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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:
ATTENTION! The guidelines outlined below do not apply for QuantSeq-Pool or LUTHOR 3' mRNA-Seq library preparation protocols.
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CORALL RNA-Seq V2 with UDI (Cat. No. 171 – 186),
QuantSeq V2 with UDI (Cat. No. 191 – 196),
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
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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. 020.96208)*, 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.
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The working stock can be aliquoted into smaller volumes and stored at -20 deg. Celciusdegrees Celsius.
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 and Reamplification Kit V2 User Guide.