We assessed the quality of nine standard primers for automated fluorescent dye terminator DNA sequencing by whether their melting temperatures (Tms) were in the optimal range for DNA sequencing, and the degree to which their sequences matched the sequences of 36 common vectors. The M13F (-21/-20), M13F (-41/-40), M13R, and T7R primers showed optimal physicochemical characteristics and were not redesigned. The M13R (-41/-40), T3, and SP6 primers showed mismatches and/or Tm values outside of the optimal range and were redesigned by these two criteria.
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