StepOne™ Real-Time PCR System, desktop - FAQs

This issue can occur if the run is interrupted. To prevent this from happening in the future, avoid ending the PCR with an infinite hold stage or interrupting the run. Please send the file to techsupport@thermofisher.com so that our software group can attempt to recover the data.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.

This is generally caused by incorrect placement of tube strips, resulting in a sample block that can't properly be lowered. We recommend contacting Instrument Services to have an engineer walk you through a manual drawer-lowering process during instrument power-up. To prevent this from happening in the future, place tubes toward the middle of the block or use a placeholder strip on the opposing side to counterbalance block pressure.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.

This issue can occur if the “Start Run” button is clicked more than once or if illegal characters are used in the experiment/file name. You can recover the data yourself by overriding the calibration file with one that represents the calibration status of the instrument when you ran that particular file. Use the toolbar at the top to navigate: Analysis then Override Calibration then Use Calibration From Another File.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.

This issue can occur if duplicate samples are added to the experiment, if illegal characters are used in the experiment/file name, or if the file extension was changed to .eds from a different extension.
Please send the file to techsupport@thermofisher.com to attempt recovery.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.

This error means that the data collection was not turned on as appropriate. If this is inadvertently turned off, no data will be collected. Thus, the data are not retrievable in such a file.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.

Please send the file to techsupport@thermofisher.com so that we can have our software group attempt to recover the data.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.

Check that all the cables are correctly plugged into the respective ports and not bent. Follow the directions on page 135 of the StepOne and StepOnePlus Maintenance Guide (https://tools.thermofisher.com/content/sfs/manuals/4376782.pdf) to check the connectivity. If the issue is still not resolved, please contact Instrument Services at 1-800-955-6288, option 3, option 1, or by email at instrumentservices@thermofisher.com for further assistance.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.

This error usually occurs when you first start your run. Reboot the instrument and restart the computer. Make sure to log into the computer as the Administrator (on the Windows OS).

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Run files will be saved to both the instrument and the connected computer. On the connected computer, files will be saved to the default data folder, unless you change it.

To find or change the default folder, 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.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.

In a touchdown PCR experiment, you will change either 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 higher in the early cycles and the efficiency in the later cycles.

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 and click ‘Save Setting'. You will then see a green triangle show up next to the parameter you changed. Your new method has now been applied.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.

Please refer to this selection table (https://www.thermofisher.com/us/en/home/life-science/pcr/pcr-plastics/real-time-pcr-plastics.html) for compatible plates, tubes, and films.

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Follow these instructions for proper loading of tubes, tube strips, and plates:

- Put the tubes or tube strips in the tray with the adapter on the 96-well support base.
- If using a plate, put the plate directly on the 96-well support base.
- Pipet your reactions into the tubes/tube strips or the wells on the plate.
- Seal the tube/tube strips with optical flat caps.
- If using a plate, seal the plate with either optical adhesive film or flat caps.

For optimal performance on the StepOnePlus instrument with partial loads:
- Load at least 16 tubes and arrange them in adjacent columns of 8 tubes, using rows A through H, or adjacent rows of 8 tubes, using columns 3 through 10.
For optimal performance on the StepOne instrument, load at least 4 tubes in the sample block.

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The StepOne and StepOnePlus Real-Time PCR Systems have a Fast block, which supports reaction volumes of 10-30 µL.

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The calibration plates can be stored and reused three times for up to one year after you receive 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 StepOne and StepOnePlus Maintenance and Administration Guide (Appendix C) (http://tools.thermofisher.com/content/sfs/manuals/4376782G.pdf) for more details.

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The RNaseP verification plate is a single-use plate that 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 that there is something wrong with the instrument, or if you want to rule out a chemistry issue, the RNaseP plate is a good way to test the system. It is also recommended to use after the instrument has been moved to a new location. For the StepOne instrument, the RNaseP plate is Cat. No. 4371439. For the StepOnePlus instrument, the RNaseP plate is Cat. No. 4351979.

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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 18 months
Spatial calibration: Every 18 months
RNaseP instrument verification: After the instrument has been moved, or as needed to verify instrument performance

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.

The StepOne, StepOnePlus, ViiA 7, and QuantStudio 12K Flex systems can all be run directly from the instrument touchscreen and don't need a separate computer to operate them.

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It is possible to import a standard curve into the Design & Analysis Software (v2.x) if you are analyzing results from one of the following Applied Biosystems real-time PCR instruments:
• 7900HT Fast Real-Time PCR System
• 7500/7500 Fast Real-Time PCR System
• StepOne/StepOne Plus Real-Time PCR System
• ViiA 7 Real Time PCR System
• QuantStudio 1 Real-Time PCR System
• QuantStudio 3 & 5 Real-Time PCR System
• QuantStudio 6 & 7 Flex Real-Time PCR System
• QuantStudio 6 & 7 Pro Real-Time PCR Systems
• QuantStudio 12K Flex Real-Time PCR system

Follow the instructions below to import a standard curve for analysis in the Design & Analysis Software (v2.x):
1. Open the data file in the Design & Analysis Software (v2.x).
2. Click on Actions.
3. Click on "Standard Curve Analysis Setting".
4. Select "External Standard Curves" and click on "Import".
5. Browse to the proper .csv file and click "Open".
6. Click "Apply".

There are two main differences between a Fast 96-well and standard 96-well block for the Applied Biosystems real-time PCR systems:
• Reaction well volume: The Fast 96-well blocks have 0.1 mL reaction wells and are compatible with 0.1 mL plates and tubes. The standard 96-well blocks have 0.2 mL reaction wells and are compatible with 0.2 mL plates and tubes.
• Ramp rate: The fast 96-well blocks have a higher maximum ramp rate than standard blocks for the same real-time PCR system. However, both the fast 96-well and standard 96-well blocks are capable of running standard and fast chemistry.

Note: For newer instruments (QuantStudio 3 and 5 Real-Time PCR Systems and later) "0.1 mL" and "0.2 mL" are being used in place of "fast" and "standard" as designations for the two 96-well block formats.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.