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查看更多产品信息 StepOnePlus™ Real-Time PCR System - FAQs (4376600)
29 个常见问题解答
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).
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.
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.
The StepOnePlus Real-Time PCR System contains six independently thermally regulated VeriFlex blocks, which can help you optimize your thermal cycling conditions. You can set a different temperature for one or more zones, or you can set one temperature for all zones in the sample blocks. [Note: The difference in temperature between adjacent zones can range from 0 to 5 degrees C, in 0.1 degrees C increments].
1.Choose ‘New Experiment' then ‘Advanced Setup' and choose the Experimental Properties as normal.
2.Under ‘Plate Setup' then ‘Assign Targets and Samples'
3.On the right-hand side under ‘View Plate Layout', check the box to ‘Enable VeriFlex Block', and click ‘Yes' to the box. You should now see the plate separated into 6 zones by red lines.
4.Click on ‘Run Method' in the left-hand column. On the Graphical View, click on the temperature that you want to change. A new window will pop up. Choose ‘Set different temperatures for one or more zones'. Adjust the temperature as needed for each zone.
5.After you click ‘OK' you will see the ‘Zones' represented on the thermal profile. If you hover over the word ‘Zones', you can see the temperatures you set.
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.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.
The threshold is the numerical value assigned for each run that reflects the average dRn (change in fluorescence) during the initial cycles of PCR (baseline). The threshold is set by determining a statistically significant point (or value) above the baseline. You can manually adjust the threshold should you desire, to the place in the geometric phase where your replicates are tightest. For more information on how to manually set a threshold, please refer to the tutorial entitled Data Analysis on the ABI PRISM 7700: Setting Baselines and Thresholds (P/N 4370923). While this tutorial is based on the ABI PRISM 7700 system, the concepts are still valid for the Applied Biosystems real-time PCR instruments. You can search the document on our website by using the part number above as the keyword.
If you choose not to manually adjust the baselines and thresholds, the SDS software on the Applied Biosystems real-time PCR instruments has an Auto Ct algorithm that can be used to automatically generate baseline and threshold values for individual detectors. The algorithm calculates baseline and threshold parameters for a detector based on the assumption that the data exhibits the "typical" amplification curve. Experimental error (i.e. contamination, pipetting inaccuracies) can produce amplification curves that deviate significantly from a typical amplification curve. The data from these irregularities can affect the Auto Ct algorithm by causing it to generate incorrect baseline and threshold parameters for the associated detector. Therefore, Applied Biosystems recommends that after analysis of experimental data, you review all baseline and threshold parameters determined by the Auto Ct algorithm.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.
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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The systems most often vary by the light source and filter sets. The 7000, 7300, 7500, and 7500 Fast systems utilize a halogen lamp as the excitation source and emission filters to detect the emission fluorescence. The 7000 and 7300 systems both have one excitation and four emission filters, while the 7500 and 7500 Fast systems have five excitation and five emission filters. Five excitation and five emission filters allow the 7500 and 7500 Fast systems to detect dyes in the far red range. The 7700 and 7900HT systems utilize an argon ion laser as the excitation source and the instrument scans across all wavelengths between 520 nm to 610 nm.
The 7500, 7500 Fast and 7900HT systems also offers the ability to upgrade to the Fast chemistry; typical real-time runs take about 30-45 minutes.
In addition, the 7900HT system offers further high throughput options such as a 384-well block and a TaqMan Low Density Array Upgrade as well as a Zymark Twister Automation Accessory that will robotically load plates on the instrument.
For further information please speak with your local Life Technologies sales representative.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.
Yes, you can run custom dyes, but the custom dye must fall into the wavelengths that are capable of being detected by a specific instrument. For optimal results on the filter-based instruments (including 7500 and 7500 Fast), the custom dye emission wavelength must fall into one of the emission filter peak detection wavelengths. For the laser-based 7900HT systems, the custom dye wavelength must fall within the overall range of detection for the instrument. Please refer to the Real-Time PCR information pages on our website under “Products & Services” for profiles of each instrument with a list of compatible standard dyes and the corresponding wavelengths supported. Please also refer to your User Manual or Installation and Maintenance Manual for instructions on how to calibrate custom dyes on your particular instrument.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.
Yes, it does. It is the TaqMan RNase P Instrument Verification Plate, Fast 96-well (Cat. No. 4351979). Please note that this verification plate is required to verify the performance of the instrument just once (see page 42 of this link: https://assets.thermofisher.com/TFS-Assets/LSG/manuals/4376782.pdf).
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.
If you have not performed another run on your StepOne Plus instrument after the run that had the error, you can try the following:
To try to retrieve the results file, transfer the .eds file directly from the instrument to an USB. Leave the USB plugged in for a minute after the instrument indicates that the data transfer has been done. Then transfer the file from the USB to your computer and try to open it as usual.
If you only need to see your results, and not necessarily to recover the file, you can upload the file to your Thermo Fisher Scientific Cloud account and view the results there.
Note: These suggestions do not guarantee recovery of your results/results file. If you are unable to retrieve your results, please contact techsupport@thermofisher.com for further assistance and possible recovery of your results/results file.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.
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.
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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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Tutorials demonstrating introductions to Real-Time PCR, guidelines for designing real-time assays using Primer Express software, and various instrument set-ups can be found under Life Technologies University on our website. User bulletins, manuals and Product inserts, protocols can be found as downloadable pdf files on our Technical Resources page.
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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.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.