Bio-Rad ddPCR Supermix for Probes User Manual

QX200™ Droplet Reader and
QuantaSoft™ Software

Instruction Manual

Catalog #186-4001, 186-4003

Preface

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.

Excel and Microsoft are trademarks of Microsoft Corporation. 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.

Practice of the polymerase chain reaction (PCR) may require a license.

Copyright © 2013 by Bio-Rad Laboratories, Inc. All rights reserved.

QX200 Droplet Reader and QuantaSoft Software Instruction Manual | i

Preface

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 61010-1:2001 (2nd ed.), EN61010-1:2001 (2nd 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

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 EC directives.

The CSA mark indicates that a product, process, or service has been tested to a Canadian or
U.S. standard and it meets the requirements of the applicable CSA standard.

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
end-user wishes to discard this product, it must be sent to separate collection facilities for
recovery and recycling.

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 adaptor that corresponds to the electrical outlets in your region. Use of
unapproved supermixes may harm the instrument and voids the warranty.

QX200 Droplet Reader and QuantaSoft Software Instruction Manualii |

PrefacePreface

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

QX200 Droplet Reader and QuantaSoft Software Instruction Manual | iii

Preface

QX200 Droplet Reader and QuantaSoft Software Instruction Manualiv |

Contents

Chapter 1 QX200™ Droplet Digital™ PCR System 1

1.1 Introduction
1

.2 System Components

1

.3 Droplet Digital PCR Workflow 4

1.4 System Setup and General Operation Instructions

1
2

4

C

hapter 2 Using the QX200 Droplet Reader

C

hapter 3 Using QuantaSoft

3.1 Setup

3.1.1 Using the Well Editor

™

Software 9

1
1

3.1.2 Using the Experiment Editor 12

3.1.3 Using the Advanced Options

3.2 Run

1
1

3.3 Analyze 14

3.3.1 Viewing Channel Data (1D Amplitude)

3.3.2 Viewing Clustering Plots (2D Amplitude)

3.3.3 Viewing Concentration Data (Concentration)

1
1
1

3.3.4 Viewing Copy Number Data (Copy Number) 19

3.3.5 Viewing Ratio Data (Ratio)

3.3.6 Viewing Events

Chapter 4 Specifications and Maintenance

4.1 Specifications

4.2 Maintenance

4.2.1 General Maintenance Procedures

4.2.2 Replacing Droplet Reader Oil and Removing Waste

Appendix A Ordering Information

2
2

2

2
2
2
2

23

5

0
1

3
3

6
8
9

0
0

1

1
2
2
2

QX200 Droplet Reader and QuantaSoft Software Instruction Manual | v

QX200 Droplet Reader and QuantaSoft Software Instruction Manualvi |

QX200™ Droplet Digital™

1

PCR System

1.1 Introduction

The QX200 Droplet Digital PCR (ddPCR™) system performs accurate and
precise digital PCR. The system consists of two instruments, the QX200 droplet
generator and the QX200 droplet reader, and their associated consumables.
The QX200 droplet generator partitions samples into 20,000 nanoliter-sized
droplets and, after PCR on a thermal cycler, droplets from each sample are
analyzed individually on the QX200 droplet reader. PCR-positive and PCR­negative droplets are counted to provide absolute quantification of target DNA
in digital form. Alternatively, amplified products can be extracted from droplets
following PCR for downstream applications, such as sequencing or cloning.

The ddPCR system lets you:



Detect rare DNA target copies with unmatched sensitivity



Determine copy number variation with unrivaled accuracy



Measure gene expression levels with precision

Applications and uses include:



Copy number variation



Rare sequence detection



Gene expression analysis



Next-generation sequencing (NGS)
library quantification



Viral load determination

For instructions on use of the QX200 droplet generator, please refer to the QX200
Droplet Generator Instruction Manual (bulletin 10031907).

