These guidelines are relevant for calculating endpoint PCR cycle numbers for 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 library prep protocolsand LUTHOR HD. Please also see consult the respective User Guides for kit-specific details.
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The final number of Endpoint PCR cycles to use is calculated by the following formula:
No. Endpoint PCR cycles = 50% MF cycle No. – 3
50% MF cycle No. = Cycle cycle number corresponding to 50% of the maximum fluorescence value that is reached at the amplification plateau.
The reason 3 cycles are subtracted from the 50 % MF cycle number is because the remaining volume of cDNA for endpoint PCR is ~17 μl, which is 10x more than the 1.7 μl input used for Endpoint PCR (17 μl of cDNA) is 10X the input used for the qPCR assay . In qPCR, 3 cycles are equivalent to a 10x difference in yield(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 that your libraries will be overcycledover-cycling.
Tips for Calculating calculating Endpoint PCR Cycle Numbers Correctly.
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cycle numbers correctly:
Setthe y-axis of the amplification plot to linear scale. to enable the cycle number to be correctly calculated.
Ensure ROX background fluorescence is turned off.
Draw a horizontal line from the amplification plateau to the y-axis and record the value as the Maximum fluorescence value.
Calculate half of this value to get the 50 % MF value.
Draw a horizontal line from this value on the y-axis to intersect the amplification curve, then draw a vertical line from the intersection point to the x-axis.
The cycle number that this vertical line intersects is the 50 % MF cycle No. Round up to the next whole number.
Subtract 3 cycles from this number to get the final Endpoint PCR cycle number.
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