Using the Rotor-Gene SYBR Green PCR Demo Kit, SYBR Green-based real-time PCR is carried out to quantify different copy numbers of a genomic DNA target. Each reaction consists of:
- Human genomic DNA template of a defined copy number
- Rotor-Gene SYBR Green PCR Master Mix
- QuantiTect Primer Assay specific for the human G protein-coupled estrogen receptor 1 (GPER) gene
The Rotor-Gene SYBR Green PCR Demo Kit handbook contains 3 protocols. Rotor-Gene users follow either the protocol for manual reaction setup or the protocol for automated reaction setup using the QIAgility. After reaction setup, users then proceed to the protocol for real-time PCR on the Rotor-Gene Q.
Manual pipetting steps can be avoided by using the QIAgility, a compact benchtop instrument that provides rapid, high-precision PCR setup. Mistakes in reaction setup due to human error are reduced and may be eliminated. The QIAgility perfectly complements the combination of the Rotor-Gene Q and Rotor-Gene Kits, enabling easy dispensing of liquids into tubes, strip tubes, and Rotor-Discs.
A standard curve is generated from the CT values obtained from a set of standards (2000, 1000, 500, 250, and 125 copies; each standard is analyzed in quadruplicate). The standard curve is then used to determine the copy number for 2 unknown samples (500 and 250 copies; 24 replicates of each unknown sample are analyzed [when performing manual reaction setup, a minimum of 4 replicates for each unknown sample can be set up instead]). In addition, 4 no template control (NTC) reactions are carried out. Thus, a total of 72 reactions are run at the same time on the Rotor-Gene Q.