and roll that side, the band will twist up into a compact form similar to a supercoiled plasmid. If you cut the band with scissors, it will no longer remain twisted.
Supercoiled plasmid bands on a gel
In gel electrophoresis of DNA, we normally consider the migration speed of a piece of DNA to depend primarily on its size (unlike proteins which have a migration speed that can also be significantly affected by the pH of the gel). However, this does not hold true with non-linear DNA fragments because their shape has an important effect on their migration speed. Because of their compact size, supercoiled plasmids may move through a gel much more rapidly than a linear fragment of DNA with the same number of basepairs. Likewise, uncut relaxed plasmids usually move at a different speed than a cut fragment of the same mass.
Fig. 5. Gel bands of cut (left) and uncut (right) plasmids. All have the same mass in basepairs, but are in different positions because their differing shape causes them to move through the gel at different speeds.
In Fig. 5, the lane on the left contains a plasmid that was digested in one place with a restriction enzyme. The lane on the right contains the same plasmid undigested. The lane with the digested sample has a single band, while the lane with the undigested sample has three bands. If the lower band had moved most rapidly because it was a smaller fragment than the others, then we would expect that it would be much dimmer than the others because, in general, less fluorescent dye is intercolated by smaller fragments. However, in this case the lower band is much brighter than the other bands, either because the supercoiled shape holds dye better or because a higher fraction of the molecules in that lane were in the supercoiled form than in other forms. A faint band is barely visible in the right lane at the same height as the band in the lane with the digested plasmid. This is probably linear plasmid molecules that were broken mechanically (no enzyme was added to that sample) - the faintness of the band is an indication that there are relatively few of these molecules. By elimination, we can infer that the upper band is uncut plasmid in the relaxed form. Some aspect of its shape causes it to move more slowly than the linear molecules. Given that all three bands in the right lane contain pieces of DNA with the same number of basepairs, it is clear that the shape of the molecule has an important effect on its migration speed.
When you examine your gel, be on the lookout for lanes which have the characteristic pattern of uncut plasmid (i.e. a brighter band lower on the gel). It may help you to interpret lanes that otherwise may not make sense on the basis of the possible fragment sizes which you have predicted.
