Short Read Length/Poor Quality Data
- Sequence provides less than 750 Q20+ bases in electropherogram (when a longer sequence is expected)
- End of the sequence is "messy"
- Signal ends abruptly before base 1000 (when a longer sequence is expected)
- Too much template DNA
- Too little DNA
- Excessive dilution of BigDye reagent
- Too much primer
- Template DNA not purified properly
- Unsequencable region reached (eg. homopolymer G)
- Check the concentration of the template DNA using a combination of gel electrophoresis and spectrophotometer readings.
- Reduce the reaction scale rather than using a higher BigDye dilution.
- Check that the oligonucleotide primer concentration is correct.
- Check that the template is clean. Consider sequencing a PCR amplified insert or switching to using a commercial plasmid miniprep kit if you are finding that many of your sequencing reactions are failing.
- Check to see if an unsequencable region has been reached. If an unsequencable or "hard stop" region is the cause then it may be possible to sequence through it by PCR amplifying the region using 7-deaza-deoxy guanosine triphosphate (7-deaza-dGTP), then sequencing the PCR product directly. The 7-deaza-dGTP analog disrupts the hoogsteen base pairing between successive guanosine bases and allows the sequencing DNA polymerase through the high G+C region.