My 7500 Fast Real-Time PCR System has been upgraded to v2.0.6, so why can't I analyze my files with HRM Software v2.0.1?
For the 7500 Fast Real-Time PCR System, if the software version is below v2.0.4, the compatible HRM Software is v2.0.1. If the 7500 Fast Real-Time PCR System has been upgraded to software version 2.0.4 or above, then you will need to get HRM Software version 3.0.1.
How do I perform the HRM calibration on my real-time PCR instrument?
The HRM calibration is a 3-step procedure. In some instrument software, the calibration process has been streamlined to a single-step procedure (applies to the ViiA7 Real-Time PCR System and QuantStudio Real-Time PCR Systems).
For the HRM calibration, the first step is a PCR reaction to generate the HRM dye plate. The reaction plate contains a DNA template, a primer pair, and the PCR mix with HRM dye. The second step is dye calibration using the PCR plate from step 1. The last step is a melt curve run, again using the same plate. For details, please refer to the HRM guides for each instrument:
- 7500 Fast Real-Time PCR System (with software version 1.4 or 2.0.4 and below) (https://tools.thermofisher.com/content/sfs/manuals/cms_050347.pdf)
- 7500 Fast Real-Time PCR System (with software version 2.0.5 and above) (https://tools.thermofisher.com/content/sfs/manuals/4457847.pdf)
- 7900HT Real-Time PCR System (https://tools.thermofisher.com/content/sfs/manuals/cms_050347.pdf)
- StepOne and StepOnePlus Real-Time PCR Systems (https://tools.thermofisher.com/content/sfs/manuals/4457847.pdf)
- ViiA 7 Real-Time PCR System (https://tools.thermofisher.com/content/sfs/manuals/4443531.pdf)
- QuantStudio 6 and 7 Real-Time PCR Systems (https://tools.thermofisher.com/content/sfs/manuals/4489823.pdf)
- QuantStudio 6 and 7 Pro Real-Time PCR Systems (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0018981_HighResolutionMelt_DA_SW_UG.pdf)
- QuantStudio 3 and 5 Real Time PCR Systems (https://tools.thermofisher.com/content/sfs/manuals/MAN0014394_HighResMeltExperiment_GSG.pdf)
- QuantStudio 12K Flex Real-Time PCR System (https://tools.thermofisher.com/content/sfs/manuals/4470692A.pdf)
Find additional tips, troubleshooting help, and resources within our High Resolution Melt Support Center.
Is my real-time PCR instrument compatible with HRM?
Among our real-time PCR instruments, the following are compatible with HRM:
- 7500 Fast Real-Time PCR System
- 7900HT Fast Real-Time PCR System
- StepOne and StepOnePlus Real-Time PCR Systems
- ViiA 7 Real-Time PCR System
- QuantStudio 6 and 7 Pro Real-Time PCR Systems
- QuantStudio 6 and 7 Flex Real-Time PCR Systems
- QuantStudio 12K Flex Real-Time PCR System
- QuantStudio 1 Real-Time PCR System
- QuantStudio 3 Real-Time PCR System
- QuantStudio 5 Real-Time PCR System
Why are there no RQ values for all the samples in my study from the 7500 or 7500 Fast Real-Time PCR System software?
The 7500 software requires that the endogenous control be run on every plate within a study. If not, you will only see RQ values for the plate that contains the endogenous control on it. The other plates will have CT values for all the wells, but the software will not calculate RQ values.
If you designed your experiments in this type of layout, you will want to do your analysis in the free DataAssist software instead. This program does not have the endogenous control requirement for every plate. Simply export the study results file from the 7500 software as a *.txt or *.csv file, and use that to create a new study in the DataAssist software. You should now be able to see RQ values for all samples, even those without an endogenous control on the same plate.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.
Why do I not see any CT or RQ values from my ddCT experiment in the software for the 7500 or 7500 Fast Real-Time PCR System?
If you have the SDS v1.4 or earlier, you will need to open the dd CT plate in a Study first in order to see the CT or RQ values. (If you have v2.0.x this error does not apply.)
1.After the initial run is complete, open, review the plots and save it. Then:
-Choose File then New
-Choose: Assay then ddCT (Relative Quantitation) Study. Set the other parameters as appropriate and click Next.
2.Click on Add Plates...', then browse to where your data file is located and select the plate (or multiple plates).
Click Finish', and you should see your study.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.