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: [phone]

Fax: [phone]

Email: [e-mail] (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.

ii  |  Automated Droplet Generator

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.

Instruction Manual  |  iii

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

iv  |  Automated Droplet Generator

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

2.1Powering On the Automated Droplet Generator . . . . . . . . . . . . 7

2.2Starting a Run on the AutoDG Instrument . . . . . . . . . . . . . . 9

2.3 Subsequent Steps . . . . . . . . . . . . . . . . .         20

2.4Advanced 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

3.2.2Software and Firmware Updates . . . . . . . . . . . . . . 28

3.2.3Cleaning 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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vi  |  Automated Droplet Generator

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

■■Single cell gene expression analysis

■■Absolute quantification

■■Rare mutant detection

■■miRNA analysis

■■NGS sample preparation

■■GMO detection

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.

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™ or QX200 Droplet Reader.

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.

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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
	oil to generate droplets in the Automated Droplet Generator
Droplet Generation Oils	Automated Droplet Generation Oil for EvaGreen	186-4112
	Automated Droplet Generation Oil for Probes	186-4110
PCR Supermixes	QX200™ ddPCR™ EvaGreen® Supermix	186-4033, 186-4034, 186-4035,
		186-4036
	ddPCR™ Supermix for Probes (no dUTP)	186-3023, 186-3024, 186-3025
	ddPCR™ Supermix for Probes	186-3026, 186-3010, 186-3027,
		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™ PCR Plate Sealer	181-4000
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.

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.

4  |  Automated Droplet Generator

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™ Plate Sealer and

C1000 Touch™ Thermal Cycler (see Table 2).

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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6  |  Automated Droplet Generator

2	Using the Automated
	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. Startup screen when instrument is powered on.

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