What computer configurations can I use with the ViiA™ 7 Fast Real-Time PCR System?

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Answer

The current recommended configuration is the Dell Gx980 tower computer running Windows® XP SP3. Note that the instrument does not require a computer to start a run. Any other computer configurations are not fully tested.

Answer Id: E2328

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a85e6c43593177fa4c71ed35083de301_FAQ

When do I need to calibrate the ViiA™ 7 Real-Time PCR System?

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Answer

The recommended maintenance schedule and calibration instructions can be found in the User Guide here: http://tools.lifetechnologies.com/content/sfs/manuals/4442661C.pdf
In brief, the background calibration is recommended to be done every month. All other calibrations (pure dye, ROI, uniformity, and normalization) are recommended to be done every six months.

Answer Id: E7183

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a89654c2b2ee0245f095c546cf9fbddb_FAQ

What are the uniformity and normalization calibrations for on the ViiA™ 7 Real-Time PCR System?

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Answer

The uniformity calibration generates data that allow the software to compensate for the physical effects of the ViiA™ 7 system filters using the ROI plate. The normalization calibration generates factors that the software uses when comparing data from multiple ViiA™ 7 instruments within a study. Two normalization plates are provided in the calibration kit, one containing FAM™/ROX™ dyes and the other with VIC®/ROX™ dyes.

Answer Id: E7184

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791370dbdc977653643ee676fd27808f_FAQ

Can I reuse my calibration plates?

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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.lifetechnologies.com/content/sfs/manuals/4442661C.pdf.

Answer Id: E7185

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031b48cfb280d589b98ef969b72fb0ac_FAQ

Can I create my own calibration plates?

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You can create your own background calibration plate using the directions in the ViiA™ 7 Real-Time PCR System Maintenance Guide (Appendix C) http://tools.lifetechnologies.com/content/sfs/manuals/4442661C.pdf. For all other calibration plates, you can purchase a calibration kit, or the individual plates, as required (http://www.lifetechnologies.com/us/en/home/life-science/pcr/real-time-pcr/real-time-pcr-reagents/real-time-calibration-kits/viia-7-system-calibration-kits.html).

Answer Id: E7187

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d62f6cd29f7af3b5141aa0f7e8f701c1_FAQ

How do I set up a touchdown PCR experiment on the ViiA™ 7 Real-Time PCR System?

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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. 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 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: E7188

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f98938edb80fb5c55f917b4928a3a770_FAQ

How do I add a custom dye to the ViiA™ 7 Real-Time PCR System?

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Answer

You will need an oligo sequence with the custom dye but without the quencher molecule. For each custom dye, prepare samples in a concentration range of 100-2,000 nM, choosing a 2-3-fold difference in dilution points. Use either the background buffer provided in the calibration kit or your own buffer to dilute the oligo. A full plate is not needed; see the example below.

1. Set up a dummy run using the ‘Standard Curve’ option. Alter the thermal profile so that it simply ramps to 60 degrees C with a 2 min hold. Ensure that the filters of interest are selected.
2. When the run is complete, export and examine the raw data. Select the concentration to use by finding the dilution that will give you an acceptable signal in the following ranges:
- For a 384-well plate: between 400,000 and 1,200,000
3.For a 96-well plate: between 1,400,000 and 4,300,000
4.Create a full plate of the dye using the selected concentration and run the custom dye calibration as normal, using 20 μL. (Refer to the ViiA™ 7 Real-Time PCR System Maintenance Guide, “Creating a custom dye plate”, for full details.)

Answer Id: E7189

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7e5119ed3331fce7f681829509d4e2cb_FAQ

How many files can the ViiA™ 7 Real-Time PCR System hold?

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Answer

The ViiA™ 7 Real-Time PCR System can store up to 100 runs on the instrument itself.

Answer Id: E7190

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298d40acfd55d8cec6f2ed2022df0f35_FAQ

What volumes can be used in my plate on the ViiA™ 7 Real-Time PCR System?

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Answer

The following volumes are supported for each instrument block:

- 96-well standard (10-100 μL reactions)
- 96-well Fast (10-30 μL reactions)
- 384-well (5-20 μL reactions)
- TaqMan® Array Micro Fluidic Cards (approximately 1 μL reactions)

Answer Id: E7191

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f8645d2cd09ad25081386552ba21b4d1_FAQ

Can I collect data at two separate steps for analysis in the ViiA™ 7 Real-Time PCR System?

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Answer

Yes. The ViiA™ 7 system can collect data at multiple steps in the amplification stage. You will need to turn on the data collection for every step of interest. In the analysis, only one set of data can be displayed at a time. To change the data set used for analysis, from your data file go to Analysis Settings then Ct Settings and change the dropdown under ‘Data Step Selection’.

Answer Id: E7192

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ca4682eba76b12cf4be72a6010db9918_FAQ

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

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Answer

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

Answer Id: E7193

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1b4b50d9c99e715046999362a7b02b0f_FAQ

What should I do if the software will not connect to the ViiA™ 7 Real-Time PCR System?

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Answer

After launching the ViiA™ 7 system software, go to the Maintenance Manager. Check if the ViiA™ 7 system icon is present in the “On the Network” window. If not, click on the “Refresh” button to the left of “Remove from My Instruments”.

If you still do not see the ViiA™ 7 icon for the following reasons:
- There is an icon but you cannot add it into the “My Instruments” area
- There is a red “X” on the instrument
- A message says “Disconnected” at the bottom of the window with a red “X” in it then you will need to add the instrument to the “My Instruments” area. Click on the wrench icon in the upper right-hand corner of the icon as pictured below. You may get a message that says that the firmware is older than the one you are trying to run. If appropriate, choose to download the new firmware and let it progress to finish. Do not turn off the instrument during this update. After the firmware update is completed, restart the instrument to check the ViiA™ 7 system icon in ‘On the Network’ area.

Answer Id: E7194

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ad61aea811e1ea377056e0be62d747de_FAQ

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

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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

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1ec5446263f71d51bd02521c26d412a8_FAQ

What does the error “Data collection incomplete and cannot be handled by analysis…” mean?

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Answer

This error is caused by a corrupt template or when the ‘Open Run Method’ button was selected to start the run. To avoid this, do not use corrupt templates or run files from previous software versions. If you have v.1.1 or earlier, please upgrade to the most current version. If you are using templates created from v.1.1 or earlier, please delete and recreate them in v1.2 or above. New run files created from v1.2 can use the ‘Open Run Method’ button.
If you have a file that gives you this error, please send your data file (*.eds) to techsupport@lifetech.com for assistance.

Answer Id: E7196

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c67a2aa599fd05c632378b8288e0a605_FAQ

Why am I not able to see/export Quantity or Ct values in the ViiA™ 7 Real-Time PCR System?

Product FAQ

Answer

After exporting data into .xls format, the Quantity or Ct values may not be present. From your file, Select ‘Export‘ and click on ‘Load Export Set’ and then ‘OK’ and ‘OK’. Then click on ‘Start Export’. The Quantity or Ct values should now be present in the exported file.

Answer Id: E7197

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e5a1894ca2d3466ce6109b88ba019aeb_FAQ