QuantStudio™ 12K Flex Real-Time PCR System, OpenArray™ block, Accufill™ System - FAQs

Please follow the instructions listed in this manual (https://licensing.appliedbiosystems.com/web/static/tools/manual.pdf).

For problems with license renewal, please send an email to instrumenthardwaresupport@thermofisher.com (for North America customers) or eurotech@thermofisher.com (for EMEA customers), with the computer ID and license key (AID-number) so you can be assisted.

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When this happens, the analysis was set to use ‘Relative Threshold' but the CRT values do not show up in the exported data. (Note: To use ‘Relative Threshold', go to “Analysis Settings”, and under the default Ct Settings tab, choose ‘Relative Threshold' under “Algorithm Settings”.)

To export the data, go to ‘Export' and make sure the ‘Results' section is checked. You will notice that the column header will still say ‘Ct' even if you have CRT in the well table like above. If you export the data but notice that the numbers still correspond to the ‘Ct' values (and not the CRT values), then you will need to use the ‘Load Export Set' button. Just click ‘OK' to the pop-up window. and the changes will be applied. The next time you export, the values should reflect the CRT values.

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Sometimes an error can occur when the file is downloaded to the computer. When this occurs you may see an abrupt break in the data. If you see this, you can repair the data by going to Analysis then Analyze from Raw Data. You should then see a fixed plot. Please note that you would need to have at least v1.2 of the QuantStudio 12K Flex software to use this feature. If you do not have this version, or if you do but the error persists, please send your data file to techsupport@thermofisher.com so that we can attempt to recover the data.

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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 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.

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Please follow the directions in this user guide (https://licensing.appliedbiosystems.com/web/static/tools/manual.pdf) to manage your license (including transfer and renewing a license).

If you are unable to resolve the issue after following these steps, please contact us at instrumentservices@thermofisher.com for further assistance.

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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 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.

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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)
- OpenArray Plate (33 nL)

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The QuantStudio 12K Flex Real-Time PCR System can store up to 100 runs on the instrument itself.

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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.

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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.

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You can find information on the QuantStudio 12K Flex instrument specifications here:
http://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/real-time-pcr-instruments.html

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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 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 QuantStudio 12K Flex Real-Time PCR System Maintenance and Administration Guide (Appendix C) for more details at http://www.thermofisher.com/content/dam/LifeTech/migration/files/pcr/pdfs.par.4982.file.dat/4470689b.pdf.

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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.

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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 annually.

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The QuantStudio 12K Flex can read FAM and VIC dyes only for the OpenArray block type.

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The QuantStudio 12K Flex software can import sample files to populate sample information into an open experiment. There are many ways to import sample names into the software. You can import samples from a text file, a plate setup file, or a sample definition file. See p.206 of the Maintenance and Administartion User Guide, or search the software menu for "import samples'' for more directions. Please visit the QuantStudio 12K Flex Support Center (https://www.thermofisher.com/us/en/home/technical-resources/technical-reference-library/real-time-digital-PCR-instruments-support-center/quantstudio-12k-flex-real-time-pcr-system-support.html) to find the Maintenance Guide.

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Please see p.22 of the QuantStudio Maintenance and Administration Guide for electrical requirements. Please visit the QuantStudio 12K Flex Support Center (https://www.thermofisher.com/us/en/home/technical-resources/technical-reference-library/real-time-digital-PCR-instruments-support-center/quantstudio-12k-flex-real-time-pcr-system-support.html) to find the Maintenance Guide.

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The QuantStudio 12K Flex software supports several import file formats that can be used to automate experiment creation and assay/sample data import. The files can be used with the command-line application or the QuantStudio 12K Flex software application programming interface (API) to integrate the QuantStudio 12K Flex system into a laboratory information management system (LIMS). Search for Cat. No. 4470689 on the website to find the Maintenance Guide for more details.

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The recommended maintenance schedule can be found on p.82 of the Maintenance and Administration User Guide. Search for Cat. No. 4470689 on the website to find the Maintenance Guide.

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No. Relative quantitation data from the QuantStudio 12K Flex system is meant to be analyzed in ExpressionSuite software. ExpressionSuite software is a free analysis program available on the Technical Resources-->Software, Patches & Updates page.

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Yes. Go to Tools > Preferences > 'non-openarray' tab and change the dropdown for block type as needed.

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Yes, on the QuantStudio 12K Flex system you can see the real time amplification plots on the touchscreen. On other systems, you will only be able to see the amplification plots in real-time on the connected computer.

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The normalization calibration generates factors that the software uses when comparing data from multiple instruments.

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The light source is a white LED. Depending on usage, it should last up to 5 years.

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Yes. We recommend that you use Optical Flat caps, such as Cat. No. N8010535. Use of domed caps may damage the heated cover.

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Yes, from the instrument touchscreen, go to Settings > About the instrument. If you are using the instrument software (when connected) go to Instrument > Instrument Console > Instrument Properties menu.

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It is possible to import a standard curve if you have either the ViiA 7 or QuantStudio 12K Flex systems. In the ViiA 7 or QuantStudio instrument software, open the data file and go to 'Analysis Settings'. Click on the 'Standard Curve Settings' tab. You will see an option to 'Use a standard curve imported from another experiment'. Choose this, then click on "Import". Browse to the proper data file and click "Select".

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Yes. You will need to match all the accessories (heated cover and plate adapter) to the block you have installed. For the QuantStudio 12K Flex system, the 96-Standard and 96-Fast blocks use the same heated cover. The 384-well and TaqMan Array Card block also use the same heated cover. The OpenArray block uses it own separate heated cover.

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The StepOne, StepOnePlus, ViiA 7, and QuantStudio 12K Flex systems can all be run directly from the instrument touchscreen and don't need a separate computer to operate them.

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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.

If the problem persists after reseating the block, please contact our Technical Support team for further assistance.

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If you are running software v1.5.1 and below on the QuantStudio 12K 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 at 10 µL/well, you will need to order two tubes, or at 20 µL/well, you will need to order three tubes. 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.

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The calibration files are stored on the QuantStudio 12K Flex 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 QuantStudio 12K Flex 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.

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Tutorials demonstrating introductions to Real-Time PCR, guidelines for designing real-time assays using Primer Express software, and various instrument set-ups can be found under Life Technologies University on our website. User bulletins, manuals and Product inserts, protocols can be found as downloadable pdf files on our Technical Resources page.

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These instructions may be found on page 79 of the ProFlex PCR System User Guide (https://tools.thermofisher.com/content/sfs/manuals/MAN0007697.pdf).

Reaction volume: 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

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These instructions may be found on page 5 of the Dual Flat Block GeneAmp PCR System 9700 User Guide (https://tools.thermofisher.com/content/sfs/manuals/cms_065075.pdf).

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

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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.
Please note that this part number does not have a product page on our website. To order, please call our customer service team at 800-955-6288, option 1, option 1.

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Additional OpenArray plates can be run off-line using a flat-block thermal cycler. The two thermal cyclers we recommend are the GeneAmp PCR System 9700 and the ProFlex PCR System.

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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.

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

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Additional Applied Biosystems OpenArray plates can be run off-line using a flat-block thermal cycler. The two thermal cyclers we recommend are the Applied Biosystems GeneAmp PCR System 9700 and the Applied Biosystems ProFlex PCR System.

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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.

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

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