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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.
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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.
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7 μl PCR Mix (PCR)
1 μl Enzyme Mix (E)
5 μl P7 primer (7000), and
1.2 μl of 2.5x SYBR Green I dye (final concentration must be 0.1x)
Make up the total volume to 30 μl using Elution Buffer (EB) or molecular biology-grade water.
IMPORTANT! The final concentration of SYBR Green I dye must not exceed 0.1x! Higher concentrations can inhibit amplification.
The qPCR is performed in a real-time PCR machine for at least 35 cycles.
Calculating the Endpoint PCR Cycle Number
The amplification curves from the qPCR (set to linear scale) are then used to determine the cycle number corresponding to 50 % of the maximum fluorescence (50 % MF cycle No.) reached at the amplification plateau (See figure below).
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