- When you look at your sequence and notice that there are no distinct peaks, you know that it has failed. This could mean that either there was a problem with the multichannel pipette and the PCR product was not transferred properly to the sequencing reaction or that the PCR itself failed. *If the E-gel image shows a distinct band, but the sequence failed, chances are that if you were to resequence it, the reaction should work.
- If it worked in one direction and not the other, then it was most likely a problem with transferring the final reaction.
- If the entire plate failed in one direction, your primer or another reagent may be no longer good.
- If there was no band on the E-gel and it didn't sequence then you will need to try another primer set. Mammal cocktail, folmer, crustacean, or another primer could be tried. If you can't get any of the primer sets to give a band, try it with 16s to see if there is even any DNA there or Nano-drop it to look at concentration. If your specimen is old the DNA may be degraded.
Return to Problems with sequences.
Return to Barcoding in the Hanner Lab Wiki.
Updated June 25 2009