QX200 Droplet Reader and QuantaSoft Software Instruction Manual | 1



Single cell gene expression analysis



Absolute quantification



Rare mutant detection



miRNA analysis



NGS sample preparation



GMO detection

Chapter 1 QX200 Droplet Digital PCR System

1.2 System Components

The system consists of two instruments and associated software, consumables, and reagents (Table 1.1):



QX200 droplet generator — utilizes proprietary reagents and microfluidics to partition samples into 20,000
nanoliter-sized droplets



QX200 droplet reader — following PCR amplification of the nucleic acid target in the droplets, this
instrument analyzes each droplet individually using a two-color detection system (set to detect FAM and
either VIC, HEX, or EvaGreen); PCR-positive and PCR-negative droplets are counted to provide absolute
quantification of target DNA in digital form using QuantaSoft

Additional materials required for performing ddPCR are listed in Table 1.2.

Table 1.1. QX200 Droplet Digital PCR system components. Items shipped with the QX200 ddPCR system
(catalog #186-4001). Catalog # refers to replacement items (quantities may be different).

Component Description Catalog #

X200 droplet generator

Q

QX200 droplet generator

™

DG8

droplet generator Microfluidic cartridge used to mix sample and oil to 186-4008

I

cartridges and gaskets (24) generate droplets; gaskets seal the cartridge to
prevent evaporation and apply pressure required
for droplet formation

Droplet generator cartridge

older

h

t

P

Power cord Connects QX200 droplet generator to power source Call technical support

nstrument used for droplet generation

ositions and holds the droplet generator cartridge in

he instrument for droplet generation

™

software

1

1

86-4002

86-3051

QX200 droplet reader

QX200 droplet reader

I

Laptop PC and QuantaSoft software
a

Droplet reader plate holders (2)

USB 2.0 cable

C

Power cord

QX200 Droplet Digital PCR system. Left, QX200 droplet reader. Right, Q X200 droplet generator.

QX200 Droplet Reader and QuantaSoft Software Instruction Manual2 |

nstrument used for droplet reading, data collection

onnects to QX200 droplet reader for data collection

C

nd analysis

P

osition 96-well PCR plate in the droplet reader

onnects QX200 droplet reader to PC

onnects QX200 droplet reader to power source

C

C

1

C

86-4003

86-3007

1

10-10608

5

all technical support

all technical support

Chapter 1 QX200 Droplet Digital PCR System

Table 1.2. Additional materials required.

Component

Q

X200 droplet generator

R

ecommended

C

atalog # and Supplier

Reagents for probe detection

PCR supermix

d

dPCR supermix for probes

D

O

roplet PCR supermix

ne-Step RT-ddPCR supermix for probes

1

1

1

86-3010, 186-3026, 186-3027,

1

86-3028

86-3023, 186-3024, 186-3025

86-3021, 186-3022

Droplet generator oil Droplet generator oil for probes 186-3030, 186-3005

Control

d

dPCR buffer control kit for probes

1

86-3052

Reagents for EvaGreen detection

PCR supermix

™

Q

X200

ddPCR™ EvaGreen® supermix 186-4033, 186-4034, 186-4035,

186-4036

Droplet generator oil

X200 droplet generator oil for EvaGreen dye

Q

86-4005, 186-4006

1

Control QX200 buffer control kit for EvaGreen dye 186-4052

Consumables and other materials

Pipets 20 μl pipet for sample loading Rainin L-20

5

8

0 μl pipet for droplet transfer

-channel, 200 μl pipet for oil

R

ainin L-50, L8-50

ainin L8-200

R

Pipet tips Filtered Rainin GP-L10F, GP-L200F

96-well PCR plates

win.tec semi-skirted 96-well plate

t

ppendorf 951020362

E

Reagent trough Any

F

oil plate seals

P

ierceable foil plate seals

1

81-4040

Plate sealer PX1™ PCR plate sealer 181-4000

8-cap strips

ny

A

QX200 droplet reader

Droplet reader oil

D

roplet reader oil

1

86-3031, 186-3004

Droplet reader waste bottle Empty waste bottle N/A