- The peaks look disordered and rough.
- The signal stops before 800 bp
- There are too little template DNA
- BigDye reagent is excessively diluted.
- There are too many primers.
- The DNA quality is poor.
- Special fragments exist which make successful sequencing unavailable.
- Check whether the DNA concentration is proper.
- Check whether the BigDye dilution concentration is proper.
- Check whether the sequencing primer concentration is correct.
- Check the DNA quality.
- Use Secondary construct reagent to solve the problem of special sequence. During the sequencing reaction, add 7-deaza-deoxy guanosine triphosphate (7-deaza-dGTP) to help the polymerase pass through GC-rich area.