Applied Biosystems™ QuantStudio™ 6 Flex Real-Time PCR System, 384-well, desktop

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 needs to have been collected for the run.

- Go to ‘Analysis Settings'
- Under the default ‘Call Settings' tab, Choose ‘Analyze Real-Time Rn 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 changes with the cycles number.
In the example below, the image on the left shows the data at a full 40 cycles. The image on the right is the same data 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.

Follow the directions in this blog post (http://solid.community.appliedbiosystems.com/community/real-time_pcr/blog/2013/03/26/transferring-a-quantstudio-12k-flex-software-license-to-another-computer) to transfer your license to another computer. (Please note that the instructions here still apply, although they are written for the QuantStudio 12K Flex Real-Time PCR System.)

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 then Tools then Preferences then 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.

Yes, the QuantStudio 6 and QuantStudio 7 Flex Real-Time PCR Systems 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 then Ct Settings and change the drop down under ‘Data Step Selection'.

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) - for QuantStudio 7 Flex Real-Time PCR System only

Each instrument can hold up to 100 experiment files.

You will need an oligo sequence with the custom dye but without the quencher molecule. Set up a plate of dye dilutions such as seen in the “Custom Dye Calibration” section of this link:
https://tools.thermofisher.com/content/sfs/manuals/4474347B.pdf
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
- 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

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. In this example we are decreasing the temperature by 0.4 degrees 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 degrees C step. Your new method has now been applied.

Please see the comparison table in this brochure (https://www.thermofisher.com/content/dam/LifeTech/migration/files/pcr/pdfs.par.56935.file.tmp/co06402%20quantstudio%20family%20brchr-final-web.pdf) for details on the differences between the two instruments.

The uniformity calibration generates data that allows the software to compensate for the physical effects of the QuantStudio system filters using the ROI (Region of Interest) 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.

The maintenance schedule can be found in the QuantStudio 12K Flex Maintenance and Administration Guide (Chapter 2). In short, the background calibration should be performed monthly, while all other calibrations should be performed every 6 months.

User Bulletin v1.2.1 has specific directions for how to renew your software license. Please search the term '4474347' on our website. The renewal directions apply to the ViiA 7, QuantStudio 6, QuantStudio 7, and QuantStudio 12K Flex systems.

You can check your license status by opening the instrument software and going to Tools --> License Central. You will see the license status and the expiration date.

The main difference between the QuantStudio 6 Flex and QuantStudio 7 Flex Real-Time PCR systems are the available blocks, filters, and automation compatibility:

QuantStudio 6 blocks: 96-well, 96-well Fast, 384-well
QuantStudio 7 blocks: 96-well, 96-well Fast, 384-well, 384-well TaqMan Array Card
QuantStudio 6 filters: 5 colors
QuantStudio 7 filters: 6 colors (21 filter combinations)
QuantStudio 6 automation: Not compatible with automation
QuantStudio 7 automation: Compatible with automation

Continuous melt curve: increases the temperature by the ramp increment (degrees C/sec) so that the temperature will change at a constant rate set in the protocol.
Step and Hold melt curve: increases the temperature by the ramp increment (degrees C) and then holds at that temperature for the time specified by the user.

The amount of data collected will depend on the filter(s) selected. The more filters you use, the longer the acquisition will take and the fewer data points will be collected.

The issue might be due to the block not being properly seated in the instrument. Please follow the steps below:

1. Remove the block from the instrument.
2. Reinsert the block into the instrument.

It is possible to import a standard curve into the Design & Analysis Software (v2.x) if you are analyzing results from one of the following Applied Biosystems real-time PCR instruments:
• 7900HT Fast Real-Time PCR System
• 7500/7500 Fast Real-Time PCR System
• StepOne/StepOne Plus Real-Time PCR System
• ViiA 7 Real Time PCR System
• QuantStudio 1 Real-Time PCR System
• QuantStudio 3 & 5 Real-Time PCR System
• QuantStudio 6 & 7 Flex Real-Time PCR System
• QuantStudio 6 & 7 Pro Real-Time PCR Systems
• QuantStudio 12K Flex Real-Time PCR system

Follow the instructions below to import a standard curve for analysis in the Design & Analysis Software (v2.x):
1. Open the data file in the Design & Analysis Software (v2.x).
2. Click on Actions.
3. Click on "Standard Curve Analysis Setting".
4. Select "External Standard Curves" and click on "Import".
5. Browse to the proper .csv file and click "Open".
6. Click "Apply".

If you are analyzing results from the ViiA 7 Real-Time PCR System, QuantStudio 6 Flex Real-Time PCR System, or QuantStudio 7 Flex Real-Time PCR System, it is possible to import a standard curve into the QuantStudio Real-Time PCR Software following the instructions below:
1. Open the data file in the QuantStudio Real-Time PCR Software.
2. Go to Analysis Settings.
3. Click on the "Standard Curve Settings" tab.
4. Select the option to "Use a standard curve imported from another experiment" and click on "Import".
5. Browse to and highlight the proper data file and click "Select".
6. Click Apply Analysis Settings.

You can get a summary of the calibrations from the QuantStudio Software by going to Instrument > Instrument Console > Manage Instrument > Information > Print Calibration Log.

There are two main differences between a Fast 96-well and standard 96-well block for the Applied Biosystems real-time PCR systems:
• Reaction well volume: The Fast 96-well blocks have 0.1 mL reaction wells and are compatible with 0.1 mL plates and tubes. The standard 96-well blocks have 0.2 mL reaction wells and are compatible with 0.2 mL plates and tubes.
• Ramp rate: The fast 96-well blocks have a higher maximum ramp rate than standard blocks for the same real-time PCR system. However, both the fast 96-well and standard 96-well blocks are capable of running standard and fast chemistry.

Note: For newer instruments (QuantStudio 3 and 5 Real-Time PCR Systems and later) "0.1 mL" and "0.2 mL" are being used in place of "fast" and "standard" as designations for the two 96-well block formats.

If you are running software v1.5.1 and below on the QuantStudio 6 Flex or QuantSudio 7 Flex Real-Time PCR instrument and using a 384-well plate, you may reach out to our Specialty Oligos team at Specialty_Oligos@thermofisher.com to request Cy5 dye calibration oligos. Each tube is provided at 800 µL, 0.2 µM. For a 384-well plate, you will need to order five tubes and aliquot 10 µL/well. Please provide your shipping address to Specialty Oligos and they will provide you with a formal quote.

If you are using a 96-well block, the Cy5 dye plate contained in either 7500 Real Time PCR Systems Spectral Calibration Kit II (Cat. No. 4351151) (Standard, 0.2 mL) or 7500 Fast Real-Time PCR Systems Spectral Calibration Kit II (Cat. No. 4362201 (Fast, 0.1 mL) can be used in place of the Cy5 dye calibration oligos.

All our real-time PCR instruments are compatible with Thermo Fisher Cloud Apps except the 7500 Fast instrument where the eds files alone are compatible with Thermo Fisher Cloud Apps.

Please uncheck and check the “Data Collection” camera icon in the Run Method, and the “Start Run” button should now be active again.

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