How many PCR cycles should I use to amplify my libraries?

The optimal number of PCR cycles for a given input amount of total RNA can vary depending on sample quality and origin. The optimal cycle number for a specific sample type should be determined using the qPCR assay.

The qPCR assay requires:

• the PCR Add-on and Reamplification Kit V2 for Illumina (Cat. No. 208)

• SYBR Green I nucleic acid stain (10,000X in DMSO, not provided in the kit). We recommend using Sigma-Aldrich, Cat. No. S9430 or ThermoFisher, Cat. No. S7585.

ATTENTION! The use of SYBR Green I-containing qPCR mastermixes from other vendors is not recommended.

Protocol

The recommended protocol for the qPCR assay is provided in the PCR Add-on and Reamplification Kit V2 User Guide.

Additional Resources

For additional information on the qPCR assay, consult the following online FAQs.