Common Problem Scenarios

The following scenarios are listed with some possible reasons why sequencing data can be less than satisfactory. Generally, most sequencing problems are related to dirty and/or poorly quantified template.

If you did not quantify ALL your templates just prior to submitting, then your first troubleshooting step should be quantifying your template if you receive results that are not satisfactory.

Your Sequencing Reaction Failed

Template Contamination

• Possible failed reaction
• We are performing small scale sequencing reactions that are very sensitive to contamination present in the template stock. We ask that all templates and primers be submitted in ddH2O for this reason. (Tris ALONE is also acceptable.)
• 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.
• QIAquick PCR Purification Kits will also remove some contamination.

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 submit a request form noting the sequencing kit to be used with a particular template.