The qPCR assay facilitates calculation of the optimal number of Endpoint PCR cycles to use for final library amplification. The assay described below is applicable for use with the following library preparation methods:
ATTENTION! The guidelines outlined below do not apply for QuantSeq-Pool or LUTHOR 3' mRNA-Seq library preparation protocols.
Instead, please consult the QuantSeq-Pool User Guide, Appendix B, and this online FAQ, and the LUTHOR 3' mRNA-Seq User Guide.
Additional Reagents Required
The qPCR assay to determine optimal PCR cycle numbers for library amplification is performed using an aliquot of purified double-stranded cDNA, prior to the final library amplification.
To perform the qPCR assay you will need these reagents in addition to your library prep kit:
The PCR Add-on Kit for Illumina (Cat. No. 020.96), and
SYBR Green I nucleic acid stain (10,000X in DMSO, not provided in the kit).
ATTENTION! The use of SYBR Green I-containing qPCR mastermixes from other vendors is not recommended.
Prepare SYBR Green I 2.5x Working Stock
SYBR Green I nucleic acid stain is provided at a 10,000x concentrated stock. We recommend using Sigma-Aldrich, Cat. No. S9430 or ThermoFisher, Cat. No. S7585.
We recommend preparing a 2.5x working stock by making a 1:4,000 dilution of SYBR Green I dye (10,000x stock) in 100% DMSO.
The working stock can be aliquoted into smaller volumes and stored at -20 deg. Celcius.
qPCR Assay Protocol
The recommended protocol for the qPCR assay is provided in the PCR Add-on Kit Instruction Manual, as well as in the Appendices of each QuantSeq 3' mRNA-Seq Library Prep Kit User Guide.