Mixed Sequence

Occasionally you'll see what is clearly mixed sequence data. This can occur with both plasmids and PCR products and is the result of usually two or even more templates being present in the sequencing reaction - resulting in two sequence ladders overlapping.

Figure: Mixed Sequence

This is characterised by the presence of (ideally) one major sequence, with a second set of peaks exactly under the first set (and clearly peaks, not just background or pull ups) - this tends to result in lots of N's being called throughout the data. To solve this just make sure you have only one clone selected during picking and inoculating clones or check the PCR conditions to be sure to get only one single product and no mispriming products whatsoever, i.e. there should be only one band present on a gel.