How many primers do I have to screen?

This depends on the sensitivity specifications of the assay. For example, if you only need to detect 1000 molecules per reaction or more, then most primer pairs will be adequate. For very high sensitivity assays, the best way to find good RPA primers is by performing a systematic screen. Typically the number of tested primer pairs will lie between 10 and 20 initially. The more oligonucleotides are tested, the greater the chance of finding good primer pairs capable of detecting single molecules with rapid kinetics.

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