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Calibration
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
Pure dye calibrations Every 6 months
ROI calibration Every 6 months
Uniformity calibration Every 6 months
Normalization calibration Every 6 months
RNaseP instrument verification After the instrument has been moved, or as needed to verify instrument performance

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.

The calibration plates can be stored and reused three times for up to 6 months after you first open them, so make sure to return them to their original packaging and return them to –20°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.

Please ask your dye manufacturer for recommendations. If there is no specific recommendation available, try TE Buffer (pH 8.0) to start.

The RNaseP verification plate 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 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. The RNaseP verification plate is a single-use plate.

You can create your own background calibration plate using the directions in the ViiA™ 7 Real-Time PCR System Maintenance Guide (Appendix C). For all other calibration plates, you can purchase a calibration kit, or the individual plates, as required.

Operation

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:

40 cycles
  • 95°C, 20 sec
  • 95°C, 3 sec
  • 72°C, 30 sec (decreasing by 0.4°C every cycle, starting at cycle 2)
  1. Go to File → New Experiment → 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. In this example we are decreasing the temperature by 0.4°C per cycle, so choose (“-“) and (0.40). Click ‘Save Setting’. You will then see a green triangle show up next to the parameter you changed, in this case next to the 72°C step. Your new method has now been applied.

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.

In this example, sample concentrations would be 100, 200, 400, 800, and 1,600 (20 µL/well). Note: The volume would be the same for a 96-well or 384-well plate.

  1. Set up a dummy run using the ‘Standard Curve’ option. Alter the thermal profile so that it simply ramps to 60°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:
    1. For a 384-well plate: between 400,000 and 1,200,000
    2. For a 96-well plate: between 1,400,000 and 4,300,000
  3. 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.)

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

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 (~1 µL reactions)

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, go to Analysis Settings → Ct Settings and change the dropdown under ‘Data Step Selection’. In this example, data collection is turned on at both 63°C and 72°C.

Data Analysis