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

Product FAQ

Answer

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.

Answer Id: E7206

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

Answer

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.

Answer Id: E6303

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

Answer

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.

Answer Id: E1824

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

Answer

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.

Answer Id: E7214

Was this answer helpful?

Yes
No
Thank you for your response

What does the error “Calibration file already exists” mean?

Product FAQ

Answer

The calibration files are stored on the ViiA™ 7 instrument; therefore, when you need to perform a new calibration, you will have to delete the old calibration files. From the instrument console, go to the ViiA™ 7 instrument touch screen and select ‘Collect Results’. You should then see a list of files stored on the instrument. Manually select a calibration file such as “Fast-96-cal” and press the ‘Delete’ button to remove the old calibration file. Occasionally, users cannot delete the file off the instrument and may encounter this error: “Some experiments did not collect results therefore cannot be deleted.” For these situations, launch the software, go to ‘maintenance’, manage files, and download the calibration file. The status of the experiment should be displayed as ‘complete’. Next, delete the old calibration file and rerun the calibration.

Answer Id: E7195

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

Answer

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.

Answer Id: E7207

Was this answer helpful?

Yes
No
Thank you for your response

Can I reuse my calibration plates?

Product FAQ

Answer

The calibration plates can be stored and reused three times for up to 1 year 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. (Note: Due to the small volumes, calibration array cards cannot be reused. Please discard them after use.) If needed, you can make your own background plate using deionized water. Please follow the directions in the ViiA™ 7 Real-Time PCR System Installation, Networking, and Maintenance Guide for more details at http://tools.thermofisher.com/content/sfs/manuals/4442661C.pdf.

Answer Id: E7185

Was this answer helpful?

Yes
No
Thank you for your response

How do I activate or renew my software license for the ViiA™ 7 Real-Time PCR System?

Product FAQ

Answer

In the ViiA™ 7 Real-Time PCR System, please follow the instructions found in this User Bulletin: How to Activate or Renew a License (http://tools.thermofisher.com/content/sfs/manuals/4474347B.pdf). If you have a license key associated with computer that is no longer in use, please contact our Instrument Services group at instrumentservices@lifetech.com with the serial number and license key so that they can reassign it for you.

Answer Id: E7199

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

Answer

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.

Answer Id: E7204

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

Answer

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

Answer Id: E7212

Was this answer helpful?

Yes
No
Thank you for your response

What can I use for data analysis on the ViiA™ 7 Real-Time PCR System?

Product FAQ

Answer

For genotyping data, we recommend TaqMan™ Genotyper Software. For relative quantitation, we recommend ExpressionSuite™ Software.

Answer Id: E7193

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

Answer

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.

Answer Id: E7215

Was this answer helpful?

Yes
No
Thank you for your response

How do I set up a custom dye calibration in the ViiA™ 7 or QuantStudio™ 12K Flex systems?

Product FAQ

Answer

Follow the directions on page 10 of this User Bulletin to add a custom dye to your ViiA™ 7 or QuantStudio™ 12K Flex system.

Answer Id: E6317

Was this answer helpful?

Yes
No
Thank you for your response

What does “Error - Cannot calculate Pure Dye Matrix” mean?

Product FAQ

Answer

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.

Answer Id: E7217

Was this answer helpful?

Yes
No
Thank you for your response

What are the extensions of files generated by the ABI PRISM™ 7000 Sequence Detection System, Applied Biosystems™ 7300, 7500, 7500Fast, 7900HT Fast Real-Time PCR System, StepOnePlus™ Real-Time PCR System, and ViiA™ 7 Real-Time PCR Systems?

Product FAQ

Answer

All run files have an .SDS or .EDS extension. A Relative Quantification study will have an .SDM or .EDM extension and a template will have an .SDT or .EDT extension. The software for StepOnePlus™, ViiA™ 7, and 7500 2.0.x software have .EDS, .EDT, or .EDM extensions; all other software have .SDS, .SDT, or .SDM extensions.

Answer Id: E2473

Was this answer helpful?

Yes
No
Thank you for your response