Protocol for using Agilent's QPCR NGS Library Quantification Kit (illumina GA) to quantify DNA sequencing libraries.
This manual provides step‑by‑step instructions for quantifying the concentration of DNA libraries prepared for the Illumina Genome Analyzer using the Agilent QPCR NGS Library Quantification Kit (illumina GA). It covers required materials, storage conditions, equipment, the QPCR workflow, data analysis, and troubleshooting tips.
The kit includes a 2× SYBR® Green QPCR Master Mix, dilution buffers, primers, and a 288‑bp DNA standard. Users are guided through plate setup, reagent preparation, running the QPCR protocol on a real‑time PCR instrument, and calculating library concentration from the standard curve.
| Component storage temperature (initial) | -20°C |
|---|---|
| 200× Dilution Buffer post‑thaw storage | 4°C |
| 2× Brilliant III Ultra‑Fast SYBR® Green QPCR Master Mix storage | 4°C for up to one month |
| Light sensitivity | QPCR Master Mix and Reference Dye are light sensitive |
| DNA standard target length | 288‑bp linear DNA |
Store the 200× Dilution Buffer at -20°C upon receipt; after thawing it may be kept at 4°C.
A real‑time PCR instrument and 96‑well QPCR plates or tubes are required.
The R‑squared value should be at least 0.98.
It can be stored at 4°C for up to one month; for longer storage keep it at -20°C.