CFX96 and CFX384 Real-Time PCR Detection Systems Instruction Manual Rev D

Copyright ©2010 Bio-Rad Laboratories, Inc. Reproduction in any form, either print or electronic, is prohibited without written permission of Bio-Rad Laboratories, Inc. Adobe and Reader are trademarks of Adobe Systems Incorporated. Cy is a trademark of GE Healthcare Group companies. CAL Fluor and Quasar are trademarks of Biosearch Technologies, Inc. SYBR® and Texas Red are trademarks of Invitrogen Corporation. Excel, Microsoft, PowerPoint, Windows, and Windows Vista are trademarks of Microsoft Corporation. EvaGreen is a trademark of Biotium, Inc. BioRad Laboratories, Inc., is licensed by Biotium, Inc., to sell reagents containing EvaGreen dye for use in real-time PCR, for research purposes only. FAM and ROX are trademarks of Applera Corporation. BioRad Laboratories, Inc. is licensed by Invitrogen Corporation to sell reagents containing SYBR® Green I for use in real-time PCR, for research purposes only.

LICENSE NOTICE TO PURCHASER Bio-Rad real-time thermal cyclers CFX96™ and CFX384™ are licensed real-time thermal cyclers under Applera's United States Patent No. 6,814,934 B1 for use in research, human in vitro diagnostics, and all other fields except veterinary diagnostics.

This CFX96 or CFX384 detection module, when combined with a C1000™ thermal cycler for which the applicable real-time thermal cycler royalty fee has been paid, constitutes a real-time thermal cycler licensed under U.S. Patent No. 6,814,934 and corresponding claims in any Canadian counterpart patent thereof owned by Applera Corporation, for use solely in research, human in vitro diagnostics, and all applied fields except veterinary in vitro diagnostics. These license rights are effective only if this detection module is combined with a Bio-Rad thermal cycler for which the applicable real-time thermal cycler royalty fee has been paid and not with any other thermal cycler. No rights are conveyed expressly, by implication or estoppel to any patents on real-time methods, including but not limited to 5' nuclease assays, or to any patent claiming a reagent or kit. For further information on purchasing license rights, contact the Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California, 94404, USA.

This product is covered by one or more of the following U.S. patents, their foreign counterparts, or their foreign patents pending, owned by Eppendorf AG: U.S. Patent Nos. 6,767, 512 and 7,074,367.

Bio-Rad Resources Table 1 lists Bio-Rad resources and how to locate what you need. Table 1. Bio-Rad resources Resource

How to Contact

Local Bio-Rad Laboratories representatives

Find local information and contacts on the Bio-Rad website by selecting your country on the home page (www.bio-rad.com). Find the nearest international office listed on the back of this manual

Technical notes and literature

Go to the Bio-Rad website (www.bio-rad.com). Type a search term in the Search box and select Literature to find links to technical notes, manuals, and other literature.

Technical specialists

Bio-Rad’s Technical Support department is staffed with experienced scientists to provide customers with practical and expert solutions. To find local technical support on the phone, contact your nearest Bio-Rad office. For technical support in the United States and Canada, call 800-424-6723 (toll-free phone) and select the technical support option.

Writing Conventions Used in This Manual This manual uses the writing conventions listed in Table 2. Table 2. Conventions used in this manual Convention

Meaning

TIP:

Provides helpful information and instructions, including information explained in further detail elsewhere in this manual

NOTE:

Provides important information, including information explained in further detail elsewhere in this manual

WARNING!

Explains very important information about something that might damage the researcher, damage an instrument, or cause data loss

X>Y

Select X and then select Y from a toolbar, menu, or software window

For information about safety labels used in this manual and on the CFX96 system or the CFX384 system, see, “Safety and Regulatory Compliance” on page iii.

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CFX96 and CFX384 Systems Instruction Manual

Safety and Regulatory Compliance For safe operation of the CFX96 system or the CFX384 system, we strongly recommend that you follow the safety specifications listed in this section and throughout this manual.

Safety Warning Labels Warning labels posted on the instrument and in this manual warn you about sources of injury or harm. Refer to Table 3 to review the meaning of each safety warning label. Table 3. Meaning of safety warning labels CAUTION: Biohazard! This symbol identifies components that may become contaminated with biohazardous material.

