Applied Biosystems™ StepOnePlus™ Real-Time PCR System

Catalog No.
43-766-00	No Computer

Catalog No. 43-766-00 Supplier Applied Biosystems™ Supplier No. 4376600

Description

Includes

No Computer

StepOnePlus™ Real-Time PCR System is a 96-well Real-Time PCR instrument perfect for both first-time and experienced users.

The StepOnePlus™ Real-Time PCR System can be setup in a variety of configurations and comes ready to use, out of the box, with intuitive data analysis and instrument control software. Utilizing robust LED based 4-color optical recording, the StepOnePlus™ Real-Time PCR System is designed to deliver precise, quantitative Real-Time PCR results for a variety of genomic research applications.

Features of the StepOnePlus™ Real-Time PCR System include:

• Advanced software and instrumentation for performing a wide array of genomic assays
• Sensitive 4-color optical LED recording system
• Intuitive and robust software perfect for both first-time and advanced users
• Simple and flexible instrument set-up and usage

New StepOne™ Software:
The StepOne™ Software included with the StepOnePlus™ Real-Time PCR System runs on the Windows™ XP operating system and provides instrument control, data collection, and data analysis capabilities (analysis software is also compatible with Windows™ 7). This latest version includes capabilities to collect melt curve data for High Resolution Melt (HRM) applications and the option to export in Real-Time PCR Data Markup Language (RDML) for compatibility with MIQE guidelines.

Rapid and Simple Assay Set-Up:
This remarkably simple Real-Time PCR system is designed with an LCD touchscreen and a user-friendly interface that brings the power of genetic studies to researchers new to Real-Time PCR (Fig 1). The intuitive software and protocol wizards help guide new users through their Real-Time PCR experiments. The StepOnePlus™ Real-Time PCR System also includes a quick-start setup so that you start a run immediately and enter plate information at a later time.

Sensitive LED-Based 4 Color Fluorescence Reading:
The StepOnePlus™ Real-Time PCR System utilizes a long-life LED-based optical system that can record fluorescence from FAM™/SYBR™ Green, VIC™/JOE™, NED™/TAMRA™, and ROX™ dyes. This cost-effective, 4-color, 96-well system delivers precise, quantitative Real-Time PCR results and saves data from all filters in every run without depending on a computer or plate setup. It can discriminate between 2 populations of 5,000 and 10,000 template copies of a TaqMan™ assay with 99.7% confidence.

Compatible with Many Genomic Analysis Techniques:
Perform a variety of standard and demanding genetic analysis research techniques on one instrument using the user friendly StepOnePlus™ Real-Time PCR System and StepOne™ Software.

The StepOnePlus™ System supports many Real-Time PCR applications, including the following:

• SNP Genotyping
• Gene Expression Analysis
• MicroRNA Expression
• Protein Expression
• Translocation Analysis
• Gene Detection
• Viral Load Analysis

The included StepOne™ Software supports a variety of analysis methods, including the following:

• Standard Curve (absolute quantitation)
• Relative Standard Curve
• Comparative Ct (ΔΔCt) (relative quantitation)
• Genotyping and Presence/Absence
• Melt Curve Analysis (as a standalone application)
• High Resolution Melting (as a standalone application)

Versatile Instrument Configuration Options:
The ultra-compact footprint of a StepOnePlus™ System can be installed in multiple distinct configurations, providing unmatched flexibility and convenience that can allow a fit in any laboratory. The StepOnePlus™ Real-Time PCR System can be used via a PC, with a PC connected to a Local Area Network (LAN), or as a stand-alone instrument (PC-free). Each StepOnePlus™ Real-Time PCR System is factory-calibrated for optical and thermal accuracy, so simply remarkable Real-Time PCR results can be obtained right out of the box.

Energy Efficiency and Space Saving:
The StepOnePlus™ Real-Time PCR System draws 38% less energy to process one sample plate (when the instrument is in a heated state), compared to the Bio-Rad CFX96™ Real-Time PCR Detection System. The StepOnePlus™ Real-Time PCR System also has advanced temperature control through use of VeriFlex™ Blocks technology and has a footprint nearly 15% smaller than Bio-Rad CFX96™, which in today's crowded laboratories helps you use your laboratory space even more efficiently. You also leave a smaller environmental footprint on the Earth.

Real-Time Data Monitoring, Dissemination, and Storage:
The system measures amplification as it occurs, allowing you to monitor the progress of your experiment cycle by cycle, either on the machine or remotely. Your data is stored on the instrument itself, and can be viewed and stored automatically via remote access, or transferred via email or a USB flash drive. Data can also be conveniently exported as PowerPoint™, Excel™, and graphical image files.

Expanded Gene Expression Capabilities:
The advanced software provided with the StepOnePlus™ Real-Time PCR System now includes the powerful Gene Expression Study Package. This software package allows for greater flexibility and accuracy in your gene expression assays through:

• Analysis of an unlimited number of plates in one study
• Sorting of data by biological or technical replicate group
• Use of multiple endogenous controls
• Assay efficiency correction

Advanced High Resolution Melting Capability:
Now with High Resolution Melt Software v3.0, you can perform post-PCR sequence variation analysis with the StepOnePlus™ Real-Time PCR System. This separately purchased software simplifies set-up by maintaining assay specific settings and accepts pasted plate layout information directly from Excel. HRM Software v3.0 also has an improved clustering algorithm for increased sensitivity and accuracy, and the ability to conduct separate analyses of multiple assays run on a single plate.

