Sequence Appearance.

• Electrophoregram has mixed peaks or is mostly blank.
• Many ‘N’s in the sequence, if bases are called at all.
• The read sequence file yields a no recognizable sequence.
• Raw data has signal intensity in the low hundreds.

Example.

Possibles Causes.
The causes for these kind of results can be several, the most common are the following ones:

• DNA Template concentration is too low.
  Important Note: measuring DNA concentration by UV absorption is often inaccurate and concentration is frequently overestimated. Agarose gels or espectrofluotometry are a much better method to estimate quality and quantity of DNA samples. In this case the Raw Data signal is low, around 100, due to poor DNA sequencing reaction.
• No correct primer was added to the reaction, so binding between the DNA and the primer cannot occur. No DNA sequencing reaction, and hence Raw Data signal usually below 100 units.
• Template purification is not working properly. Sometimes the sample preparation is contaminated with ethanol or other molecules that inhibit the DNA Polymerase, yielding no sequencing reaction.

Treatment.

• Check the concentration of the template by agarose gel or espectrofluorometry to ensure that it is in the ranges described in the Sample Submission Guidelines.
• Check the primer sequence against the template sequence to ensure that there is a proper binding site.
• Improve the quality of the template preparation, mainly for the primers making fresh dilution each time in case problems were observed.