QuantStudio™ 12K Flex Real-Time PCR System - Getting Started

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.

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.

We recommend performing the calibration for the OpenArray™ block every 12 months. This can be done using the QuantStudio™ 12K Flex OpenArray™ Calibration Kit (Cat. No. 4478601).

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. 
   
   
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. 

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:
   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.

To get log files from the QuantStudio™ 12K Flex Real-Time PCR instrument,

1. Insert a USB cord into the instrument.
2. Follow the instructions in the “Tools” menu on the touch-screen.
3. Download all 5 log files at once to the USB.

To get log files from the QuantStudio™ 12K Flex Real-Time PCR software on the client computer,

1. Navigate to this path: C:\Applied Biosystems\AppData\QuantStudio 12K Flex Software\UserData\data\log
2. Zip the entire contents of this directory.
3. Navigate to this path: C:\Applied Biosystems\AppData\QuantStudio 12K Flex Software\MetaData\logs
4. Zip the entire contents of this directory.

You need an OpenArray® Plate Adaptor, Cat. No. 4472306. You do not need a compression pad. This part number works with both the GeneAmp® PCR System 9700 and the ProFlex™ PCR System.

You need an OpenArray® Plate Adaptor, Cat. No. 4426824, and a compression pad, Cat. No. 442825.

• Position the adaptor.
  
  1. Insert the two adaptor pins into the circular slots on the module.
  2. Be sure the adaptor sits flat.
• Position the TaqMan® OpenArray® plates.
  1. Orient each plate with the barcode facing up and along the edge of the block, away from the center of the adaptor.
  2. Fill up the right sample block first when placing the adaptor on the module (right sample block is the primary control block).
• Fill up the center slots before any edge slots.
  1. (Recommended) Insert unused TaqMan® OpenArray® plates into any empty slots.
• Place the compression pad over the TaqMan® OpenArray® plates (only needed for NT-OpenArray® plates, not for QuantStudio™ 12K), with the Teflon coating (smooth side) facing upwards and away from the plates.
• Slide the heated cover over the compression pad, then pull down the handle.

For more detailed instructions, refer to the Dual Flat Block GeneAmp® PCR System 9700 User Guide.

These instructions may be found on page 5 of the Dual Flat Block GeneAmp® PCR System 9700 User Guide.

93°C for 10 min, ramp rate 100%

50 cycles of:
95°C for 45 sec, 84% ramp rate
94°C for 13 sec, 100% ramp rate
53°C for 2 min 14 sec, 44% ramp rate

25°C for 2 min, 100% ramp rate

Optional: Add a post-PCR overnight hold as shown below:
4°C for 99 min 59 sec, 100% ramp rate

These instructions may be found on page 79 of the ProFlex™ PCR System User Guide.

反応容量: 33 nL
Cover temperature: 70°C

94°C for 600 sec, ramp rate 1.3

50 cycles of:
99°C for 30 seconds, ramp rate 1.3
54°C for 21 seconds, ramp rate 1.1
57°C for 90 seconds, ramp rate 0.1

To perform pre-read and post-read, choose “Get Plate IDs” and input the setup file either with Genotyping.edt or .spf for your runs.

Check the box for “Include Pre-read” and uncheck “Include Amplification”.

Edit the file location and the file name and start the run.

After your pre-read run has ended, you can either keep the run console on to continue with the post-read, or you can close it and perform other pre-read runs.

If you closed the run console and wish to perform the post-read, put the OpenArray in the same position as it was when you did the pre-read and choose “Get Plate IDs” again. Select the .eds file that you had performed pre-read as the setup file and start the run. Note: it is important here that you select the .eds file, NOT the .spf or .edt.

If you have placed the array at the wrong position, the software will prompt you to select the correct position.

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.
• 

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.

You can download your SPF plate files from here.