7500 Real-Time PCR System, laptop - FAQs

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.

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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.

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You may encounter this error message, which will abort the run before it even starts. If so, go into the software under Setup then Run Method and check how much time has been set for the data collection step. The instrument has a minimum collection time, which is dependent on the number of filters being used. (This is true in both the 1.x and 2.0.x versions of the 7500 software.) Increase the extension time to 30 sec and restart the run. It should now proceed as normal. If the error persists, please contact us at techsupport@thermofisher.com for further assistance.

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This error can be caused by the wrong plastics being used in the block, such as the wrong Precision Plate Holder being used in the tray. This can give an error message such as “Block up switch not activating” and will not allow the run to start. Open the drawer, make sure the correct plate/tubes and plate/tube holder are being used and in the correct orientation. Turn off the instrument and open the front cover to make sure the heated cover door is closed. Restart the instrument and retry the run. If the error still persists, please contact us at instrumentservices@thermofisher.com for further assistance.

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If you see this error message, please check the following:

1. Verify that the USB connection is secure between the instrument and computer.
2. Verify that the computer user power save settings are set to Never for all users (in Windows under Control Panel, Power Options).
3. Is there a screen saver in use? If so, set the screen saver so that it will not come on during a run.
4. Is there antivirus or other network software in use? If so, disconnect from the network while running the instrument.
If the error persists, please contact us at instrumentservices@thermofisher.com for further assistance.

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Sometime there is an error such that the ROI will not calibrate all 96 wells. If you run into this problem check the following:

- Is the ROI calibration plate old or expired?
- Spin down the plate and visually inspect the wells. Is the volume consistent and correct across all wells? If not, try a new ROI calibration plate.
- Try the manual calibration and increase the exposure time.
- Check the status of the halogen lamp. If it is over 2,000 hours, replace the bulb and try again.

If you have tried all the steps above but the ROI still will not calibrate, please contact us at instrumentservices@thermofisher.com for further assistance.

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You may get a message that the heated cover is not reaching the set temperature. If you get this error, or notice this issue on your own, please contact us at instrumentservices@thermofisher.com.

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“Fatal Error 1420” is related to the door or heated cover not being fully closed on the instrument itself. We recommend opening the front access door using a small screwdriver, ensuring that all inner components are correctly seated, then closing the door completely. Please then reboot the instrument and attempt to restart your run. Please see page 60 of the Installation and Maintenance Manual for more information: https://tools.thermofisher.com/content/sfs/manuals/cms_041437.pdf

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Click on the ‘Group By' button and choose ‘Well Position' (Column). Then highlight the plate and print the report.


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Go to ‘View Well Table' from the Amp B117Plot screen.


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Yes. If you have the newer version of the software (v2.0.1 or later), which creates *.eds files, your data will be directly compatible with our Protein Thermal Shift Software (https://www.thermofisher.com/order/catalog/product/4466038?ICID=search-product). If you have the older software (v 1.x), you will have to program the software differently (see below) and analyze the results independently. For more details on the analysis, you can refer to this paper: “The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability.” Nat Protoc 2007;2(9):2212-21.

For an experiment using SDS v1.x, follow the directions below:
1.Create a new experiment, choosing Absolute Quantification assay type. Click ‘Next'.
2.Go to ‘Select Detectors'. If there is no predefined detector for this application, create a new one. Make sure the Reporter Dye is ROX, and Quencher Dye is none.
3.Assign the detector to the plate. Select none for the Passive Reference

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In a touchdown PCR experiment, you will either change the temperature or the time of a particular PCR step with every cycle. Most commonly, the annealing temperature is adjusted throughout the experiment, such that the specificity is increased in the early cycles and the efficiency in the later cycles.

