The sequencing templates prepared by the method in the manual are single-stranded (approx. 7250 nucleotides), and as a result will not line up with double-stranded markers of the same length. The apparent size will vary depending on the applied voltage, ethidium and agarose concentration in the gel, and whether TBE or TAE is used as running buffer. We strongly recommend using single-stranded M13 DNA (e.g. single-stranded M13mp18, NEB #N4040) as a marker.