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Primer Dimers
Identification
- The trace signal is mixed in the early regions (normally before base 400) but not in the late regions
- The signal drops from very high early in the trace (usually in the first 100 bases) to much lower levels
- The total quality scores counts are generally low to moderate depending on the region affected
Causes
- Contamination of the template, primer stock or other sequencing reagents with primer dimers
- Too low an annealing temperature
- Two primer binding sites present
Overcoming Problem
- Change all the sequencing reagents and water for a fresh batch. Clean the pipettes as they can also be the cause of primer dimer contamination
- Raise the annealing temperature and/or use a shorter annealing time
- Check the sequence of the vector to ensure that two primer binding sites are not present
