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These guidelines are relevant for calculating endpoint PCR cycle numbers for QuantSeq V2 with UDIs, QuantSeq-Pool, and CORALL RNA-Seq V2 library prep protocols. Please also see consultthe respective User Guides for kit-specific details.

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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 used for the qPCR assay. In qPCR, 3 cycles are equivalent to a 10x difference in yield.

NOTE: It is usually safe to add one additional cycle to the calculated No. Endpoint PCR cycles without risking that your libraries will be overcycled.

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Tips for Calculating Endpoint PCR Cycle Numbers Correctly.

• We recommend setting 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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