Sequencing failed

 

Status descriptions

  • The image shows disordered wave peak.
  • Except the intensive dye signal at 70 bp and 110 bp, there are only low signal values in other places.
  • The average signal intensity is lower than 100.
  • The signals do not conform to the expected sequence or do not correspond to any sequence in the GeneBank.

Possible reasons

  • Poor DNA quality
  • There is DNA loss during the alcohol precipitating purification
  • The template DNA is scanty
  • Use the wrong primer  
  • The water used for sequencing reaction is polluted and the polluted water contains substances which can suppress the reaction.
  • The primer synthesis fails or the primer degrades. The primer is not successfully synthesized, so it can not be used for the sequencing.
  • The sequencing reagent loses activity. If the BigDye reagent is preserved for a too long time or preserved in inappropriate environment, the Taq will lose activity and the fluorescence of the fluorescent labeling nucleotides will degenerate.
  • The capillary is blocked.

Solutions

  • The DNA quality is not good. Use purification reagent to make purification again to remove the impurities from the DNA.
  • During the alcohol precipitating purification, DNA loss happens. Replace the alcohol precipitating purification method with purification reagent sold in the market, and this will often produce a good effect, but the cost is relatively higher.
  • The template DNA is scanty. Before the sequencing reaction, run the template DNA on the agarose gel to confirm the DNA concentration and by the way check whether the sample is polluted by genomic DNA and RNA.
  • Use the wrong primer. Reconfirm the primer sequence and its correctness.
  • The water used for sequencing reaction is polluted; please use clean and unpolluted water.
  • The primer degrades. Please synthesize the primer again and dissolve it and preserve it in appropriate environment.
  • The sequencing reagent loses activity. The internal reagent of the company is strictly managed, so such situation seldom happens.