
The quality of the DNA template is one of the most critical factors in automated DNA sequencing. Please ensure that the template is at the correct concentration (see Concentration of Template DNA and Primers, that it is free of contaminants and dissolved in distilled water (especially no EDTA present), and that the DNA is not degraded.
If you have enough template DNA, always determine its concentration by UV absorption. However, most of the time, you cannot reliably quantitate a mini-prep using a spectrophotometer!. See Why can't I use a spectrophotometer to check my miniprep?.
Some comments about getting accurate spec readings on DNA:
If you do not have enough to measure spectrophotometrically, then run a small aliquot on a gel and estimate the amount of DNA by comparison with standards of similar size and known concentration.
If you are unable to determine the concentration accurately, you should consider not proceeding until you have enough DNA to measure accurately.
After determining the concentration, dilute the template to the correct final concentration (see table) using *distilled water* (please no TE or other EDTA-containing buffer), and avoid adding any divalent cations (i.e. Mg, Ca, Mn).