Realtime PCR system of PCRmax

Date: 25/02/2016    View: 1498 Use in Biological Technology

Introduction:

-   The PCRmax Eco 48 real time PCR system is a high specification, economically priced real time thermal cycler that accommodates a unique 48-well polypropylene PCR plate utilising the same geometry as standard 384-well plates, but only 1/8 of the size. This enables users to dramatically reduce the qPCR reagent volumes compared to traditional 96-well instruments, saving users precious sample, whilst still producing a strong fluorescence signal. Minimizing the plate size also significantly improves thermal uniformity. A minimum volume of 5μl is validated, resulting in a more efficient use of expensive and ‘hard to acquire template DNA samples.

Key features:

-   MIQE compliant.

-   HRM functionality is provided as standard and can discriminate class IV SNP 99.9% of the time.

-   The Eco 48 can utilise four colours for easy multiplexing.

-   Industry leading ±0.1ºC temperature uniformity (recorded at 95ºC no settle time).

-   Suitable for both single labs and core facilities.

-   High uniformity provides high quality data.

-   Fast cycling enables several experiments per day, all at an economical price.

-   Fastest block-based real-time PCR system with the ability to run 40 cycles in 20 minutes (or less when optimised).

-   The PCRmax Eco 48 is an open platform that can utilise any chemistry, dye or PCR reagent.

-   Calibrated for SYBRÒ, FAMTM, HEXTM, VICTM,, ROXTM  and CyÒ 5 fluorescent dyes.

-   Easy to use software, streamlined for novices and experts.

-   No need to run triplicates, to compensate for poor thermal uniformity of block.

-   The Eco 48 format provides a small 34.5cm x 31cm (13.6” × 12.2”) footprint that fits anywhere.

The Eco 48 Workflow: The Eco 48 workflow is based on three simple steps:

-   Load samples into the 48-well plate using the backlit EcoDock and place the plate into the Eco 48.

-   Run experiment.

-   The Eco 48 software provides instrument control, data collection, and data analysis. The most common qPCR parameters are automatically in place. These can be easily changed to meet specific experimental requirements.

Eco 48 Thermal Technology

-   qPCR specificity and efficiency relies on precise temperature control during the denaturation and annealing steps. For the highest accuracy, temperature must remain uniform across the entire heat block, ensuring that all samples are processed equally. The unique thermal block design of Eco 48 achieves this with a unique heating and cooling system that provides accurate ±0.1ºC temperature control and quickly cycles from one temperature to the next

Eco 48 Thermal System

-   To achieve true temperature control the Eco 48 thermal system consists of a precisely electroformed 48-well hollow silver block containing conductive fluid. The block is heated and cooled by a single Peltier device, with an agitator assembly consisting of two paddles driven by electromagnetic motors. During PCR cycling, the paddles move rapidly, circulating the fluid across the 48 wells, allowing the block to achieve high ramp rates, thus reducing the overall experiment time.

-   This unique design delivers industry leading thermal stability of ±0.1°C virtually eliminating thermal non-uniformity and preventing edge effect. The result is higher qPCR performance, tighter Cq, greater PCR efficiency, higher R2 and the ability to perform demanding HRM applications.

-   Fast, uniform temperature control is important because accurate dwell temperatures ensure primers bind most efficiently and polymerase enzymes work optimally, generating the maximum yield of target DNA. The hermetically sealed hollow block contains a conductive fluid and two opposing agitators driven by electromagnetic motors. During PCR cycling, these agitators rapidly circulate fluid throughout the hollow block, transferring heat from the peltier quickly and evenly.

Optical Technology

-   The Eco 48 Real Time PCR system contains an advanced high-performance optical system that delivers precise and sensitive fluorescence detection, facilitating all 4 colour multiplex applications.

-   The system is provided factory-calibrated for use with SYBRÒ, FAMTM, HEXTM, VICTM,, ROXTM  and CyÒ5 and can also be used with any qPCR fluorophores overlapping with these calibrated dyes. For excitation, two panels of 48 fixed LEDs provide excitation energy of distinct spectra, enabling broad range excitation. Each of the 48 LEDs illuminates a specific well location, eliminating the optical distortion created by most stationary optical systems. HRM analysis protocols are supported by continuous data acquisition during the melt for increased data collection and reduced run times, HRM of a full plate is less than 10 minutes. The user can change the plate setup and perform data analysis after the run is complete.

Adaptive LED Control (ALC)

ALC normalises variation in fluorescence across all wells at each cycle of a run and provides specific tuning for each LED following each PCR cycle:

-   Speeds up data collection because only 1 image is required to cover the full sample dynamic range.

-   Expands the linear range of detection by reducing LED exposure to high emission wells and preventing premature detector saturation.

-   LED adjustment following each amplification allows Eco 48 to reduce exposure time as signal intensity increases, maximising the linear range of emission detection.

