Overview and example chromatograms

On this page we compiled a collection of typical failure chromatograms, so, you can start trouble shooting by just comparing images. Your data probably look much better than the examples below but you might detect some kind of pattern and use it to further improve the process and get even better results! Basically the data should be as in figure 1 below, nice sharp peaks with just a single colour represented in each (unless a heterozygote is present), with no pull-up peaks showing underneath. As shown below many factors might be responsible for results of unsatisfactory quality. Please note that different zooms are applied to the different chromatograms shown below to make the respective effects more obvious. Sometimes it is difficult or even impossible to pinpoint one reason for a failure, as several factors are involved at the same time or one is extremely severe and the chromatogram shows more or less nothing. F. i. this is true if the the DNA pellet was lost during precipitation or if there is much too much salt present in the sample or if the primers do not bind for some reason. In these cases you might only be able to start trouble shooting via the "theoretical" approach. Very commonly used programs for viewing the sequencing data are CHROMAS (for PC/Windows) and TraceViewer (for other systems as well). You can get a free copy of CHROMAS (http://www.technelysium.com.au/chromas.html) or TraceViewer (http://www.codoncode.com/TraceViewer/install.htm) from the web.

Fig.1: High Quality Read Fig.2: Just Failure? Fig.3: Dirty DNA Template Fig.4: Mixed Sequence Fig.5: Wrong amount of DNA Fig.6: Sequence dep. difficulties Fig.7: PCR Products