Failed DNA sequencing reaction

Cause

• Bad DNA quality
• Wrong primer

Solution

• Use template DNA after cleanup
• Verify that a primer site exists in the template DNA

Noisy or weak DNA sequencing traces

Cause

• DNA concentration is too high or too low
• Primer concentration is too low or the primer is degraded

Solution

• Use template at the appropriate concentration as determined by agarose gel electrophoresis and spectrophotometer measurement
• Use primers at the appropriate concentration and avoid repeated freeze-thaw cycles

Mixed signal in the trace

Cause

• Two or more templates with the same primer site are mixed
• A single template contains two or more identical primer sites

Solution

• Confirm that the DNA template is a single sample by agarose gel
• Check the template sequence or perform sequencing with a different primer

Sudden ending of DNA sequence signal

Cause

• Highly stable secondary structure within the DNA template
• Long G or C stretch

Solution

• Perform sequencing with a reverse primer
• Proceed with the Difficult Sequencing service

Slippage sequencing

Cause

• Repetitive sequence or homopolymeric sequence
• Alleles containing insertion or deletion sequences

Solution

• Perform sequencing with a reverse primer
• Perform sequencing of each allele with allele-specific primers

Blurry trace chromatogram peaks

Cause

• Capillary overload
• High sequencing run voltages

Solution

• Rerun with the appropriate amount and conditions

Sharp peaks in the trace signal

Cause

• Small gas bubbles inside the capillary

Solution

• Do not shake the buffer vigorously; degas if necessary, then rerun

Pull up peak

Cause

• Appears in short PCR products; high DNA concentration during the run

Solution

• Dilute and rerun