In this example, we will set the method to do the following:
1. Go to File then New Experiment then Advanced Setup. Fill out the relevant options as you normally would.
2. Go to the Run Method under the ‘Setup' section and you should see the Graphical View of your thermal profile. Check the box next to ‘Enable AutoDelta'. You should see some grey triangles appear next to the Temperature and Time at every step in the Cycling Stage. (Note: If you want to start the changes at a later cycle, set this here under ‘Starting Cycles'.)
3. A new window called ‘AutoDelta Settings' will open up. Select the appropriate options. In this example we are decreasing the temperature by 0.4 degrees C per cycle, so choose (“-“) and (0.40). Click ‘Save Setting'. You will then see a green triangle show up next to the parameter you changed, in this case next to the 72 degrees C step. Your new method has now been applied

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Run files will be saved to a default folder on the connected computer, unless you change it. To find or change the default folder (in SDS v2.0.1 or later), go to Tools then Preferences then Defaults. Here you will see a Data Folder and an Import Folder. The default location is shown. If you want files to be saved to (or open from) a different location, click ‘Browse' and choose the new folder.

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No. The software will use the last data collection step in the cycling stage for all amplification plots and Ct analysis. So even if you were to set two separate steps with “Data Collection On”, you would only be able to view and analysis data from the latter step.

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The following volumes are supported for each instrument block:
-7500: 20-100 µL reactions
-7500 Fast: 10-30 µL reactions

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Using the instrument computer when a run is in progress is not recommended, as this poses a risk of corrupting the data.

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The 7500 and 7500 Fast Real-Time PCR Systems can be used to run assays designed with custom dyes (dyes not manufactured by Life Technologies). Custom dyes must fluoresce within the 520-650 nm spectral range measured by the 7500 or 7500 Fast instrument. To use a custom dye, you must first determine what the right concentration of the dye is. You will need to order an oligo with a 5' custom dye but no quencher. Make up a plate with different concentrations of this oligo (approximately 25-3,200 nM) and use the ROI Inspector to assess fluorescence. Choose the concentration that displays the brightest possible signal without saturation in all filters. Once you have found the correct concentration, create a full plate of custom dye at this concentration and perform the custom dye calibration. See Appendix B in the 7500 and 7500 Fast Real-Time PCR System Maintenance Guide (http://tools.thermofisher.com/content/sfs/manuals/cms_077749.pdf) for full details.

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The Applied Biosystems 7500 and 7500 Fast Real-Time PCR Systems use the following dye sets for calibration: Cy3, Cy5, FAM, JOE, NED, ROX, SYBR Green, TAMRA, Texas Red, and VIC dyes. Custom dyes that are read between 520 and 650 nm can also be used, although you will have to calibrate the system first for any new dye.

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Instrument Specifications
Block Options (Fixed): 7500: 96-well (standard); 7500F: 96-well (Fast)
Sensitivity: Down to 1 copy
Dynamic Range: 9 logs of linear dynamic range
Calibrated Dyes: FAM, SYBR, VIC, ROX, NED, TAMRA dyes (Cy3, Cy5, and Texas Red dyes - 7500F only)
Detection Method: SYBR dye, primer-probe detection
Resolution: Detect changes as little as 1.5-fold
Reaction Volume Range: 20-100 µL (7500); 10-30 µL (7500F)
Reaction Speed: Fast or standard (7500 has standard mode only)
Optics: Tungsten-halogen lamp, 5 excitation filters, 5 emission filters, CCD Camera
Temperature Range: 4-99.9 degrees C
Run Time: <2 hr (standard mode); ~35 min (Fast mode - 7500F only)
Temperature Accuracy: ±0.25 degrees C (between 35 degrees C and 95 degrees C, after 3 min)
Temperature Uniformity: ±0.5 degrees C (after 30 sec)
Thermal Cycling System: Peltier-based system
Available Applications: Gene expression, genotyping, copy number variation, HRM, protein thermal shift, protein detection, mutation detection, miRNA, presence/absence
Dimensions: 34 cm (W) x 45 cm (D) x 49 cm (H)
Weight: 34 kg (75 lb)
Remote Monitoring: No
On-Board Memory: No
Setup Configurations: PC-controlled only

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Follow the directions in the 7500 and 7500 Fast Real-Time PCR System Maintenance Guide, Chapter 6 (http://tools.thermofisher.com/content/sfs/manuals/cms_077749.pdf), to decontaminate the sample block. You can also watch this helpful video demonstration (https://www.youtube.com/watch?feature=player_embedded&v=dJyolVGhpjk).

