Bio-Rad QX200 User Manual
Automated Droplet Generator
Instruction Manual
Catalog #186-4101
Bio-Rad Technical Support
For help and technical advice, please contact the Bio-Rad Technical Support department. In the United
States, the Technical Support department is open Monday–Friday, 5:00 AM–5:00 PM, Pacific time.
Phone: 1-800-424-6723
Fax: 1-510 -741-5802
Email: LSG_TechServ_US@bio-rad.com (for U.S. and international customers)
Online technical support and worldwide contact information are available at www.consult.bio-rad.com.
Legal Notices
No part of this publication may be reproduced or transmitted in any form or by any means, electronic or
mechanical, including photocopy, recording, or any information storage or retrieval system, without permission
in writing from Bio-Rad Laboratories.
Bio-Rad reserves the right to modify its products and services at any time. This instruction manual is subject
to change without notice. Although prepared to ensure accuracy, Bio-Rad assumes no liability for errors, or for
any damages resulting from the application or use of this information.
FAM and VIC are trademarks of Applera Corporation.
TaqMan is a trademark of Roche Molecular Systems, Inc.
twin.tec is a trademark of Eppendorf, AG.
EvaGreen is a trademark of Biotium, Inc. Bio-Rad Laboratories, Inc. is licensed by Biotium, Inc. to sell reagents
containing EvaGreen dye for use in real-time PCR, for research purposes only.
Bio-Rad’s thermal cyclers and real-time thermal cyclers are covered by one or more of the following U.S.
patents or their foreign counterparts owned by Eppendorf AG: U.S. Patent Numbers 6,767,512 and 7,074,367.
This product and/or its use is covered by claims of U.S. patents, and/or pending U.S. and non-U.S. patent
applications owned by or under license to Bio-Rad Laboratories, Inc. Purchase of the product includes a
limited, non-transferable right under such intellectual property for use of the product for internal research
purposes only. No rights are granted for diagnostic uses. No rights are granted for use of the product for
commercial applications of any kind, including but not limited to manufacturing, quality control, or commercial
services, such as contract services or fee for services. Information concerning a license for such uses can be
obtained from Bio-Rad Laboratories. It is the responsibility of the purchaser/end user to acquire any additional
intellectual property rights that may be required.
Copyright © 2014 by Bio-Rad Laboratories, Inc. All rights reserved.
Instruction Manual | i
Safety and Regulatory Compliance
This instrument has been tested and found to be in compliance with all applicable requirements of the
following safety and electromagnetic standards:
IEC 61010-1:2010 (3rd ed.), EN61010-1:2010 (3rd ed). Electrical Equipment for Measurement, Control,
and Laboratory Use — Part 1: General requirements
EN 61326-1:2006 (Class A). Electrical equipment for measurement, control, and laboratory use. EMC
requirements, Part 1: General requirements
UL 61010-1:2004, Laboratory equipment, Test & Measurement Equipment and Industrial Process Controls
CAN/CSA 22.2 No 61010-1-04, Safety Requirements for Electrical. Equipment for Measurement,
Control, and Laboratory Use, Part I: General. Requirements
This equipment generates, uses, and can radiate radiofrequency energy and, if not installed and used in
accordance with the instruction manual, may cause harmful interference to radio communications. Operation
of this equipment in a residential area is likely to cause harmful interference, in which case the user will be
required to correct the interference at his own expense.
The CE mark indicates that the manufacturer ensures the product conforms with the essential
requirements of the applicable EN directives.
The CSA mark indicates that the product has been tested to Canadian and U.S. standards,
and it meets the requirements of those applicable standards.
This equipment has been tested and found to comply with the limits for a Class A digital
device pursuant to Part 15 of the FCC Rules. These limits are designed to provide
reasonable protection against harmful interference when the equipment is operated in a
commercial environment.
The Waste Electrical and Electronic Equipment Directive symbol indicates that when the enduser wishes to discard this product, it must be sent to separate collection facilities for recovery
and recycling.
This instrument is for use only by trained personnel.
Do not position the equipment so that it is difficult to operate the plug of the power supply.
The plug of the power supply is the disconnect device.
No serviceable parts inside.
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Instrument Safety Warnings
Alteration of this instrument voids the warranty and safety certification and creates a potential safety hazard.
This instrument is intended for laboratory use only. Bio-Rad Laboratories is not responsible for any injury
or damage caused by use of this instrument for purposes other than those for which it is intended, or by
modifications of the instrument not performed by Bio-Rad Laboratories or an authorized agent. Follow the
safety specifications listed here and throughout this manual. Use only the power cord supplied with the
instrument, using only the plug adapter that corresponds to the electrical outlets in your region. Use of
unapproved supermixes may harm the instrument and voids the warranty.
