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Troubleshooting page

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
Perform an instrument self test Every month
Pure dye calibrations Every 12 months
ROI calibration Every 12 months
Uniformity calibration Every 12 months
Normalization calibration Every 12 months
RNase P instrument verification After the instrument has been moved, or as needed to verify instrument performance

The uniformity calibration generates data that allows the software to compensate for the physical effects of the QuantStudio™ 12K Flex Real-Time PCR System filters using the ROI plate. The normalization calibration generates factors that the software uses when comparing data from multiple QuantStudio™ instruments within a study. There are 2 normalization plates provided in the calibration kit, one containing FAM™/ROX™ dye and the other with VIC®/ROX™ dye.

All 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 QuantStudio™ 12K Flex Real-Time PCR System Maintenance and Administration Guide (Appendix C) for more details.

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.

Operation

Instrument Specifications
Block Options (Interchangeable) 96-well (standard), 96-well (Fast), 384-well, TaqMan® Array Card, OpenArray® plate (4 plates)
Sensitivity Down to 1 copy
Dynamic Range 9 logs of linear dynamic range
Calibrated Dyes FAM™, SYBR®, VIC®, ROX™, NED™, TAMRA™ dyes
Detection Method SYBR®, primer-probe detection
Resolution Detect changes as little as 1.5-fold
Reaction Volume Range 10–100 µL (96-well standard) 15–30 µL (96-well Fast) 5–20 µL (384-well) ~1 µL (TaqMan® Array Card) 33 nL (OpenArray®Plate)
Reaction Speed Fast or standard
Optics 6 excitation filters (450–670 nm), OptiFlex™ system (enhanced fluorescence detection), 6 emission filters (500–720 nm)
Temperature Range 4–99.9°C
Run Time 2 hr (standard mode) ~35 min (Fast mode)
Regulatory Statement For Research Use Only. Not for use in diagnostic procedures.
Temperature Accuracy ±0.25°C (between 35°C and 95°C, after 3 min)
Temperature Uniformity ±0.5°C (after 30 sec)
Thermal Cycling System Peltier-based system
Available Applications Gene expression, genotyping, copy number variation, HRM, protein thermal shift, protein detection, mutation detection, miRNA, presence/absence, digital PCR
Dimensions 50.5 cm (W) x 67.2 cm (D) x 73.8 cm (H)
Weight 69.5 kg (152 lb)
Remote Monitoring Yes (up to 10 instruments)
On-Board Memory Yes (up to 100 experiments)
Setup Configurations PC-controlled or run directly from touch screen

The QuantStudio™ 12K Flex Real-Time PCR System features a 6-color filter set that supports all Life Technologies™ dyes. Custom dyes that excite between 455 and 672 nm and read between 505 and 723 nm can also be used, although you will have to calibrate the system first for any new dye.

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. touchdown 1

  4. 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.

  5. touchdown 3

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.

custom dye

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:
    For a 384-well plate: between 400,000 and 1,200,000
    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 QuantStudio™ 12K Flex Real-Time PCR System Maintenance and Administration Manual, Appendix C, “Creating a custom dye plate”, for full details.)

The QuantStudio™ 12K Flex 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)
  • OpenArray® Plate (33 nL)

Yes. The QuantStudio™ 12K Flex Real-Time PCR 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’.

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 → Preferences → 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.

Follow the directions in this blog post to transfer your license to another computer.

Data Analysis

For genotyping data, we recommend to use TaqMan® Genotyper™ software. For relative Quantitation, we recommend to use ExpressionSuite software.

The ‘reveal traces’ feature for genotyping runs allows you to trace the clusters throughout the PCR process. For some assays you may find better cluster discrimination at, say, cycle 40, as opposed to cycle 50. To use this feature, the amplification data need to have been collected for the run.

  • Go to 'Analysis Settings'.
  • Under the default ‘Call Settings’ tab, Choose 'Analyze Real-Time Run (?) Data'.
  • Click ‘Apply Analysis Settings’.
  • Under the Allelic Discrimination Plot, check the box next to ‘Reveal Traces’. You can then move the slide bar to see how the data change with the cycle number.
  • QS12K

The plot on the left shows a genotyping data set at a full 40 cycles. The plot on the right is the same data set after revealing traces (grey lines). The clusters can be traced back by moving the cycle bar. Notice that one point is no longer called at the earlier cycle set point.