QX200 Droplet Digital PCR System: Instrument Familiarization and Standard Operation
1 Purpose
This procedure orients new personnel to the Bio-Rad QX200 Droplet Digital PCR (ddPCR) system and describes its safe, standard operation: the components of the system, the consumables and reagents, the four-stage workflow (droplet generation, plate sealing, thermal cycling, and droplet reading), basic use of QuantaSoft software, routine maintenance, and common troubleshooting.
It is a familiarization and operation SOP. It does not cover the science of assay or primer design (see SOP-L701) or the analysis and interpretation of results (see SOP-L702 Applications Guide). The workflow described here follows Bio-Rad’s published guidance for the platform (Bio-Rad Laboratories, 2018, 2024), which itself implements the droplet-counting principle of absolute quantification (Hindson et al., 2011).
2 Scope
- Applies to: Operation of the QX200 ddPCR system in the lab, comprising the QX200 Droplet Generator, the PX1 PCR Plate Sealer, the C1000 Touch Thermal Cycler with 96-deep-well reaction module, and the QX200 Droplet Reader, together with QuantaSoft software.
- Does not apply to: Assay/primer design (SOP-L701), data analysis and interpretation (SOP-L702), or DNA extraction (SOP-L700).
- Prerequisite: A prepared, validated reaction mix and plate plan from SOP-L701.
This instrument SOP is numbered L214, placing it ahead of the L700-series analysis SOPs in the lab scheme. Conceptually it precedes analysis: complete this familiarization before running samples for SOP-L702.
3 Responsibilities
| Role | Responsibility |
|---|---|
| Lab PI | Approve instrument access; oversee major maintenance |
| Lab Manager | Maintain consumable stock, oil, and service records |
| Trained Personnel | Operate the system per this SOP; log each run |
| New Students | Complete familiarization and supervised runs before independent use |
4 Definitions
- Amplitude: The fluorescence signal intensity of a droplet; positive droplets (containing target DNA) have high amplitude; negative droplets have low amplitude; a clear separation between positive and negative populations is required for accurate quantification.
- Droplet digital PCR (ddPCR): A method of absolute quantification of nucleic acids in which the sample is partitioned into ~20,000 nanoliter-sized water-in-oil droplets, each containing zero or one target molecule; after PCR amplification, the fraction of fluorescent positive droplets is used to calculate target concentration using Poisson statistics.
- FAM: A green fluorescent dye (fluorescein amidite) used to label one probe or primer in a duplex ddPCR assay; detected in the FAM channel (channel 1) by the QX200.
- HEX: A yellow-orange fluorescent dye used to label a second probe in a duplex ddPCR assay, or as an alternative to VIC; detected in channel 2 by the QX200.
- Poisson correction: A statistical adjustment applied by the DropletQuant software to account for the probability that some droplets contain more than one target molecule; necessary when target concentrations are high.
- Copies per microliter (copies/µL): The absolute concentration of the target DNA in the final ddPCR reaction, as reported by DropletQuant; must be back-calculated to the original sample concentration using the appropriate dilution factor.
- Threshold: The fluorescence amplitude boundary separating positive from negative droplets; set manually or automatically in the analysis software; incorrect threshold setting is the most common source of ddPCR quantification error.
5 Safety and Hazards
- Droplet generation oil and reader oil. Use the oil specified for the chemistry (probe vs. EvaGreen). Avoid skin and eye contact; wear gloves and eye protection; consult the SDS for each oil. Clean spills promptly.
- Reagents. ddPCR supermixes and any sample DNA are handled with gloves. Environmental soil DNA is low biohazard risk, but treat all extracts with standard lab precautions.
- Moving parts. The droplet reader autosampler moves during a run. Keep hands clear and the lid closed while running.
- Electrical and thermal. Standard bench-instrument electrical safety. The thermal cycler block reaches high temperatures; do not touch the block during or immediately after a run.
- Sharps / glass. None in normal operation; cartridges and plates are single-use plastics.
ddPCR requires Bio-Rad’s proprietary droplet-generation reagents, cartridges, and gaskets. Substituting non-specified consumables will produce unusable droplets (Bio-Rad Laboratories, 2018). Match the oil to the supermix chemistry.
6 Equipment, Consumables, and Reagents
6.1 Instruments
| Item | Role in workflow |
|---|---|
| QX200 Droplet Generator | Partitions each 20 uL reaction into ~20,000 droplets |
| DG8 cartridge holder | Holds the DG8 cartridge during generation |
| PX1 PCR Plate Sealer | Heat-seals droplets into the 96-well plate with pierceable foil |
| C1000 Touch Thermal Cycler (96-deep-well module) | Endpoint PCR amplification within droplets |
| QX200 Droplet Reader | Streams droplets single-file and reads two-color fluorescence |
| Instrument computer (QuantaSoft) | Plate setup, run control, thresholding, export |
6.2 Consumables and reagents
| Item | Notes |
|---|---|
| DG8 cartridges and gaskets | Single-use; one cartridge = 8 samples |
| Droplet generation oil (probes) or QX200 EvaGreen droplet oil | Match to supermix chemistry |
| Droplet reader oil | Loaded in the reader |
| ddPCR Supermix for Probes or QX200 ddPCR EvaGreen Supermix | Per assay (SOP-L701) |
| ddPCR 96-well plates and pierceable foil heat seals | For PX1 sealing and reading |
| Filtered pipette tips, low-bind tubes | Standard molecular-biology consumables |
Keep a stock log for cartridges, gaskets, oils, plates, and foil. The droplet generator and reader each draw oil from onboard bottles; check levels before every session.
