How is the cycle number of the endpoint PCR determined?

The number of Endpoint PCR cycles to use is calculated by the following formula:

No. Endpoint PCR cycles = 80% MF cycle No.*

80% MF cycle No. = cycle number corresponding to 80% of the maximum fluorescence value that is reached at the amplification plateau.

IMPORTANT: This formula is to be applied only if the same volume of cDNA for the endpoint PCR and the qPCR assay are used. If more cDNA is utilized for the endpoint PCR than for the qPCR, the cycle number may need to be adjusted. For example, if 2 µl of cDNA is used for the qPCR assay, and 9 µl will be used for endpoint PCR, subtract 2 cycles to account for using ~4x more cDNA as template for the final cDNA amplification. See Appendix B of the TeloPrime V2 User Guide for more details.

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 80 % 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 80 % MF cycle No. Round up to the next whole number.

NOTE: The qPCR assay requires SYBR Green I dye (final concentration of 0.1x in the reaction mix) and a real-time PCR machine. The TeloPrime Full-Length cDNA Amplification V2 Kits (Cat. No. 013) contain sufficient reagents to perform a qPCR and endpoint PCR for each sample. Lexogen also offers a TeloPrime PCR Add-on V2 Kit (Cat. No. 018) for 16 reactions if additional PCRs are required.