CAUTION: Risk of danger! This symbol identifies components that pose a risk of personal injury or damage to the instrument if improperly handled. Wherever this symbol appears, consult the manual for further information before proceeding CAUTION: Hot surface! This symbol identifies components that pose a risk of personal injury due to excessive heat if improperly handled

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Instrument Safety Warnings The warning labels shown in Table 4 also display on the instrument and refer directly to the safe use of the CFX96 system or the CFX384 system. Table 4. Instrument safety warning labels Icon

Meaning Warning about risk of harm to body or equipment. Operating the CFX96 or CFX384 real-time PCR detection system before reading this manual can constitute a personal injury hazard. For safe use, do not operate this instrument in any manner unspecified in this manual. Only qualified laboratory personnel trained in the safe use of electrical equipment should operate this instrument. Always handle all components of the system with care and with clean, dry hands Warning about handling biohazardous materials. When handling biohazardous samples, adhere to the recommended precautions and guidelines, and comply with any local guidelines specific to your laboratory and location. Warning about risk of burning. A thermal cycler generates enough heat to cause serious burns. Wear safety goggles or other eye protection at all times during operation. Always allow the sample block to return to idle temperature before opening the lid and removing samples. Always allow maximum clearance to avoid accidental skin burns Warning about risk of explosion. The sample blocks can become hot enough during the course of normal operation to cause liquids to boil and explode

NOTE: For information about the C1000™ thermal cycler, refer to the C1000 thermal cycler instruction manual.

Safe Use Specifications and Compliance Table 5 lists the safe use specifications for the CFX96 system and the CFX384 systems. Shielded cables (supplied) must be used with this unit to ensure compliance with the Class A FCC limits. Table 5. Safe use specifications Safe Use Requirements

Specifications

Temperature

Ambient temperature of 15 - 31oC. Relative humidity maximum of 80% noncondensing

Indoor use

Altitude

Up to 2,000 meters above sea level

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 • IEC 61010-2-010:2005, EN61010-2-010:2003. Safety requirements for electrical equipment for measurement, control, and laboratory use. Part 2-010: Particular requirements for laboratory equipment for the heating of materials • IEC 61010-2-081:2001+A1, EN61010-2-081:2002+A1. Safety requirements for electrical equipment for measurement, control, and laboratory use. Part 2-081: Particular requirements

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CFX96 and CFX384 Systems Instruction Manual

for automatic and semi-automatic laboratory equipment for analysis and other purposes (includes Amendment 1) • 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 radio frequency 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.

Hazards The CFX96 and CFX384 real-time PCR detection systems are designed to operate safely when used in the manner prescribed by the manufacturer. If the CFX96 or CFX384 system or any of its associated components are used in a manner not specified by the manufacturer, the inherent protection provided by the instrument may be impaired. Bio-Rad Laboratories, Inc. is not liable for any injury or damage caused by the use of this equipment in any unspecified manner, or by modifications to the instrument not performed by Bio-Rad or an authorized agent. Service of the CFX96 or CFX384 system should be performed only by Bio-Rad personnel.

Biohazards The CFX96 and CFX384 systems are laboratory products. However, if biohazardous samples are present, adhere to the following guidelines and comply with any local guidelines specific to your laboratory and location.

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

SURFACE DECONTAMINATION WARNING! To prevent electrical shock, always turn off and unplug the instrument prior to performing decontamination procedures.

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The following areas can be cleaned with any hospital-grade bactericide, virucide, or fungicide disinfectant: • Outer lid and chassis • Inner reaction block surface and reaction block wells • Control panel and display To prepare and apply the disinfectant, refer to the instructions provided by the product manufacturer. Always rinse the reaction block and reaction block wells several times with water after applying a disinfectant. Thoroughly dry the reaction block and reaction block wells after rinsing with water. WARNING! Do not use abrasive or corrosive detergents or strong alkaline solutions. These agents can scratch surfaces and damage the reaction block, resulting in loss of precise thermal control.

DISPOSAL OF BIOHAZARDOUS MATERIAL The CFX96 or CFX384 system contains no potentially hazardous chemical materials. Dispose of the following potentially contaminated materials in accordance with laboratory local, regional, and national regulations: • Clinical samples • Reagents • Used reaction vessels or other consumables that may be contaminated

Chemical Hazards The CFX96 or CFX384 system contains no potentially hazardous chemical materials.

Explosive or Flammability Hazards The CFX96 or CFX384 system poses no uncommon hazard related to flammability or explosion when used in a proper manner as specified by Bio-Rad Laboratories.

Electrical Hazards The CFX96 or CFX384 system poses no uncommon electrical hazard to operators if installed and operated properly without physical modification and connected to a power source of proper specification.

