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I am currently trying to prepare mutant allele fraction (MAF) solutions for an allele discrimination qPCR assay. I began with plasmids cloned with the different desired alleles. I amplified the the plasmids via PCR using primers flanking the mutation site. The amplicons were purified and quantified using qPCR with primers that flank the mutation site. The quantification was done with an standard curve of Ct values plotted against the log dilution factor of the solutions, with an efficiency of 84.18%.

Based on the Ct values obtained, the mutant allele and wild type allele amplicon solutions were then diluted to the log dilution factor that would give a specific Ct value (using the standard curve).

When I amplified the 100% mutant and 0% mutant solutions, I obtained roughly the same Ct values (values differed by no more than 0.5). When I mixed the 100% and 0% MAF solutions to prepare 5%, 1%, 0.1% MAF solutions and amplified them with the same primers that flank the mutation site, the Ct values from each MAF solution differed by up to 2, when it should differ only very slightly since the total DNA content for each of the different MAF solutions are the same.

Does anyone have any idea why this is happening?

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