# How do I calculate endpoint cycle numbers?

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, CORALL RNA-Seq V2 and LUTHOR HD. Please 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 number corresponding to 50% of the maximum fluorescence value that is reached at the amplification plateau.*

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 over-cycling.

**Tips for calculating Endpoint PCR cycle numbers correctly:**

Set the y-axis of the amplification plot to linear scale.

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.

*Figure: Example of Endpoint PCR cycle number calculation from qPCR amplification curve (linear scale).*