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The RNaseP verification plate contains template, master mix, and a TaqMan assay for RNaseP. It is used to verify that the instrument is performing to specifications. If you have reason to suspect there is something wrong with the instrument, if the instrument has been moved, or if you want to rule out a chemistry issue, the RNaseP plate is a good way to test the system. The RNaseP verification plate is a single-use plate.

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The calibration plates can be stored and reused three times for up to 12 months after you first open them, so make sure to return them to their original packaging and return them to -20 degrees C storage until the next use. If needed, you can make your own background plate using deionized water. Please follow the directions in the 7500 and 7500 Fast Real-Time PCR System Maintenance Guide (Appendix C) (http://tools.thermofisher.com/content/sfs/manuals/cms_077749.pdf) for more details.

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Recommended Maintenance Schedule

Power on/off the computer controlling the instrument: Weekly
Check computer disk space. If necessary, archive or back up your experiment files and instrument settings: Weekly
Background calibration: Every month
Run disk cleanup and disk defragmentation: Every month
Perform an instrument self test: Every month
Pure dye calibrations: Every 6 months
ROI calibration: Every 6 months
Optical calibration: Every 6 months
RNaseP instrument verification: After the instrument has been moved, or as needed to verify instrument performance

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TET dye is not recommended to be used on the following real-time PCR instrument models: 7000, 7300, 7500, 7500 FAST, StepOne, and StepOnePlus Systems.

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Yes. you can choose either standard or fast ramp on the 7500 Fast System for software versions 2x or greater, and a 9600 emulation thermal cycling mode is available for customers who wish to run assays based on adjusted ramp rate. However, you should be aware that we cannot guarantee identical performance to the same modes run on a standard 7500 system block.

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Runs can be compared between standard and fast mode, as the data collected are collected in all five filters in the instrument. However, keep in mind that these data may not always show identical results and may have added variability due to the data collection processes and differences in ramp rate between standard and fast mode.

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Yes, older 7500 or 7500 fast data files are compatible with the current version of the SDS software. However, there is one exception: please note that the SDS v1.3 software will not allow RQ Studies to be created from RQ Plates from different instrument types, different thermal modes (i.e., fast and standard modes) or different numbers of thermal cycles.

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Yes, the standard 7500 instrument has a 96 x 0.2 mL block, whereas the 7500 Fast instrument has a 96 x 0.1 mL block.

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All Applied Biosystems instrument systems are qualified to detect a two-fold change. Scientifically speaking, there is a statistical calculation that predicts the probability of detecting one molecule in any given sample called the Poisson Distribution. The Poisson Distribution predicts that one molecule will be detected 66% of the time. This is assuming that the assay has been optimized to the fullest capacity and that there are no aliquoting or pipetting errors involved. Please refer to the following paper concerning one-copy detection: Lockey, C., E. Otto, and Z. Long. "Real-time fluorescence detection of a single DNA molecule." BioTechniques 24 (1998): 744-746.

Each assay designed on one of our platforms needs to be optimized to determine the Linear Dynamic Range of the assay. An Applied Biosystems Sequence Detection System (Real-Time PCR instrument) may be able to detect one copy in a fully optimized assay. As stated above, one copy can only be detected 66% of the time in a fully optimized system.

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All our real-time PCR instruments are compatible with Thermo Fisher Cloud Apps except the 7500 Fast instrument where the eds files alone are compatible with Thermo Fisher Cloud Apps.

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