QuantStudio™ 12K Flex Real-Time PCR System, 384-well block, desktop - FAQs

What is the difference between continuous melt curves and step and hold melt curves on the Applied Biosystems Real-Time PCR Instruments?

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.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.

I recently switched the block in my real-time PCR instrument. Now it shows the status as 'not calibrated' for all the calibration plates, even though my calibrations are current. What is causing this issue?

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.

Can I import a standard curve for my real-time PCR data into the Design & Analysis Software (v2.x)?

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".

Can I import a standard curve for my real-time PCR data into the QuantStudio 12K Flex Software?

If you are analyzing results from the QuantStudio 12K Flex Real-Time PCR System, it is possible to import a standard curve into the QuantStudio 12K Flex Software following the instructions below:
1. Open the data file in the QuantStudio 12K Flex 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.

What is the difference between a Fast 96-well and standard 96-well block?

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.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.

Do you offer Cy5 dye and Texas Red calibration plates for the QuantStudio 12K Flex Real-Time PCR System 384-well block?

We do not offer Cy5 or Texas Red 384-well calibration plates for the QuantStudio 12K Flex Real-Time PCR System. This would require a custom dye calibration. Please contact our specialty oligos team at specialty_oligos@thermofisher.com to obtain the Cy5 and Texas Red dye used in the custom dye calibration. Upon receiving the dye, please add 10 µL of the dye to each well, seal the plate, and follow the instruction beginning at the bottom of page 7 in the User Bulletin (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/CustomDyeCalibration_AB_RealTimePCR_UB.pdf).

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.

What type of Real-Time PCR (Sequence Detection System) information is available from Applied Biosystems?

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.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.