The optimization of the primer concentration is essential. Each set of primers works best at a different concentration. Primer concentration is usually determined to be optimal when the specific amplification relative to primer-dimers is maximal, in a positive versus negative control experiment. However, the recent development of melting curve analysis for PCR products has made the dimers issue obsolete (see below and also the next chapter). One major limitation in primer optimization is, however, the availability of a good positive template for optimization. Finding a tissue that definitely expresses the gene of interest, cloning a rearrangement or a translocation, etc, remain essential to the optimization process. Again primer optimization differs from one type of assay to another.
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