Presence of Salt

The presence of salt in an automated sequencing reaction is now thankfully a lot less common than it used to be. Of course it still occurs, and is very detrimental to the quality of the data obtained as a result of this sort of contamination. A characteristic 'full view' chromatogram from sequencing in the presence of a high level of salt, very much looks like the 'ski-jump' profile seen from too much DNA. As a difference usually the peaks at the beginning are lower. The presence of salt inhibits the sequencing reaction - it can be demonstrated that the higher the salt concentrations, the less data is produced, up to a point where the reaction is completely inhibited by excess salt. The salt is usually introduced from the DNA template and/or the primers. This usually occurs upon elution of the DNA from an anion exchage column with a high salt buffer (cf. section about dirty template DNA) or failure to remove most of the salt by washing after precipitation of DNA. If the DNA is kept in either a 10mM Tris-HCl pH 8.0 buffer or, even better, in water, then it should be fine for automated sequencing. Please be aware that drying a volume of 50 µl DNA in 10mM Tris-HCl delivers a considerable amont of salt and buffer. Therefore pure water is preferable to dissolve DNA. Salt very much affects the efficiency of loading the DNA to the capillaries used in automated sequencing machines. This loading process ("electrokinetic injection") is driven by an electric field and because salt ions migrate much faster then the bulky DNA molecules this loading is inefficient in the presence of salt. The sequencing is less sensitive in the end.