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The sequencing facility

Troubleshooting

Here are some guidelines for troubleshooting failed or unsatisfactory sequences.

Your Sequencing Reaction Failed


Template Contamination

  • Possible failed reaction
  • Not all plasmid preps are created equal. Some are better for sequencing than others. Generally the purer your template is, the more likely you will have success with sequencing. Kits that say they work well with ABI BigDye sequencing are best. Your sales representative or company technical support should know this information.
  • Generally precipitating the DNA, washing the pellet, and resusupending the DNA in ddH2O is sufficient.
  • Column PCR purification kits will also remove some contamination.
  • Exo Sap is ok.


Primer Problems

  • The primer sequence is not contained within the template
  • The annealing temperature is out-of-range for the cycle sequencing reaction.
  • The primer forms secondary structure.
  • Insufficient amount of primer in the sequencing reaction.
  • Degraded primer


Template Problems

  • Template concentration is too low.
  • The priming site has some secondary structure preventing the primer from annealing to it.
  • Occasionally, specific cell lines do not produce good sequencing data.
  • Degraded template

 

Your Sequencing Data is Not Clean (AKA "noisy")

  • Template contamination
  • Template concentration is too low resulting in a low signal-to-noise ratio.
  • Template concentration is too high (resulting in detector saturation).
  • Template contains a sequence that does not allow the enzyme to operate efficiently*
  • Primer is annealing to two or more sites within the same template.
  • Insufficient amount of primer in the sequencing reaction.
  • Degraded template


Your Sequence Signal Stops Prematurely in an Abrupt Manner

  • Possible secondary structure*
  • Template contains a sequence that does not allow the enzyme to operate efficiently*
  • Sometimes linearizing a plasmid will solve a secondary structure problem.

 

Your Sequence Signal Gradually Tapers Off

  • Possible top-heavy sequence 
  • Template contamination
  • Template contains a sequence that does not allow the enzyme to operate efficiently*
  • Template concentration is too low.
  • Insufficient amount of primer in the sequencing reaction.
  • Possible secondary structure*
  • Degraded template
  • Template is too concentrated.


*Problem may be resolved by requesting an alternate sequencing kit. Please contact us.