PCRmax® Eco 48 Real-Time qPCR System FAQs

Frequently Asked Questions for Eco 48 qPCR System

Q. What dyes and specific wavelengths does the Eco 48 qPCR system support?

A. The following table details the excitation and emission wavelengths of the Eco 48 qPCR system in each channel. The emission filters combined with spectral deconvolution algorithms effectively minimize crosstalk between dyes. The ECO 48 qPCR system is factory-calibrated for SYBR Green I, FAM, HEX, VIC, ROX, Quasar670, and Cy5. You can use other dyes if they are within the wavelength range of the emission filters.

Excitation and Emission Wavelength in Eco 48 qPCR System (per channel)

Channel Excitation (nm) Emission (nm) Example fluorophores detected
1 452 to 486 505 to 545 SYBR Green I, FAM
2 542 to 582 604 to 644 ROX
3 452 to 486 562 to 596 HEX, VIC
4 542 to 582 665 to 705 Cy5, Quasar670

Q. What is High-Resolution Melt (HRM) curve analysis?

A. HRM is a recent advancement to the traditional melt curve analysis that significantly increases the amount of detail and information that can be captured. It is sensitive enough to be able to differentiate sequence differences within PCR amplicons down to a single nucleotide. Mutations within amplicons are detected as either a shift in the Tm of the product or a change in the shape of the melting curve.

Q. How does the Eco 48 PCR system achieve higher HRM performance than other block-based systems?

A. Temperature control and uniformity are the most challenging factors affecting HRM. The Tm shift for a single base change can be as small as 0.2°C for challenging Class IV A to T single nucleotide polymorphisms. Most current block-based instruments report temperature uniformity specifications in the range of ± 0.25°C to ± 0.5°C, generally considered too high to reliably differentiate a Class IV SNP. Block-based instruments that claim to perform HRM do so by extensive calibration and software compensation or by employing specialized analysis methods, such as temperature shifting, to overcome the thermal non-uniformity across the block. The Eco 48 qPCR system has a unique thermal block that provides thermal uniformity of ± 0.1°C, well above the industry standard for a block-based system. This extreme thermal uniformity allows the Eco48 qPCR system to perform HRM without software corrections. The thermal uniformity of the Eco 48 qPCR system supports genotyping of even the most challenging Class IV SNPs.

Q. How do you analyze data generated with the Eco 48 qPCR system ?

A. The Eco 48 qPCR system comes with its own analysis software called Eco Study. This will allow for detailed analysis, interpretation of the data, and exporting raw and normalized data to various formats including Power Point, Excel, CSV and TSV. Eco software has unlimited licenses.

Q. How long will the Eco 48 qPCR system take to complete my run? How fast is an Eco 48 qPCR system?

A. We specify that a standard protocol of 40 cycles will be completed in 40 minutes. Optimized protocols can perform 40 cycles in under 30 minutes using standard plastics and chemistries due to the Eco 48 thermal control and ramp rates.

Q. How do I increase the throughput of the Eco 48 qPCR system beyond 48 wells?

A. Eco Study software enables data from multiple plates to be combined and analyzed as a single study, effectively increasing sample throughput. Using Eco Study, a standard curve from one plate, or even a previous experiment, can be applied across all plates in a study, eliminating the need for each plate to carry its own standards and making additional wells available for samples. The Eco 48 qPCR system provides exceptional repeatability from plate to plate, making this possible.

Q. How does the Eco 48 qPCR system provide a significantly higher level of thermal uniformity compared to other block-based systems?

A. The Eco 48 qPCR system hollow has a thermal block constructed of highly conductive silver and filled with a thermal conductive fluid. The fluid is rapidly circulated through the block by paired agitators that are powered by high-efficiency electromagnetic motors. The constant circulation of fluid evenly distributes the heat throughout the block to remove the hot or cold spots typical of block-based instruments. This design allows for rapid temperature ramps and extreme thermal uniformity (± 0.1°C recorded at 95°C) well to well across the plate, with no edge effect.

Q. I am using a kit that includes an internal control, and so I need to select two channels for reading. Where do I select the channels?

A. On the Plate Layout tab, click the assays button to open the Assays dialog box. Add two assays, one for the target and another for the IPC. Assign reporter dyes from different channels to each assay. Regardless of the plate setup, the Eco 48 qPCR system acquires data in all four channels for all 48 wells. The dyes enable you to focus on the desired information during analysis.

Q. On how many computers can I install the Eco 48 qPCR system software?

The Eco 48 qPCR system comes with unlimited licenses for the system software, so that you are not constrained to perform the data analysis on the included workstation. You can install the Eco Study software from the USB drive that is provided with each Eco 48 qPCR system.

Q. Can I export my Eco 48 qPCR system run data for use in other software packages?

You can export your data as multiple *.csv files or as a single Excel file with multiple worksheets. In addition, you can export a report as a PDF or PowerPoint file.