These guidelines are relevant for calculating the correct number of cycles to use for endpoint PCR with the following kits: QuantSeq V2 with UDIs, QuantSeq-Pool, and CORALL RNA-Seq V2 and LUTHOR HD. Please consult the respective User Guides for kit-specific details.
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3 cycles are subtracted from the 50 % MF cycle number because the input used for Endpoint PCR (17 μl of cDNA) is 10X the input used for the qPCR assay (1.7 μl of cDNA). This corresponds to a difference in ~ 3 PCR cycles.
NOTE: It is usually safe to add one additional cycle to the calculated No. Endpoint PCR cycles without risking overcyclingover-cycling.
Tips for calculating Endpoint PCR cycle numbers correctly:
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