Notice:

We are obtaining a Thermo Fisher QuantStudio Absolute Q dPCR System in mid-July for multi-users and will be retiring our Bio-Rad QX200 Droplet Digital system as a service in the near future. If you are starting a new digital PCR project contact Anne-Marie Girard  for more information about setting up your project for the new system.

Information Pertinent to the Bio-Rad System Below

The Bio-Rad QX200™ AutoDG™ Droplet Digital™ PCR System is a technology that provides high-precision with absolute quantification of nucleic acid targets.  Droplet Digital PCR (ddPCR) affords scientists the ability to quantify template molecules that may be undetectable using traditional techniques.  Researchers are utilizing ddPCR to quantify genomic alterations, such as copy number variation, detect rare sequences, and determine absolute quantification of target DNA copies without the need for a standard curve.

Bio-Rad has more information available:

  • Overview page
  • Webinars
  • Applications
    • Liquid Biopsy
    • Copy Number Variation (CNV)
    • Rare Sequence Detection
    • Gene Expression
    • Single-Cell Analysis
    • Pathogen Detection and Microbiome Analysis
    • Next Generation Sequencing (NGS)
  • ddPCR Assays

The system includes an Automated Droplet Generator (ADG) that generates 20,000 droplets from one ddPCR reaction containing your target or background DNA. Samples are processed 8 at a time. Hydrolysis probes (Taqman) or EvaGreen (SYBR-like) chemistry can be used on the system.  After PCR the samples are transferred to a QX 200 Droplet Reader where individual droplet intensities are read.

The Bio-Rad QX200™ AutoDG™ Droplet Digital™ PCR System is located in ALS 3012. The system can be operated as a Multi-user instrument, or Full service with sample dropoff for CQLS to run. Run times need to be coordinated with CQLS Core staff during lab open hours.

Service maintained by Anne-Marie Girard.

Process of ddPCR

Set up ddPCR reaction mix with primers or probes, sample, Bio-Rad supermix (probes or EvaGreen).  More information about setting up reactions.

Load sample plate on Bio-Rad Automated Droplet Generator.  20,000 droplets are generated in each well distributing target and background DNA into droplets and placed in a new plate. Oil type corresponds to either Probes or EvaGreen chemistry.

Seal plate immediately with foil in plate sealer to prevent evaporation of oil.

Run sample reactions on PCR using a slow ramp time of no more than 2-2.5°C/second.

Load PCR plate in QX200 Droplet reader. All droplets are read and counted. Analyze concentrations with QuantaSoft™ software. A copy of the software for use in your lab can be requested from CQLS staff. 

Services

There are three different ways the instrument can be run:

Full Service - Core Reaction plus Run: Investigator drops off samples along with primers and probes.  CQLS staff set up the reactions and run them on the ADG, PCR and droplet reader

Partial Service – Core Run:  Investigator sets up reactions and hands plate to CQLS staff to run on the ADG, our PCR and droplet reader with CSV file of information related to samples. Time of run must be coordinated with Staff and instrument availability.

Multi-User Service - Multiuser Run: Investigator reserves time to run system, sets up reactions, receives enough consumables to run ADG with their samples (oil, DG32™ Automated Droplet Generator Cartridges, specific tips for ADG, plate, foil), sets up droplets on ADG, seals plate, runs PCR on their own thermal cycler, reads droplets on QX200 Digital Droplet Reader. User must be trained by CQLS staff before using system.

Setup Information

Very Important Consumable Requirements

The instrument requires very specific consumables, plates, tips, oil, Supermix.  Not using the required supplies can damage the instrumentation severely.

Absolutely no SYBR!    It will destroy the droplet reader!

CQLS supplies the required plastics and oil which is recovered in the fees.  Supermix is included in the full service fee structure.  For multi-user service you may purchase the supermix yourself.  Consult with staff to make sure you have the correct reagents ahead of time. 

Multi-User Run

After scheduling your run with CQLS staff and submitting an order to Web Order, you may pick up your consumables to run your samples. Check in with staff before the run to let them know the number of samples you will be running and to start access to the instrument. 

Reserve time to use instrument on the calendar. Time requested will show up after approved. 

You must receive training by CQLS staff prior to operating this instrument independently.  Please contact staff to schedule training.

Service maintained by Anne-Marie Girard.

Example Sample Setup

Here is an example of setup of samples, NTC, positive control setup, to be run in triplicate. ddPCR – Sample Preparation Outline.

 Reaction Considerations

  • Samples should be arranged in columns and in an Eppendorf twin.tec semi-skirted 96 well plate. (example: catalog #951020362).  Other plates will not work with the ADG. Plates are available from CQLS online store on an individual basis if needed.
  • Set up of ddPCR reactions similar to qPCR in initial experimental setup parameters.
  • Cut the DNA to make it easier for the enzyme to find templates. You can try reactions without cutting first, but cutting often will increase “yield”. Is also recommended for regular qPCR. Use NEB website to search for sites that don’t cut your region of interest.
  • Run should include: No Template Control, Negative Control, Positive control (aim for 100,000 copies of Target) in triplicate.
  • Set up positive control spatially far from NTC and Negative control in the plate.
  • Run dilutions of target to determine optimal concentration. Run triplicates (to control for user pipetting error and instrumentation variability.) Dilutions get rid of inhibitors and can make ddPCR more sensitive than qPCR. Optimal target concentration is 1-5 copies/droplet. May be able to get rid of triplicates once best dilution determined for assay.
  • Make as big a master mix as possible. Avoid pipetting 1 µl. Never make individual reactions.
  • Very important to thoroughly mix master mix. Vortex master mix and vortex after samples added. Thermal mixing in tube will not distribute master mix as in regular PCR.
  • Droplet reader uses negative control to do calculation, set up threshold
  • You must use Bio-Rad reagents! Do not use SYBR green! Its fluorescence will destroy the droplet reader and your PI will be responsible for replacement. Use EvaGreen instead.
  • You may set up your reactions in your own lab and walk them over here.
  • Strongly suggest Rainin tips or some other high quality tip. Low quality tips have sheared ends which can have small fragments that interfere with the droplet generation.
  • Recommended amplicon length is 60-200 bp. BioRad has gotten 1.5KB EvaGreen and 4.4KB Taqman probes to work in-house. Out in the field have seen 2500KB EvaGreen and 5000KB Probes.
  • Use FAM and HEX, or FAM and VIC. Probe is hydrolyzed during hybridization
  • ddPCR™ Supermix for Probes (No dUTP) gives stronger reactions that the version with dUTP.
  • Optimization of reaction: Annealing temperature could be changed +/- 10°C from the 60°C. Also can change primer and probes. Make serial dilutions to find the optimal concentration of template.
  • SYBR based chemistry (EvaGreen) with primers constructed 100-200 bases apart may possibly be optimized to see different populations in the results.
  • Assay design: Default concentration for primer 900nm to drive reaction to completion.

Data Analysis

Analyze concentrations with QuantaSoft™ software. A copy of the software for use in your lab can be requested from CQLS staff.

Bio-Rad also has a list of digital PCR publications.

Some Guides

Droplet Digital™ PCR  Applications Guide 

Rare Mutation Detection Best Practices Guidelines 

QuantaSoft Manual 

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