Question

Assume that you have cut λ DNA with the restriction enzyme HindIII. You separate the fragments on an agarose gel and stain the DNA with ethidium bromide. You notice that the intensity of the stain is less in the bands that have migrated closer to the "+" pole. Give an explanation for this finding.

Answer 1

$\lambda$ DNA is used for the digestion with the restriction enzyme HindIII to generate different DNA fragments of different sizes that move on the agarose gel electrophoresis band from the negative terminal towards the positive terminal of the gel. The smaller fragments move towards faster towards the + terminal of the agarose apparatus gel whereas the larger size bands in the electrophoresis gel assembly buffer system move at a slower velocity. The smaller size fragments bind to the ethidium bromide with less intensity as the number of the binding sites of the stain are less in the smaller size bands that have migrated closer to the + pole. Therefore, the intensity of the smaller bands after visualization on the agarose gel under the UV transilluminator appears to be of lower intensity as compared to the illumination intensity of the larger size bands on the gel.