Sequencing chromatogram shows double / mixed peaks throughout (Sanger Sequencing)

Question

Accepted Answer

From the start of the read the trace shows overlapping double peaks; bases cannot be called confidently. Common causes: 1) Picked a mixed colony (two different inserts in the same prep) 2) Two plasmid templates contaminated the same tube 3) Sequencing primer is not specific and binds two sites 4) Template purity is poor or template is partially degraded Solutions: 1) Re-streak the colony to isolate a true single colony, re-prep, and re-sequence 2) Re-prep with fresh culture from a single colony 3) Switch to a more specific sequencing primer 4) Verify A260/A280 and gel-check template quality before submitting