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Star Republic: Guide for Biologists

Sequencing troubleshooting

Dye Termination

  • Gel compression: Peaks on the elctropherogram appear to be overlapping or are unevenly spaced because of secondary structures, common for GC-rich areas. To reduce gel compression, use 7-deaza dGTP or dITP in place of dGTP. See also Sequencing of GC-rich DNA.
  • Flat line: low and noisy signal---the sequencing reaction didn't work. The problem is frequently poor template quality (too much contamination) or quantity (too little), poor primer annealing (low primer concentration), and mistakes in handling of samples or programming PCR machine.
  • Noise data: The most common cause is dirty DNA template. Two or more DNA templates may be present or primers anneal to more than one site. Enzyme slippage can also occur in homopolymer regions. Poor gel preparation or insufficient sample denaturation before electrophoresis.
  • Loss of the G signal: Dideoxy G label is more photosensitive than the other dyes. Excessive exposure to light may result in loss of signal from this dye.

Dideoxy Chain Termination

  • No signal: component missing from reaction, template concentration too low, or wrong primer.
  • Signal disappears after 100 bases: low template concentration or low enzyme activity.
  • Peaks in all four lanes: template dirty, low enzyme activity, poor denaturation. The template may also GC-rich or CT-rich. For GC-rich area, add 3 ml of DMSO to the annealing reaction. For CT-rich area, more T7 enzyme is needed.
  • Noise data: The most common cause is dirty DNA template. Two or more DNA templates may be present or primers anneal to more than one site. Enzyme slippage can also occur in homopolymer regions. Poor gel preparation or insufficient sample denaturation before electrophoresis.
  • Missing bands: termination reactions too long.