Order supermixes, droplet oils, cartridges, plates, and validated PrimePCR ddPCR assays from the Bio-Rad digital assay catalog: https://www.bio-rad.com/digital-assays. Confirm chemistry (probe vs EvaGreen) before ordering so the supermix and matching oil are consistent.
7 Procedure
7.1 System Overview
The QX200 system performs absolute quantification by partitioning, amplifying, and counting (Bio-Rad Laboratories, 2018; Hindson et al., 2011). A single 20 uL reaction is split into roughly 20,000 uniform water-in-oil droplets. Each droplet is a tiny independent PCR. After endpoint amplification, the reader passes droplets one at a time through a two-color optical detector (channel 1 for FAM, channel 2 for HEX or VIC), scoring each droplet as positive or negative. QuantaSoft fits the positive fraction to a Poisson model to return target concentration in copies per microliter.
The work proceeds through four instruments in sequence:
- Droplet Generator (Section 6.4) makes the droplets.
- PX1 Plate Sealer (Section 6.5) seals them into a plate.
- C1000 Touch Thermal Cycler (Section 6.6) amplifies.
- Droplet Reader (Section 6.7) counts.
7.2 Daily Startup
- Power on the instrument computer and the instruments to be used.
- Launch QuantaSoft and confirm it connects to the droplet generator and/or reader.
- Check oil levels in the droplet generator and the reader; top up or replace bottles per the manual (Bio-Rad Laboratories, 2024).
- Empty the reader’s waste bottle if near full.
- Confirm consumable stock for the planned run (cartridges, gaskets, oil, plate, foil).
7.3 Consumables Orientation
Before the first run, identify each consumable and where it loads:
- DG8 cartridge seats in the cartridge holder. The middle row of wells receives sample; the bottom row receives generation oil; the top row collects the generated droplets.
- Gasket snaps over the loaded cartridge to seal it for generation.
- Generation oil is chemistry-specific. Using probe oil with EvaGreen supermix (or vice versa) will fail.
- Pierceable foil is the only seal type compatible with the reader’s autosampler; do not use hard-seal films.
7.4 Droplet Generation
- Prepare reactions per SOP-L701 and load into the cartridge holder with a DG8 cartridge.
- Pipette each sample into the sample wells (avoid bubbles; pipette to the first stop).
- Pipette the matched generation oil into the oil wells.
- Place the gasket over the cartridge.
- Load the holder into the QX200 Droplet Generator and close the lid; the run takes about two minutes per cartridge.
- After generation, slowly transfer the droplets from the top row of the cartridge to the corresponding wells of a ddPCR 96-well plate using a multichannel pipette. Pipette gently and at an angle; droplets are fragile and shearing them lowers the usable count.
Damaged or sheared droplets reduce accepted-droplet counts and can fail the QC threshold (see SOP-L702, Section 6.2). Pipette droplets slowly and avoid introducing air.
7.5 Plate Sealing (PX1)
- Place the plate and a sheet of pierceable foil in the PX1 Plate Sealer.
- Seal at the manufacturer-specified temperature and time (commonly 180 C for 5 s; confirm against the current manual (Bio-Rad Laboratories, 2024)).
- Inspect the seal: the foil should be uniformly bonded with no lifted edges.
7.6 PCR Amplification
- Transfer the sealed plate to the C1000 Touch with the 96-deep-well module.
- Run the validated cycling program for the assay (from SOP-L701).
- Set the ramp rate to about 2 C/s. This slower ramp is important for even amplification across the droplet population on this platform (Bio-Rad Laboratories, 2018).
- Include a final hold and store at 4 C if not reading immediately; read within the window specified for the chemistry.
A generic template program is given in Appendix B; the assay-specific anneal temperature and cycle number come from SOP-L701.
7.7 Droplet Reading
- Place the amplified, sealed plate in the QX200 Droplet Reader plate holder with the retaining clamp.
- In QuantaSoft, set up the plate layout: sample names, assay/target names, fluorophores (FAM, HEX/VIC), experiment type (e.g., absolute quantification), and supermix/chemistry.
- Start the run. The autosampler aspirates droplets well by well and the detector reads two channels per droplet.
- Do not open the reader or disturb the plate while running.
7.8 QuantaSoft Orientation (Reading the Output)
This is orientation only; thresholding decisions and full analysis are covered in SOP-L702.