When lifting, two people are required. Grip from the underside, one person on each opposite side.
After unpacking for placement of equipment, transport per lifting instructions; place near grounded outlet with
plug of power supply accessible.
PPE (Personal Protective Equipment) Training
Proper use of gloves is recommended with use of oils and sample plates. OSHA requirements for PPE are set
forth in the Code of Federal Regulations (CFR) at 29 CFR 1910.132 (General requirements); 29 CFR 1910.138
(Hand protection); 29 CFR 1926.95 (Criteria for standard personal protective equipment). Any gloves with
impaired protective ability should be discarded and replaced. Consider the toxicity of the chemicals and
factors such as duration of exposure, storage, and temperature when deciding to reuse chemically exposed
gloves. Features to aid glove selection for handling of machines, assays, oils, and cleaning solvents:
Butyl gloves are made of a synthetic rubber and protect against peroxide, hydrofluoric acid, strong
bases, alcohols, aldehydes, and ketones
Natural (latex) rubber gloves are comfortable to wear and feature outstanding tensile strength, elasticity,
and temperature resistance
Neoprene gloves are made of synthetic rubber and offer good pliability, finger dexterity, high density,
and tear resistance; they protect against alcohols, organic acids, and alkalis
Nitrile gloves are made of copolymer and provide protection from chlorinated solvents such as
trichloroethylene and tetrachloroethene; they offer protection when working with oils, greases, acids, and
caustic substances
Transporting the Instrument
Avoid dropping or subjecting the instrument to shock when transporting.
Biohazards
If biohazardous samples are present, adhere to the following guidelines and comply with any local guidelines
specific to your laboratory and location.
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General Precautions
Always wear laboratory gloves, coats, and safety glasses with side shields or goggles
Keep your hands away from your mouth, nose, and eyes
Completely protect any cut or abrasion before working with potentially infectious materials
Wash your hands thoroughly with soap and water after working with any potentially infectious material
before leaving the laboratory
Remove wristwatches and jewelry before working at the bench
Store all infectious or potentially infectious material in unbreakable leak-proof containers
Before leaving the laboratory, remove protective clothing
Do not use a gloved hand to write, answer the telephone, turn on a light switch, or touch anything that
other people may touch without gloves
Change gloves frequently. Remove gloves immediately when they are visibly contaminated
Do not expose materials that cannot be properly decontaminated to potentially infectious material
Upon completion of the operation involving biohazardous material, decontaminate the work area with an
appropriate disinfectant (for example, a 1:10 dilution of household bleach)
No biohazardous substances are exhausted during normal operations of this instrument
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Table of Contents
Chapter 1. QX200™ AutoDG™ Droplet Digital™ PCR System ...........1
1.1 Introduction ................................................... 1
1.2 System Components ........................................... 2
1.3 Installation and General Operation ................................. 4
1.4 System Setup and General Operation Instructions .....................5
Chapter 2. Using the Automated Droplet Generator .................7
2.1 Powering On the Automated Droplet Generator ....................... 7
2.2 Starting a Run on the AutoDG Instrument ........................... 9
2.3 Subsequent Steps ............................................ 20
2.4 Advanced Loading and Switching of Automated Droplet Generation Oils ... 20
2.4.1 Loading a Bottle of Oil before Configuring a Plate ................ 21
2.4.2 Changing between AutoDG Oil Types ......................... 23
2.5 Accessing Run Logs ........................................... 24
Chapter 3. Specifications and Maintenance ......................27
3.1 Specifications ................................................27
3.2 Maintenance .................................................28
3.2.1 General Care ............................................ 28
3.2.2 Software and Firmware Updates ............................. 28
3.2.3 Cleaning the Oil Waste Reservoir............................. 29
Chapter 4. Automated Droplet Generator Troubleshooting ..........31
4.1 Restarting a Run after an Error ................................... 31
4.2 Restarting a Run after a System Error ............................. 32
4.3 Exporting Logs for Technical Support..............................33
4.4 Additional Troubleshooting ...................................... 34
Ordering Information .........................................35
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1
QX200™ AutoDG™ Droplet Digital™ PCR System
1.1 Introduction
Bio-Rad’s QX200 AutoDG Droplet Digital PCR (ddPCR™) system combines
water-oil emulsion droplet technology with microfluidics to perform accurate and
precise digital PCR. The system consists of two instruments — an Automated
Droplet Generator and a Droplet Reader — and associated consumables. The
Automated Droplet Generator partitions each sample into ~20,000 uniform
nanoliter-sized droplets in which nucleic acid molecules are distributed in a
random fashion. Each droplet serves to partition the reactions. The 96-well PCR
plate of droplets is removed from the AutoDG Instrument, sealed, and PCR
is performed to end point in a thermal cycler. Droplets from each sample are
analyzed individually on the QX200 Droplet Reader; the droplets stream single
file through the reader for fluorescence analysis.