For Research Use Only. Not for use in diagnostic procedures.

Specifications

• Format: 96-well Plate, 8-tube Strips, 0.1 mL Tubes
• Thermal Accuracy: 0.25°C (35°C to 95°C) of Display Temperature
• Sample Ramp Rate: Fast Mode: ± 2.2°C/s, Standard mode: ± 1.6°C/s
• Sensitivity: 1 Copy
• Temperature Range (Metric): 4 to 100°C
• Reaction Volume Range: 10–30 μL (Standard Curve Experiments: 40 μL in Standard Mode is Validated)
• Passive Reference Dye: ROX (Separate Tube), No ROX, ROX (Pre-mixed)
• Dynamic Range: Linear Dynamic Range greater than 9 Log Units (Detection)
• Calibrated Dye: VIC™, SYBR™ Green I, ROX™, JOE™, NED, FAM™, TAMRA™
• High-throughput Compatibility: Multiplexing
• For Use With (Equipment): StepOnePlus™
• Thermal Uniformity: 0.25°C (35°C to 95°C) of Setpoint/Display Temperature
• Reaction Speed: Fast, Standard
• Detection Method: SYBR, Primer-Probe Detection
• Run Time: <2 hrs/run (Standard Mode), <40 min/run (Fast Mode)
• Optics: 4 Emission Filters, Blue LED Excitation source, photodiode
• Thermal Cycling System: Peltier-based System
• Dimensions: 24.6 cm/9.7 in.(W) x 42.7 cm/16.8 in.(D) x 51.2 cm/20.2 in.(H)
• Capacity: 96 x 0.1 mL Tubes, 12 x 8-tube Strips, 1 x 96-well Plate
• Product Line: StepOnePlus
• Peak Block Ramp Rate: 4.6°C/s
• Display Type: LCD Touchscreen
• Quantity: 1 instrument
• Weight: 24 kg (52 lb.)

Frequently Asked Questions (FAQs)

What does "Error - Cannot calculate Pure Dye Matrix" mean?

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.

What does "Error - Fatal Error Number 0x8158" mean?

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.

What does "Error - Java.lang.IllegalArgumentException: One of the raw spectra is null" mean?

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.

What does "Error - Cannot open the file because file contents are not recognized" mean?

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.

What does "Error - Analysis failed due to GExSession doesn't exist in context" mean?

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.

What does "Error - Analysis failed due to: 1" mean on the StepOne or StepOnePlus Real-Time PCR System?

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

What should I do when I get the error "Error - Cannot connect to the instrument" on the StepOne or StepOnePlus Real-Time PCR System?

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.

What does "Error - Failure to acquire needed controlling rights" mean?

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).

Where are my files saved to on the StepOne and StepOnePlus Real-Time PCR Systems?

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.

How do I set up a touchdown PCR on the StepOne and StepOnePlus Real-Time PCR Systems?

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.

How do I use the VeriFlex blocks for PCR gradients in the StepOnePlus Real-Time PCR System?

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.

What plates or tubes can I use with the StepOne and StepOnePlus Real-Time PCR Systems?

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.

How do I load tube strips on the StepOne and StepOnePlus Real-Time PCR Systems?

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.

What volumes can be used in my plate for the StepOne or StepOnePlus Real-Time PCR Systems?

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

Can I reuse my calibration plates for the StepOne or StepOnePlus Real-Time PCR Systems?

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.

When do I need to run the RNaseP calibration for the StepOne or StepOnePlus Real-Time PCR Systems?

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.

When do I need to calibrate the StepOne or StepOnePlus Real-Time PCR System?

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

What real-time instruments can be run independently from a computer?

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.

What is the threshold on the Applied Biosystems real-time PCR instruments and how do I set it?

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.

Can TET dye be used on the Applied Biosystems real-time PCR instruments?

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.

What are some of the main differences between Applied Biosystems Real-Time PCR Systems, like the 7500, 7900HT, 7700 or 7000 Sequence Detection System instruments?

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.

Can I use a custom dye on my Applied Biosystems 7500, 7500 Fast, 7900HT Fast or other Real-Time PCR System?

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.

Does the StepOnePlus Real-Time PCR System (Cat. No.: 4376600) include an RNAse P calibration plate?

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).

During the final step of my run on the StepOne Plus Real-Time PCR System, I encountered the error "Cannot calculate Pure Dye Matrix" and cannot export my results. Can I recover my results and/or the results file?

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.

Can I import a standard curve for my real-time PCR data into the Design & Analysis Software (v2.x)?

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".

What is the difference between a Fast 96-well and standard 96-well block?

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.

Can any of the Applied Biosystems Real-Time PCR Systems detect one copy?

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.

What type of Real-Time PCR (Sequence Detection System) information is available from Applied Biosystems?

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.

What qPCR instruments are compatible with Thermo Fisher Cloud?

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.

For Research Use Only. Not for use in diagnostic procedures.