Troubleshooting

• Contaminants: salts (EDTA,NaCl,NaAc,Kac,KCl), proteins, detergents (SDS,Triton X-100), RNA, chromosomal DNA, organic chemicals (ethanol, chloroform, phenol), divalent cations (Mg,Ca,Mn), and excess PCR primers, dNTPs, enzyme, and buffer components from PCR. Be sure to check the quality of the DNA before submitting for sequencing.
• Incorrect template or primer concentration
• Sub-optimal primer selection or annealing
• Secondary structure of template

Good sequencing

The sequencing electropherogram will show machine model and analysis software version in the left hand corner. The sample name and investigator will appear to the middle left. On the middle right you will see notation showing signal strength. Typical good signal will show GATC values 100-600. Peaks are sharper for the first 500 bases, resolution of peaks drops off from that point, but are still readable out to 750 bases.

Bad and Not-So-Good sequencing
The following shows some examples of problems in sequencing and what the possible reason for failure may be. This list is by no means complete or all-inclusive of reasons for failures. If you notice any obvious omissions or alternate solutions, please let us know.

• Complete failure - No signal present at all
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  Notice poly C from dye front and one to two spikes	See section below on low signal strength for suggested solutions
  Insufficient template	Quantitate the double stranded DNA by fluorimeter, Double check gel quantitation. Increase the amount of DNA
  Inhibitor contaminating template such as salt or ethanol	Clean up template. Spin columns
  Insufficient primer	Make sure you diluted primer to correct concentration. 12 pmol primer per reaction *NOT 12 picomolar*
  Primer has no annealing site, poor primer design (low melting temp) or incorrect primer sequence	Pick good sequencing area. If there are any mismatches between the primer and template the primer will not anneal correctly. Do not make primer in areas of ambiguity.

  
  
  
• Low signal strength of G A T C values less than 50
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  Insufficient template	Quantitate the double stranded DNA by fluorimeter, Double check gel quantitation. Increase the amount of DNA
  Inhibitor contaminating template such as salt or ethanol	Clean up template. Spin columns
  Insufficient primer	Make sure you diluted primer to correct concentration. 12 pmol primer per reaction *NOT 12 picomolar*
  Primer has no annealing site, poor primer design (low melting temp) or incorrect primer sequence	Pick good sequencing area. If there are any mismatches between the primer and template the primer will not anneal correctly. Do not make primer in areas of ambiguity.

  
  
  
• Primer-dimer
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  Primer dimer caused by self binding primer or primer binding to other primers present in template mixture (PCR). Short fragment is predominately amplified. There may or may not be any sequencing following such an artifact.

  Analyze primer used for sequencing. Could template be contaminated with other primers to cause primer dimers? Is point of drop-off at the cloning site?

  
  
  
• Abrupt signal loss
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  PCR product	This is normal
  Plasmid insert with secondary structure	sequence opposite strand, try a primer that anneals in a different position, Ask CSL staff to add DMSO or try other techniques

  
  
  
• Noisy Sequencing
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  Probable cause: Low signal strength due to low template/primer or impurities such as salt or ethanol
  
  
  Probable cause: Multiple templates/primers/priming sites or high signal strength
  
  
  Probable cause: Mixed plasmid prep, two inserts
  
  

  low signal strength	Low signal strength may cause background to appear even higher. Check for impurities. Increase primer and/or template concentration
  high signal strength (saturating detector)	Decrease template concentration
  Contaminated template (impurities)	Purify template with spin columns
  Mixed plasmid prep, vector portion is clean 2 inserts seen	streak for isolation 2X
  Multiple templates/primers/priming sites	Streak plasmids for isolation 2X, Are there possible multiple priming sites? N-1 primer? If a PCR product make sure you removed original primers, also one primer may have generated both ends, or primers may amplify more than one product of DNA.

  
  
  
• Slow drop-off of read / Early signal loss
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  Low template or primer concentration	Increase either primer or template concentration
  Contamination with salt, ethanol, or other impurity	Purify template with spin columns
  High GC content	See next section
  High GT content	Talk to CSL staff about what we can do
  Secondary structure	sequence opposite strand, try a primer that anneals in a different position, Ask CSL staff to add DMSO or try other techniques

  
  
  
• High GC content with short read
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  Talk to CSL staff to determine alternate sequencing strategies such as adding DMSO, increasing denaturation temperatures, alternate sequencing chemistries
  
  
• Waves due to enzyme slippage at poly T region
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  The DNA polymerase may slip during sequencing reaction when it reaches a poly base region. A partially synthesized strand will rebind a base ahead or behind where it should and a series of products are generated with slightly different lengths of poly base regions. This generates a wave pattern as seen above.

  Use an anchored primer or alternate sequencing chemistry such as dRhodamine.