- When you get double peaks, it means that there are at least two copies of the gene you are trying to sequence. This could mean that there is contamination or that there are multiple alleles of the gene. Either way, you cannot really use a double sequence.
- If you had been using a cocktail (like C_Fish or Mammal cocktail) try using the different primer sets from the cocktail. For example, C_Fish uses FishF2, VF2_t1, FishR2, and FR1d_t1. Because there are two reverse primers and two forward primers, they may be sitting down on your DNA in slightly different places. So try running four PCRs on one sample (FishF2 and FishR2, FishF2 and FR1d_t1,VF2_t1 and FishR2, VF2_t1 and FR1d_t1) and which ever set of primers gives the nicest band, sequence off that.
- If trying different primer sets does not resolve the double peak problem and the sample is of particular importance, you can always try Cloning.
Return to Problems with sequences.
Return to Barcoding in the Hanner Lab Wiki.
Updated June 25 2009