How can I check the optimal PCR cycle number if I cannot perform the qPCR assay?

The qPCR assay provides the fastest way to determine the optimal cycle number for endpoint cDNA amplification by PCR. It is important not to overcycle libraries so as to avoid reduced complexity of the final libraries. If qPCR cannot be performed (i.e., no access to a qPCR instrument, or SYBR Green I dye cannot be obtained), then endpoint testing can be performed instead.

This will require additional PCR reactions and purifications, therefore the TeloPrime PCR Add-on V2 Kit would be required (Cat. No. 018.16), along with additional purification reagents (The Magnetic Bead Purification Module Cat. No. 022.96, or 1x Ampure PB® Beads from PacBio can be used; alternatively pooling the PCR products and purifying using columns included in the TeloPrime V2 kits can be performed, however this will use up the provided columns provided).

Using 1-2 µl aliquots of the purified cDNA obtained at step 29, perform parallel endpoint PCRs for a range of cycle numbers: e.g., 14 – 20 cycles (increasing increments of 2 cycles can be used). For best evaluation in detail, purify the PCR products and run an aliquot on Bioanalyzer (or similar) to check the size distribution profile and yield.