PIXO - Real-Time PCR System with HRM Illumina Helixis makes high performance research tools universally accessible to individual researchers or large laboratories. This Real-Time PCR system is built on novel technologies from the Calltech labs of Nobel Prize Laureate David Baltimore and Axel Scherer and offers high performance, flexibility and ease of use for both experienced and novice Real-Time PCR users. Meeting the demanding requirements for high performance and flexibility by scientists with many years experience using advanced qPCR techniques, PIXO offers 4-color multiplex detection and supports all qPCR applications and chemistries. Young investigators, just starting their first project, will find this instrument easy to use right out of the box. Now every scientist can have access to this powerful research tool and generate high quality, reproducible data every time. helixis presentation polymerase chain reactions réaction en chaîne polymérase PCR temps réel real-time realtime real time thermocyclers thermocycleurs HRM high resolution melt melting melts fusion à haute résolution courbes curves

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Product Information

• Features
• Thermal System
• Optical System
• Software
• Specifications

Features

• Low cost, priced for the individual user
• Temperature control and uniformity far exceeding more costly instruments
• High performance optics
• Uniquely improved user interface for easy set up and data analysis
• Compact size for a "personal scale" Real-Time PCR
• All chemistries and applications supported, including HRM and multiplexing
• Plug and play set up, no calibration required

• The Real-Time PCR instrument
• Laptop computer with free software
• USB key with unlimited license software enables data analysis at other computers
• Dock easily loads 48 well reaction plates
• Starter set: 10 plates, 40 seals, 1 evaluation plate and master mix

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Thermal System

The PIXO™ Real-Time PCR instrument provides best in class thermal uniformity. The thermal block design is based on Helixis IP developed in the labs of Nobel Laureate David Baltimore and Professor Axel Scherer at California Institute of Technology. This unique design provides a new level of PCR data quality, reproducibility and enables high performance applications such as HRM due to precision thermal control and uniformity. The efficiency and specificity of PCR reactions are highly dependent on precise temperature control for the denaturation and annealing steps during thermocycling. In addition well to well thermal uniformity across the thermal block ensures that sample data within an experiment can be accurately compared for analysis. Most Real-Time PCR systems contain a solid metal thermal block which requires significant power and four linked Peltier heaters to heat and cool between the different PCR cycling temperatures. In addition about 15 seconds are required for temperature equilibration of these blocks at each step of the PCR thermal protocol, adding to the time required to complete each experiment. Real-Time PCR instruments using this thermal system design claim +/- 0.5°C well to well thermal uniformity across the block. This design delivers a new level of thermal performance of +/- 0.1°C well to well uniformity across the 48 well plate (FIGURE 1). This enables higher PCR performance (Cq, PCR Efficiency, R2) and demanding applications such as High Resolution Melt (HRM).

Each thermal system includes a precisely electroformed hollow silver block that is heated and cooled by a single Peltier device. The hollow block is hermetically sealed and contains a conductive fluid and two opposing agitators driven by electromagnetic motors. During PCR cycling these agitators rapidly circulate the fluid throughout the hollow block, transferring heat from the Peltier evenly across the block. This unique design virtually eliminates thermal non-uniformity and block edge effects, significantly enhancing thermal efficiency and uniformity performance compared to other block design systems.

PIXO systems consistently deliver the best thermal uniformity performance, providing ± 0.1°C well to well uniformity. This uniformity is also demonstrated across multiple instruments

The high performance thermal uniformity provides reproducible quality data as shown by the % coefficient of variation (CV) of Cq values across replicate samples at <.25% (Table 2). 48 replicate reactions were run using standard PCR thermal protocol and analysis. This unique instrument block design can also reduce overall PCR run times with increased thermal ramp rates up to 5.5°C/sec that require minimal equilibration time at each temperature. Typical run time for a 40 cycle PCR protocol is less than 40 minutes.

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Optical System

This Real-Time PCR system contains an advanced high performance optical system that delivers precise and sensitive fluorescence detection. On the excitation side, fixed 48 LED arrays provide fluorescent dye excitation over a broad spectrum. Each of the 48 wells is individually illuminated by dynamically controlled LEDs to minimize cross-talk between wells. On the detection side, the optical system detects up to four targets in a single reaction. Four emission filters in a linear filter slide and a high performance CCD camera detect the fluorescence from each sample target at each cycle. The excitation LED arrays and emission filter combinations are optimized for the most commonly used real-time PCR dyes.

Raw fluorescence data from four emission filters is automatically analyzed using spectral de-convolution software to effectively minimize cross-talk between dyes. Additional dyes in the wavelength range of the optical system emission filters are supported with no additional calibration required.

At the end of each cycle fluorescence data is collected in all four channels in all 48 wells regardless of plate setup. This approach prevents any data loss and allows changes to the plate setup and data analysis even after the run is completed. Standard melt curve and HRM analysis protocols support continuous data acquisition in a single dye channel during the melt for increased data collection and reduced run times. The PIXO™ optical system is factory calibrated for the following dyes: FAM™, HEX™, ROX™, Cy5® and DNA binding dyes.

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Software

The software coming with the instrument is a completely integrated software solution providing instrument control, real-time data collection and advanced analysis. The system uses a unique icon driven user interface to simplify experimental design and setup.

The software supports all real time PCR applications:

• Absolute quantification by standard curve
• Relative quantification using the ΔΔCq method including support for multiple reference gene normalization
• Allelic Discrimination by end-point fluorescence
• Gene scanning and genotyping by High Resolution Melt curve analysis

To make data analysis and submission for publication review more efficient, the software offers a MIQE compliant Real-Time PCR analysis software.

Pre-set defaults for plate set-up and thermal profile are provided for the most commonly used experimental protocols to enable fast and efficient run set-up (Figure 1). Experiment templates can be customized and saved for future use. All qPCR chemistries and starting materials are supported. All standard Real-Time PCR applications can be selected, and the software provides High Resolution Melt (HRM) analysis as a standard option. When running a Quantification experiment, you can select the Relative Quantification or Standard Curve analysis options.

Figure 1. Software Home Screen

Plate set-up is easily completed by selecting icons corresponding to each sample type (Standard, Control, Unknown, Amplicon) (Figure 2). After set-up options are entered, the thermal profile tab is clicked at the top of the screen, or Start Run is selected to confirm the default thermal profile for the run.

Figure 2. Simplified Plate Layout Tab

The thermal profile screen is shown with smart default thermal protocol parameters based on the type of experiment selected on the home page. Temperature and time for each protocol step can easily be changed by using click and drag action with the mouse. (Figure 3).

Figure 3. Thermal Profile Tab

After the thermal profile for the selected experiment type is either confirmed or changed, the Start Run icon is clicked to start the thermal cycling. As the run progresses, the progress of the fluorescence generation can be monitored in real time by selecting the Monitor Run view. At the completion of the PCR thermal cycling the data is available for analysis using the Analyze Data option. This data analysis interface allows viewing the raw data, amplification plot, melt curve analysis and the analyzed results including PCR efficiency, R2, Y intercept and Cq values. Data can be exported into Excel and custom reports can be generated directly into PDF. High resolution images suitable for presentations can be exported in multiple image formats.

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Specifications

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