PCR-positive and PCR-negative droplets are counted to provide absolute
quantification of target DNA in digital form. Positive droplets, which contain
at least one copy of the target DNA or RNA molecule, exhibit increased
fluorescence compared to negative droplets. The fraction of PCR-positive
droplets enables the target to be quantified according to the Poisson
distribution. Alternatively, amplified products can be extracted from droplets
following PCR for downstream applications, such as sequencing or cloning.
The QX200 System works with both hydrolysis probes and EvaGreen
fluorescence detection chemistries, and its flexible design allows for high
throughput and ultra-sensitive detection.
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QX200 AutoDG Droplet Digital PCR System
The ddPCR System lets you:
Achieve absolute quantification without the use of a standard curve
Detect rare DNA target copies with unmatched sensitivity
Determine copy number variation with unrivaled accuracy
Measure gene expression levels with precision
Design scalable assays for high sensitivity or high throughput
Expand applications using flexible ddPCR chemistry — EvaGreen or probes
Applications and uses include:
Copy number variation
Rare sequence detection
Gene expression analysis
Next-generation sequencing (NGS) library
quantification
Viral load determination
This manual covers use of the Automated Droplet Generator and preparation for PCR. For information on the QX200
Droplet Reader, please refer to the QX200 Droplet Reader Instruction Manual, part number 10031906.
Single cell gene expression analysis
Absolute quantification
Rare mutant detection
miRNA analysis
NGS sample preparation
GMO detection
1.2 System Components
Bio-Rad’s Automated Droplet Generator simplifies the Droplet Digital PCR workflow, making digital PCR both
scalable and practical. The AutoDG Instrument prepares droplets for up to 96 samples at a time with minimal
hands-on time required. In less than 45 minutes, a plate of droplets can be generated. The plate can then be
sealed, thermal cycled, and ddPCR analysis can be performed on either a QX100
Automated droplet generation eliminates user-to-user variability that can be associated with manual droplet
generation. The AutoDG Instrument’s HEPA-filtered enclosure reduces contamination during droplet
generation. Consumables are automation-friendly with SBS standard access.
2 | Automated Droplet Generator
™
or QX200 Droplet Reader.
1.2 System Components
The system’s guided quick load allows users to get started faster. The large, color touch screen provides simple
setup and plate tracking. Select the desired number of columns across a 96-well plate, load Droplet Generation
Oil and only the consumables you need as prompted by the system, and start the run. A countdown timer
will display the time remaining so users can maximize their time in the lab. When the plate is ready, users are
notified and a time-elapsed counter begins. Every run is stored in an exportable log file for future reference.
Following reaction preparation using ddPCR supermix, 22 µl of each of up to 96 prepared samples (or blanks) in
a 96-well plate are loaded into the Automated Droplet Generator. A bottle of the Automated Droplet Generation
Oil is loaded into the instrument, along with DG32
™
Cartridges and Pipet Tips for the AutoDG Instrument. There,
the samples and oil are combined within the microchannels of the cartridge to create an emulsion of ~20,000
monodisperse, nanoliter-sized droplets for each of the samples. Following automated droplet generation, the
96-well PCR plate of droplets is sealed, and PCR is performed to end point in a thermal cycler.
When cycling is complete, the plate is loaded into the QX200 Droplet Reader. The droplet reader sips each
sample, singulates the droplets, and streams them in single file past a two-color detector. The detector reads
each droplet and determines which contain a target (+) and which do not (–). If quantification of droplets is not
required, PCR products can be extracted from droplets following thermal cycling for downstream applications,
such as sequencing or cloning.
The Automated Droplet Generator includes the components listed in Table 1. Additional requirements for the
Droplet Digital PCR workflow (automated droplet generation, sealing, cycling, and reading) are listed in Table 2.
Table 1. Automated Droplet Generator components. Items shipped with the Automated Droplet Generator
(catalog #186-4101).
Component Description Catalog #
Automated Droplet Generator Instrument used for Automatic Droplet Generation 186-4101
Power cord Connects Automated Droplet Generator to power source Call technical support
Cooling block accessory Prevents evaporation during droplet generation Call technical support
Oil waste reservoir Collects oil waste from priming and flushing Call technical support
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QX200 AutoDG Droplet Digital PCR System
Table 2. Additional materials required for Droplet Digital PCR.
