How is the cycle number of the endpoint PCR determined?

The qPCR assay requires the PCR Add-on and Reamplification Kit (Cat. no. 018), SYBR Green I dye (final concentration of 0.1x in the reaction mix), a real-time PCR machine.

Determine the cycle number at which the fluorescence reaches at 80% of the maximum. If using the same volume of cDNA for the endpoint PCR, this is the cycle number to be used for the endpoint PCR.

If more cDNA is used 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.