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Instrument and Consumables

For instrument specifications, please see Appendix D, “Specifications and Layout”, in the QuantStudio™ 3D Digital PCR System User Guide for more details.

The QuantStudio™ 3D Digital PCR 20K Chip is a 10 mm2 high-density reaction plate that consists of a single array of 20,000 reaction wells, where each well can accommodate an 865 pL reaction. Hydrophobic coatings on the chip surface enable loading and isolation of independent PCR reactions within the reaction wells. The image below is taken from the QuantStudio™ 3D Digital PCR System User Guide (page 9).

The QuantStudio™ 3D Digital PCR System features a filter set that is optimized for the FAM™, VIC®, and ROX™ dyes available from Life Technologies.

Use all of the immersion fluid within 60 minutes of uncapping the syringe. Once a syringe is opened, you cannot reattach the cap for later use.

The Tilt Base inclines the thermal cycler sample block by 11°, the angle required for optimal performance of the QuantStudio™ 3D Digital PCR 20K Chips.

You can configure the QuantStudio™ 3D instrument to upload the imaging data to a USB drive, a network file server, or the cloud data storage service. See Appendix B, “Networking”, in the QuantStudio™ 3D Digital PCR System User Guide for more information on setting up network destinations. Also, the QuantStudio™ 3D instrument retains a copy of the analyzed data from the imaged QuantStudio™ 3D Digital PCR 20K Chip that you can access from the Run History screen. Note: The results will remain in the QuantStudio™ 3D instrument cache for approximately 300 readings. After 300 chips, the instrument removes the oldest data file in the cache to store each new reading.

The UV glue is usable for 3 months after first opening, when stored properly. Keep it stored in an amber bag while using it on the bench, and in an amber bag in a dark place between uses.

No, the current v1 Master Mix can only be used with the older Chips.

No, the v2 Master Mix can only be used with the v2 Chips.

We recommend you upgrade the firmware to v3.0 in order to read the 2D barcode; otherwise the .eds files will not have a Chip ID.

No. No matter which version of Chip Lid is being used, v2 Chips require Firmware v3.0 for proper data analysis.

Yes, Firmware v3.0 will work with all versions of Chips and Lids.

Yes, internal studies have shown comparable performance between v1 and v2 Chips.

At low concentrations, there is no impact on the data. At higher concentrations (>2000 copies/L), there is a slight impact. Using the Poisson Plus algorithm will correct for well-to-well volume variation.

Operation

The QuantStudio™ 3D Digital PCR Chips are meant to be used on a Dual Flat Block GeneAmp® 9700 PCR System or the ProFlex™ 2x Flat PCR System.

The QuantStudio™ 3D instrument only performs the imaging and primary analysis of the digital chips. The chips themselves must be cycled offline on a Dual Flat Block GeneAmp® 9700 PCR System or the ProFlex™ 2x Flat PCR System.

The system is meant to be used with the QuantStudio™ 3D Digital Master Mix only.

Yes, The GeneAmp® 9700 PCR System must be running firmware version 3.12 or later to cycle QuantStudio™ 3D Digital PCR 20K Chips. The firmware version of your GeneAmp® 9700 PCR System is shown on the main display after you first turn on the instrument. If needed, download and upgrade the firmware.

For the ProFlex™ 2x Flat PCR System, make sure you have v1.4.1 or later, so that you have the correct program to run the QuantStudio™ 3D Digital PCR 20K Chips. If needed, download and upgrade the firmware.

Yes. Never handle QuantStudio™ 3D Digital PCR 20K Chips or Chip Case Lids without gloves. Oils from your hands can contaminate the components and interfere with thermal cycling and imaging. For more instructions, please see the guidelines in Chapter 2 in the QuantStudio™ 3D Digital PCR System User Guide on loading the digital chips.

Using the Chip Carrier on either the ProFlex™ 2x Flat PCR System or the GeneAmp® 9700 Dual Flat Block, up to 24 QuantStudio™ 3D Digital Chips can be run at one time. Please refer to Chapter 3 in the QuantStudio™ 3D Digital PCR System User Guide for more details on loading the chips for thermal cycling.

After thermal cycling, the QuantStudio™ 3D Digital PCR 20K Chips can remain on the sample block for up to 24 hours. Read the QuantStudio™ 3D Digital PCR 20K Chips within 1 hour after you remove them from the GeneAmp® 9700 PCR System sample block.

The QuantStudio™ 3D Digital PCR System can read the digital chip in less than 1 minute. In fact, if you are running multiple chips, you can run the next chip in the series when the instrument touch screen displays the Analyzing Chip screen.

Please follow the procedures described starting in Appendix B of the QuantStudio™ 3D Digital PCR System User Guide to connect the instrument to a network.

You should use the Poisson Plus algorithm for projects that contain QuantStudio™ 3D Chips with target quantities >2000 copies/μL.

The Poisson Plus algorithm corrects for well-to-well load volume variation, on a per Chip basis. This becomes important at higher target concentrations.

Yes, there is an option to export the Chip data as XML on the Export tab in AnalysisSuite™ software v3.0.

Sample Preparation

The current QuantStudio™ 3D Digital PCR Chip allows for one sample per chip; however, duplexing allows analysis of two targets per chip.

Please see the section on Sample Dilution in Chapter 2 of the QuantStudio™ 3D Digital PCR System User Guide for more details on how to determine the target copy number per genome.

This will depend on the concentration range of the sample used. The QuantStudio™ 3D Digital PCR System has application- and concentration-specific requirements for technical replicates. At a low concentration, more replicates will be needed to have confidence in the data. Also, due to the nature of the application, replicates would be needed when studying rare mutations.

The answer is that sample prep for digital PCR is no different from real-time PCR when using the QuantStudio™ 3D Digital PCR System. If you have samples in your freezer that you’ve run on real-time PCR, then you’re ready to run them with digital PCR.