Wrong Amount of DNA

A basic mistake in quantification is to overestimate the amount of DNA that is destined for sequencing. This will result in a fairly obvious weak trace on the gel (signal strength values will be correspondingly low), the major problem with this lies in the fact that the peaks will be re-scaled once analysed and so the background noise associated with the probe will also be re-scaled. This results in a characteristic "noisy" sample. If you are using this sample for heterozygote detection be very careful! Many false results may be seen.

Figure: Insufficient DNA

The peak heights should be nice and even throughout the length of the run. If the peaks at the start of the sample are all off scale and then the data suddenly drops down in strength, finally dying off only 300-400 bases into a run it probably means that the reaction has been overloaded with template DNA (a so called 'ski-jump' profile when looked at in the Full View image). When the sequencing reaction becomes overloaded with template DNA the majority of the dye label is incorporated within the first few rounds on the cycle sequencing. This leads to the formation of many short extension products and usually peaks that are off scale right at the start of the analysed data. Simply reduce template quantities for sequencing.

Figure: Too much DNA

It must be noted however that this profile can also be seen when salt is present in a cycle sequencing reaction - this also leads to more dye incorporation in the first few rounds of the cycling, although usually it is not associated with the peaks that are off scale at the start of the trace. In general, it is best to quantitate your DNA on a gel by comparing the band with a band of a DNA marker of known quantity. If you are using a spectrophotometer try to use a salt buffer and not water to dilute your samples as this gives more consistent results!