How far should RPA primers be apart?

This depends on the application and need. In general we recommend that under the conditions of the standard TwistAmp® kits the amplicon generated by two RPA primers should typically be no longer than about 500bp. There is probably no lower limit to the size of RPA products, but the minimum size of RPA primers requires that amplicon will typically be longer than about 80bp. For the most rapid real-time kinetics the final amplicons should ideally be 100-200 base pairs. Under specialised conditions amplification products of as much as 2 kilobases have been generated. However, the standard TwistAmp® kits do not readily permit the generation of such large amplicons. See assay design manual for details about primer design and approaches to achieve larger amplicon sizes.

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