What do I need for the qPCR Assay?
The qPCR assay facilitates calculation of the optimal number of Endpoint PCR cycles to use for final library amplification. The assay described below (using the PCR Add-on and Reamplification Kit; Cat. No. 208) is applicable for use with the following library preparation methods:
CORALL RNA-Seq V2 with UDI (Cat. No. 171 – 186),
QuantSeq V2 with UDI (Cat. No. 191 – 196, 225),
QuantSeq-Pool (Cat. No. 139),
LUTHOR HD (Cat. No. 204, 221).
NOTE: kit is not recommended for qPCR assays with older versions of QuantSeq 3’ mRNA-Seq and CORALL RNA-Seq.
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 and Reamplification Kit V2 for Illumina (Cat. No. 208)*, and
SYBR Green I nucleic acid stain (10,000X in DMSO, not provided in the kit).
*For QuantSeq-Pool, if you have >7 pools, the PCR Add-on and Reamplification Kit V2 is required for qPCR. If you have <6 pools, the provided kit solutions for Library Amplification (PM, P5, P7, PE) are sufficient for qPCR and final amplification.
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 degrees Celsius.
qPCR Assay Protocol
The recommended protocol for the qPCR assay is provided in the PCR Add-on and Reamplification Kit V2 User Guide.