- 1-D plot: fluorescence amplitude vs. droplet (event) number for one channel. Positive droplets sit above the threshold line; negatives below.
- 2-D plot: channel 1 (FAM) vs. channel 2 (HEX/VIC) amplitude. In a duplex assay, droplets fall into four clusters: double-negative, FAM-only, HEX-only, and double-positive (Bio-Rad Laboratories, 2018).
- Thresholding: the boundary between positive and negative clusters. Review it for every well; adjust manually in QuantaSoft Analysis Pro where the automatic call is poor.
- Well merging: replicate wells of the same sample/target may be merged to increase the effective partition count before concentration is calculated.
- Concentration: QuantaSoft reports copies per microliter from the Poisson fit. Export as described in SOP-L702, Section 6.1.
7.9 Shutdown
- Close the experiment and export/back up data (SOP-L702).
- Remove and discard the plate and used cartridges/gaskets (Section 8).
- Check and, if needed, empty the reader waste bottle.
- Power down per the manual; leave the system in the state your lab specifies for the reader oil lines.
7.10 Maintenance and Care
- Each session: check oil levels and the waste bottle; inspect for leaks.
- Routine: keep the reader’s fluidics maintained per the manual; do not let oil bottles run dry mid-run.
- Do not: force cartridges or gaskets, reuse single-use consumables, mix oil chemistries, or use non-pierceable seals.
- Records: log each run (date, operator, assay, plate, instrument notes) and any maintenance or service in the instrument logbook.
7.11 Troubleshooting
| Symptom | Likely cause | Action |
|---|---|---|
| Low accepted-droplet count | Sheared droplets; pipetting too fast; bubbles | Pipette droplets slowly at an angle; avoid air; re-generate |
| Few or no droplets generated | Wrong/insufficient oil; cartridge/gasket seated poorly | Use matched oil; reseat cartridge and gasket |
| “Rain” (droplets between clusters) | Suboptimal anneal temperature; degraded sample | Optimize anneal (SOP-L701); check DNA quality (SOP-L700) |
| Reader error / clog | Low oil; debris; fluidics issue | Check oil and waste; run reader maintenance; see manual |
| High NTC positives | Contamination | Rerun with fresh reagents; review setup (SOP-L702, Section 6.2) |
8 Quality Control and Acceptance Criteria
- Each well should yield at least 10,000 accepted droplets (target ~12,000 to 20,000); wells below 10,000 are excluded in analysis (SOP-L702).
- Run the controls defined in SOP-L702 (NTC, extraction blank, positive control) on every plate.
- Confirm uniform plate sealing and correct plate orientation before reading.
- Log instrument run metadata to support digital-PCR reporting standards (The dMIQE Group & Huggett, 2020).
9 Waste Disposal
- Used DG8 cartridges, gaskets, plates, foil, and tips: dispose as standard laboratory plastic/biohazard waste per the lab’s waste plan.
- Generation and reader oil and oily waste: collect and dispose per the SDS and the lab’s chemical-waste procedures; do not pour oil down the drain.
- Reagent containers: rinse and dispose per local guidance.
10 References
For peer-reviewed studies that used ddPCR, including environmental and microbial applications useful when validating an assay or interpreting low target concentrations, search Bio-Rad’s ddPCR publication database: https://www.bio-rad.com/ddPCR/publications.
11 Training Record
Before operating the QX200 system independently, personnel must:
- Read this SOP and review the current Bio-Rad QX200 manual (Bio-Rad Laboratories, 2024) and Applications Guide (Bio-Rad Laboratories, 2018).
- Read SOP-L701 Primer Design and SOP-L702 Applications Guide for context on what they are running and why.
- Observe a full run (generation, sealing, cycling, reading) performed by trained personnel.
- Complete at least one full run under supervision, including QuantaSoft plate setup and export.
- Be signed off by the PI or lab manager.
| Trainee | Date observed | Date supervised run | Signed off by | Date |
|---|---|---|---|---|
12 Appendix A: Pre-Run Checklist
13 Appendix B: Generic Thermal Cycling Template
Anneal/extension temperature and cycle number are assay-specific and come from SOP-L701. The structure below is a starting template for probe-based ddPCR; confirm against the supermix insert and current manual (Bio-Rad Laboratories, 2024).
| Step | Temperature | Time | Cycles | Ramp |
|---|---|---|---|---|
| Enzyme activation | 95 C | 10 min | 1 | ~2 C/s |
| Denaturation | 94 C | 30 s | 39–40 | ~2 C/s |
| Anneal/extension | (assay) | 1 min | 39–40 | ~2 C/s |
| Enzyme deactivation | 98 C | 10 min | 1 | ~2 C/s |
| Hold | 4 C | hold | 1 | ~2 C/s |
EvaGreen chemistry uses a different supermix, oil, and cycling profile than probe chemistry. If the lab runs EvaGreen assays, add a separate template here once the standard profile is fixed in SOP-L701.