Component Description Catalog #
Automated Droplet Generator
DG32 Cartridges Microfluidic cartridge with gasket used to mix sample and 186-4108, 186-4109
Droplet Generation Oils Automated Droplet Generation Oil for EvaGreen 186-4112
Automated Droplet Generation Oil for Probes 186-4110
PCR Supermixes QX200
186-4036
ddPCR™ Supermix for Probes (no dUTP) 186-3023, 186-3024, 186-3025
ddPCR
186-3028
One-Step RT-ddPCR Kit for Probes 186-3021, 186-3022
Controls QX200 Buffer Control Kit for EvaGreen 186-4052
QX200 Buffer Control Kit for Probes 186-3052
Pipet Tips Pipet Tips for Automated Droplet Generator 186-4120, 186-4121
Pipet Tip Bin Pipet Tip Waste Bins for AutoDG 186-4125
96-well PCR Plates twin.tec 96-well semi-skirted plates Eppendorf 951020362
Sealer and Thermal Cycler
Foil Seal Pierceable Foil Heat Seal 181-4040
Plate Sealer PX1
Thermal Cycler C1000 Touch™ Thermal Cycler with 96-deep well 185-1197
reaction module
QX200 Droplet Reader*
Droplet Reader Oil ddPCR Droplet Reader Oil 186-3004
* For information on components shipped with QX200 Droplet Reader, see part number 10031906.
oil to generate droplets in the Automated Droplet Generator
™
ddPCR™ EvaGreen® Supermix 186-4033, 186-4034, 186-4035,
™
Supermix for Probes 186-3026, 186-3010, 186-3027,
™
PCR Plate Sealer 181-4000
1.3 Installation and General Operation
Automated Droplet Digital PCR involves the following steps (4.5–5 hours for the complete workflow):
1. Prepare PCR-ready samples — combine nucleic acid sample (DNA or RNA), primers, and probes (FAM,
VIC, or HEX) or intercalating dye (EvaGreen) with Bio-Rad ddPCR supermix (see Table 2).
2. Make droplets — load 22 μl of the ddPCR reaction into a 96-well PCR plate, then load the plate and required
consumables into the Automated Droplet Generator to partition the sample into droplets. The Automated
Droplet Generator uses microfluidics to combine oil and aqueous sample to generate the nanoliter-sized
droplets required for ddPCR analysis. It processes up to 96 samples at a time in less than 45 min.
1.4 System Setup and General Operation Instructions
3. Perform PCR — remove the 96-well PCR plate containing droplets from the Automated Droplet Generator,
seal the plate with foil, and perform PCR to end point (~40 cycles) using a PX1
C1000 Touch
™
Thermal Cycler (see Table 2).
™
Plate Sealer and
4. Read droplets — load the plate into the QX200 (or QX100) Droplet Reader and start your run. The droplet
reader sips each sample, singulates the droplets, and streams them in single file past a two-color detector.
The detector reads the droplets to determine which contain a target (+) and which do not (–).
If reading or quantifying droplets and recovering material from droplets in parallel, prepare two sets of
reactions, one for each application. For example, a set of eight wells of droplets can be generated: four of
these will be read after thermal cycling, and four will not be read.
5. Analyze results — the droplet reader connects to a laptop computer running QuantaSoft
™
Software. The
software provides a complete set of tools for setting up and naming samples, running and controlling
the instrument, and analyzing results. It also reads the positive and negative droplets in each sample and
plots the fluorescence, droplet by droplet. The fraction of positive droplets in a sample determines the
concentration of target in copies/μl.
The QX200 ddPCR System is compatible with hydrolysis probe (TaqMan) chemistry and can detect up to
two fluorophores and up to four targets at a time (FAM/VIC or FAM/HEX). It is also compatible with EvaGreen
chemistry. Use only the approved Bio-Rad supermixes listed in Table 2 with this system; using unapproved
supermixes may harm the instrument and voids the warranty.
The QX100 ddPCR System does not support EvaGreen chemistry; running this chemistry will damage
the instrument.
1.4 System Setup and General Operation Instructions
Connect the Automated Droplet Generator to a power source using the power cord and power adapter
provided. Leave 2" (~5 cm) clear space behind and 5" (~13 cm) clear to the right and left of the instrument
for proper ventilation. Position the instrument such that it can be easily disconnected from the power
source, should that become necessary for servicing the equipment.
The Automated Droplet Generator is powered on by plugging it into a power source.
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Using the Automated
2
Droplet Generator
2.1 Powering On the Automated Droplet Generator
The Automated Droplet Generator (AutoDG™) is designed to remain powered
on in order to preserve positive airflow inside of the instrument and track
consumable use. The instrument stays in an idle state when not being used.
Please see Section 2.2 for instructions on starting a run from idle mode.
1. When powering on the Automated Droplet Generator, you will see a startup
screen while the instrument powers on and performs a self-check. Please
note that the door will automatically close.
Fig. 1. Star tup screen when instrument is powered on.
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