Why Did My Seq Fail?

Frequent reasons for a DNA sequencing reaction resulting in
"No analyzed data"

  • "No analyzed data" results when there is an insufficient level of fluorescent termination products for the computer software to call bases.
  • Poor quality template DNA. Qiagen ion exchange resin (tip-20, tip-100, etc.) or Qiawells seem to generate the best quality plasmid template DNA for the widest number of researchers. Template DNA must be free of residual ethanol and salt. Please refer to our sheet on plasmid template preparation for more information.
  • Insufficient amount of template DNA. For double-stranded plasmids, 250-300 ng of template DNA are required per reaction. We rely on the investigator's quantitation of template DNA to set up the reactions. In general, the more accurate the measurement of template concentration, the better the quality of data that will be returned (Too much DNA can be almost as bad as too little).
  • Insufficient primer concentration. We require primers at 4 µM concentration which for a 20mer is equivalent to a about 27 ng/µl.
  • Primer and/or template was not added to the reaction. Occasionally, either the primer or template is accidentally left out of a reaction. We try to immediately identify and repeat such reactions.
  • The tm of the primer is << 50oC.The annealing temperature in our cycle sequencing reactions is 50oC, therefore, all primers should have a melting temperature Ñ 52oC. Melting temperature of primers should be determined by a program (Oligo or Primerselect) which uses the nearest neighbor summation of thermodynamic parameters to calculate an accurate tm. We recommend that all sequencing primers be between 20 and 30 nucleotides in length.
  • The template does not contain a sequence complementary to the primer. For example, the pgem series of plasmid vectors do not contain a T3 primer site, but instead contain an SP6 site.
  • For PCR products, occasionally one of the primers used to generate the PCR product does not work in fluorescent cycle sequencing. It is likely that such a primer, although sufficiently competent in the exponential PCR process, is very inefficient in the linear amplification of cycle sequencing.