I've designed a single-step nested PCR in which two sets of nested primers amplify an outer and an inner amplicon (see attachment). By using a lower inner primer concentration I obtain a large majority of outer amplicons.I'm an now interested in the dynamics of this PCR. I would like to see when the outer primers actually start taking over the amplification from the inner primers and see what influence primer concentration, Tm etc. have on this reaction.
I thought of using probes which are specific for the outer amplicon and one which is non-specific in order to calculate a ratio of each amplicon after each cycle. But designing a specific probe for the outer amplicon proves difficult. Does anybody have an idea how to solve this?
Edited by ForEpi, 14 June 2013 - 02:41 AM.