-   Minimise optical artefacts by keeping each well brightness similar to the CCD. This reduces the influence of a highly fluorescing sample on adjacent wells and avoids ‘blooming’, no artificats from adjacent wells.

-   Maximise sensitivity by permitting an appropriate exposure level for each well, by dye, rather than compromise with a universal setting

Applications:

The PCRmax Eco 48 is a high performance instrument for demanding applications.

-   Absolute & Relative Quantitation

+      Absolute quantification standard curves with efficiency calculation.

+      Relative quantification using the ΔΔCq method with support for multiple reference gene normalization.

+      Relative gene expression with efficiency control.

+      Next Generation Sequencing (NGS) library quantification.

+      Copy Number Variation: insertions, deletions, inversions.

+      RNA Characterisation: siRNA and miRNA.

+      Viral load.

-   Genotyping and High resolution melting

+      Allelic Discrimination.

+      DNA Methylation.

+      SNP Verification

+      Microbiology: pathogen detection and identification.

-   High resolution melting (HRM)

+      Genotyping, methylation studies and mutation screening.

-   Library quantification

+      qPCR is the most precise method for quantifying libraries prior to cluster generation.

+      Works at concentrations below the detection threshold of conventional spectrophotometric methods.

-   Data

+      Validation of array and sequencing quality data.

-   Designed for:

+      Academic, Government and Corporate laboratories.

+      Human research, AgriGenomics, Translational, Forensics and many more markets.

+      Probe, DNA Binding Dye and HRM chemistries.

+      Gene expression, Viral detection, Sequencing Library

+      Preparation, Genotyping and Mutation Screening applications

+      Eco 48 is for research use only.

Rapid cycling

-   The Eco 48 has rapid heating and cooling rates. Average ramp rates of 5.5°C/sec reduce overall PCR run times. The typical run time for a 40 cycle PCR protocol is under 40 minutes.

-   In testing, the quickest optimised programme enabled a 40 cycle qPCR experiment to complete in just 15 minutes.

High throughput

-   Eco 48 thermal control is superior to traditional 96-well peltier systems where thermal efficiency decreases as distance from the peltier increases. With Eco 48, faster and more precise cycling increases sample throughput because the need for multiple replicates is reduced by increased uniformity across the block. Eco 48 well-to-well, plate-to-plate and instrument-to-instrument results are as consistent as running a single experiment. To take advantage of this, Eco 48 has a function in the software where different experiments are reliably combined. High uniformity reduces the need for experimental replicates which saves on samples, eliminates the need for more expensive instruments, reduces reagent running costs and ensures the entire plate of data is valuable.

-   Eco 48 wells HIGH uniformity - Run duplicates. 24 samples Run time 40 minutes.

-   36 samples per hour: Fewer replicates and faster cycling allows Eco 48 to process more samples than a standard 96 well system.

ECO 48 Software

The Eco 48 system comes with two distinct software elements. The first, Eco Control, drives the units set up and experimental runs while Eco Study is the analysis package. The Eco 48 software are both open licence, you can install them on as many PCs as you like, and are MIQE compliant.

-   EcoControl software.

+      User-installable, intuitive, easy to use software that integrates user control, real-time data collection, and advanced data analysis.

+      Supplied fully featured including HRM capability.

+      New software version 5 enables a widened chemistry library and contains new thermal profiles.

+      Facilitates rapid thermal cycling: 40 cycles in 40 minutes.

+      Supplied on a USB stick.

-   EcoStudy software.

+      Data analysis and Multi-Experiment Analysis.

+      Allows higher throughput.

+      Limitless sample volume.

+      Supplied on a USB stick.

-   Easy-to-Use Interface

+      Eco 48 software uses a unique icon-driven user interface to simplify experimental design and setup.

+      Pre-set thermal profile defaults are provided for the most commonly used experimental protocols.

+      Temperature and time for each protocol step can easily be changed by click-and-drag action with the mouse.

+      Experiment templates can be customized and saved for future use.

-   Data Analysis

+      With the Eco 48 system and software, data collection is monitored in real time, allowing researchers to access run viability immediately.

+      The user-friendly data analysis interface also allows researchers to easily view the amplification plot, melt curve analysis, and the analysed results, including Cq values, PCR efficiency, R2 and Y-intercept.

+      Data can be exported into Excel or CSV and custom reports generated directly into PowerPoint or PDF formats.

+      High-resolution images can be directly exported in multiple image formats, ready to use in any presentation.

+      Conforms to Minimum Information for Publication of

+      Quantitative Real-Time PCR Experiments (MIQE) guidelines, making data analysis and submission for publication review more efficient.

-   High-Performance Results

Due to unmatched temperature control and an optical system designed for individual well monitoring, the Eco 48 system produces highly accurate, reliable results with PCR efficiencies between 90% and 110% and R2> 0.99 when using standard optimized assays

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