To provide increased intensity for the smaller bands and the reference bands, multiple fragments of the same size have been cloned into the plasmids used for many of the DNA ladders. These fragments, identical in size, are indistinguishable on agarose gels, but, on acrylamide gels, even slight differences in DNA sequence can lead to noticeably different migration rates. Fragments of the same size do not always run the same on acrylamide.
For the 100bp DNA Ladder, the 500 and 517bp fragments, which run as a close doublet on agarose, separate very clearly on acrylamide, with the 517bp band running around midway between the 500 and 600bp fragments. This “anomalous” migration is an inherent characteristic of acrylamide gel electrophoresis and does not indicate any error in the stated size of the DNA fragments in our ladder.

For the 50bp DNA Ladder and the Low Molecular Weight Ladder, two or more of the bands comprising the 200bp reference band tend to run differently, resulting in one or more extra bands detectable around the 200bp range. As with the 100bp DNA Ladder, this is attributed more to the limitations of acrylamide gel technology than to a problem with the ladder composition. As defined in most molecular biology lab manuals (Maniatis’ Cold Springs Harbor Molecular Cloning Manual, 2nd edition) and as acknowledged by the manufacturers of acrylamide gels, applications requiring precise sizing of DNA fragments should be performed using agarose gels whenever possible.