No recognizable sequence

 

No recognizable sequence - raw - website

No signal, except for the unincorporated dye peak at the beginning of the raw data trace. Split base line.

 

 

No recognizable sequence - electropherogram - website

Indistinguishable peaks, with no base calling.

 

 

Possible causes

Recommendations

Insufficient template

Quantify the DNA template. Ensure that the concentration is in the range specified on the “Sample Preparation and Submission” TAGC web page.

Poor quality template:

  • degraded by repeated freeze-thaw cycles or UV exposure.
  • contaminated with sequencing inhibitors (EDTA, salts, detergents, ethanol, phenol, chlorophorm), RNA or chromosomal DNA.

 

Verify the OD260/A280 ratio:

  • 1.7 to 1.9: good quality DNA
  • Below 1.7: DNA contaminated by proteins
  • Above 1.9: DNA contaminated by chloroform

Run an agarose gel to detect any contaminating DNA or RNA.

Clean up the template.

Dilute the template in 10 mM Tris, pH 7.5 or water, not TE.

 

Insufficient primer

Quantify the primer. Concentration must be 3.2 uM (3.2 pmol/uL). For large plasmids (>8 kB) and bacterial genomic DNA, primer concentration must be 10 mM.

Low primer binding affinity or no annealing site

PCR the template before submitting to sequencing. Sequence in both directions to determine which primer gives the best results.

Degraded primer

Prepare a fresh stock. Repeated freeze/thaw cycles reduce primer quality.