For ultrafast, real-time PCR and two-step RT-PCR using sequence-specific probes
Sensitive detection of even low copy numbers
Accurate detection of a wide range of template amounts
Optimized for ultrafast, reliable results on Rotor-Gene cyclers
Specially formulated, ready-to-use master mix for fast cycling
The Rotor-Gene Probe PCR Kit is designed for use with the Rotor-Gene Q and other Rotor-Gene cyclers, providing ultrafast, highly sensitive quantification of gDNA and cDNA targets with real-time PCR and two-step RT-PCR using sequence-specific probes. Outstanding performance is achieved through the combination of a specially optimized master mix and the unique Rotor-Gene cycler. For convenience, the master mix can be stored at 2–8°C.
For 400 x 25 µl reactions: 3 x 1.7 ml 2x Rotor-Gene Probe PCR Master Mix, 2 x 2 ml RNase-Free Water
The Rotor-Gene Probe PCR Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
Real-time PCR was carried out using two-fold dilutions of human genomic DNA (30 ng to 3.75 ng) and a self-designed TaqMan assay for IL1R2 (interleukin 1 receptor, type II). Reactions were run using either [A] the Rotor-Gene Probe PCR Kit and Rotor-Gene Q or [B] a kit and cycler from Supplier AII (5 replicates per template dilution). The Rotor-Gene system provided lower CT values, greater reproducibility within replicates, and greater resolution of different template dilutions.
High precision in real-time PCR.
Real-time PCR analysis of human genomic DNA was carried out using the Rotor-Gene Probe PCR Kit and Rotor-Gene Q. [A] Analysis of two-fold dilutions of DNA (from 30 ng [5000 copies] to 0.06 ng [20 copies]) using a self-designed TaqMan assay for IL1R2 (interleukin 1 receptor, type II); 5 replicates per template dilution. The average difference in CT value between adjacent template dilutions was 1.07 cycles. [B] Analysis of 5 ng DNA using a self-designed TaqMan assay for IL1R2. From 100 replicates, the standard deviation was 0.21. [C] Analysis of 20 ng DNA using a self-designed TaqMan assay for ACTB (actin, beta). From 100 replicates, the standard deviation was 0.29.
Reliable detection down to 1 cell.
Real-time PCR was carried out using fourfold dilutions of human genomic DNA (equivalent to 16, 666 cells down to 1 cell) and a self-designed TaqMan assay for ACTB (actin, beta). Reactions were run in triplicate using the Rotor-Gene Probe PCR Kit and Rotor-Gene Q. The Rotor-Gene system provided reliable detection over the entire range of template dilutions, with high reproducibility within each set of triplicates.
Wide dynamic range.
Real-time PCR was carried out using ten-fold dilutions of plasmid DNA (109 to 10 copies) and a self-designed TaqMan assay for PPIA (cyclophilin A). Reactions were run in triplicate using the Rotor-Gene Probe PCR Kit and Rotor-Gene Q. The Rotor-Gene system provided reliable detection over the entire range of template dilutions as well as highly reproducible CT values within each set of triplicates.
Real-time PCR was carried out using ten-fold dilutions of plasmid DNA (109 to 10 copies) and a self-designed TaqMan® assay for PPIA (cyclophilin A).
Principle
The Rotor-Gene Probe PCR Kit enables reliable real-time two-step RT-PCR quantification on the Rotor-Gene Q without the need for optimization of reaction and cycling conditions. The kit is designed for use with hydrolysis probes (e.g., TaqMan® probes). Highly specific amplification is assured through a balanced combination of K+ and NH4+ ions, which promote specific primer annealing, enabling high PCR specificity and sensitivity (see figure "Specific primer annealing"). Fast cycling without compromising performance is achieved using Q-Bond, a novel PCR additive that enables cycler run times of as low as 45 minutes (see figure "Fast primer annealing").
Components of 2x Rotor-Gene Probe PCR Kit*
Component
Features
Benefits
HotStarTaq Plus DNA Polymerase
5 min activation at 95ºC
Set up of qPCR reactions at room temperature
Rotor-Gene Probe PCR Buffer
Balanced combination of NH4+ and K+ ions
Specific primer annealing ensures reliable qPCR results
Unique Q-Bond additive
Faster PCR run times enable faster results and more reactions per day
* Also contains dNTP mix (dATP, dCTP, dGTP, dTTP).
Procedure
The ready-to-use Rotor-Gene Probe PCR Master Mix eliminates the need for optimization of reaction and cycling conditions. Just add template DNA, primers, and probe to the master mix and program the cycler. Instructions are provided in the detailed handbook supplied with the kit.
Hydrolysis probes (e.g., TaqMan® probes) can be used in combination with the Rotor-Gene Probe PCR Kit on the Rotor-Gene Q for fast and sensitive quantification — simply add the primer-probe mix and template to the master mix.
For optimal results in real-time two-step RT-PCR, we recommend synthesizing cDNA using the QuantiTect Reverse Transcription Kit. The kit provides fast cDNA synthesis in just 20 minutes with integrated removal of genomic DNA contamination.
Applications
The Rotor-Gene Probe PCR Kit is for fast, real-time PCR and two-step RT-PCR of gDNA or cDNA targets using sequence-specific probes on the Rotor-Gene Q. It is also compatible with Rotor-Gene 3000 and the Rotor-Gene 6000 cyclers.
For ultrafast, one-step qRT-PCR gene expression analysis of RNA targets using sequence-specific probes on Rotor-Gene cyclers, use the Rotor-Gene Probe RT-PCR Kit.
Features
Specifications
Applications
Real-time quantification of genomic DNA or cDNA targets
Description
For ultrafast quantitative real-time PCR and two-step RT-PCR using sequence-specific probes