Transport Before moving or shipping the C1000™ thermal cycler, or CFX96 or CFX384 optical reaction module, decontamination procedures must be performed. Always move or ship the C1000 thermal cycler chassis and CFX96 or CFX384 optical reaction module in separate containers with the supplied packaging materials which will protect the instrument from damage. If appropriate containers cannot be found, contact your local Bio-Rad office.

Storage The CFX96 or CFX384 system can be stored under the following conditions: • Temperature range: –20 to 60oC • Relative humidity: maximum 80%

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Disposal The CFX96 or CFX384 real-time PCR detection system contains electrical or electrical materials; it should be disposed of as unsorted waste and must be collected separately, according to European Union Directive 2002/96/CE on waste and electronic equipment - WEEE Directive. Before disposal, contact your local Bio-Rad representative for country-specific instructions.

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Table of Contents Bio-Rad Resources... ii Writing Conventions Used in This Manual... ii Safety and Regulatory Compliance... iii Hazards... v

Table of Contents... ix Chapter 1. System Installation... 1 Unpacking the Optical Reaction Module... 1 System Requirements... 1 System Overview... 2 Setting up the system... 4 Installing CFX Manager Software... 6 Software Files... 8 Running Experiments... 8

Chapter 2. CFX Manager™ Software... 11 Main Software Window... 12 Startup Wizard... 15 Detected Instruments Pane... 15 Status Bar... 17 Instrument Properties Window... 17 Master Mix Calculator... 20 Scheduler... 21

Chapter 3. Performing Runs... 25 Run Setup Window... 25 Protocol Tab... 26 Plate Tab... 27 Start Run Tab... 27 Run Details Window... 28 Instrument Summary Window... 31

Chapter 4. Protocols... 33 Protocol Editor Window... 33 Protocol Editor Controls... 35 Temperature Control Mode... 38 Protocol AutoWriter... 39

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Chapter 5. Plates... 41 Plate Editor Window... 41 Select Fluorophores Window... 45 Well Loading Controls... 45 Experiment Settings Window... 48 Well Selector Right-Click Menu Items... 50 Well Groups Manager Window... 50 Plate Spreadsheet View Window... 52

Chapter 6. Stand-Alone Operation... 53 Control Panel... 53 Main Menu... 54 Creating a New Run... 55 Exporting Data for Analysis... 61 Creating a Data File... 63 Setting up Email... 64

Chapter 7. Data Analysis Overview... 69 Data Analysis Window... 69 Quantification Tab... 72 Data Analysis Settings... 73 Well Selectors... 77 Charts... 79 Spreadsheets... 80 Export... 81

Chapter 8. Data Analysis Windows... 83 Quantification Tab... 83 Quantification Data Tab... 87 Melt Curve Tab... 89 Melt Curve Data Tab... 91 End Point Tab... 93 Allelic Discrimination Tab... 95 Custom Data View Tab... 97 QC Tab... 98 Run Information Tab... 99 Data File Reports... 100 Well Group Reports... 103

Chapter 9. Gene Expression Analysis... 105 Gene Expression... 105 Plate Setup for Gene Expression Analysis... 106 Gene Expression Tab... 106 Experiment Settings Window... 111 Gene Study... 113 Gene Study Report Window... 117 Gene Expression Calculations... 119

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CFX96 and CFX384 Systems Instruction Manual

Chapter 10. Users and Preferences... 125 Log in or Select User... 125 User Preferences Window... 126 Email Tab... 127 Files Tab... 128 Protocol Tab... 128 Plate Tab... 129 Data Analysis Tab... 130 Gene Expression Tab... 131 QC Tab... 132 Custom Export Tab... 133 User Administration... 134

Chapter 11. Resources... 137 LIMS Integration... 137 Calibration Wizard... 142 Instrument Maintenance... 144 Application Log... 146 Troubleshooting... 146 References... 149

Index... 151

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System Installation Read this chapter for information about setting up the CFX96™ or CFX384™ system: • Unpacking the optical reaction module (page 1) • System requirements (page 1) • System overview (page 2) • Setting up the system (page 4) • Installing CFX ManagerTM software (page 6) • Software files (page 8) • Running experiments (page 8)

Unpacking the Optical Reaction Module Your CFX96 or CFX384 optical reaction module shipment includes these components: • Optical reaction module • USB cable • CFX Manager software installation CD • Instruction manual • CFX Manager software quick guides for system installation, protocol, plate, data analysis, gene expression analysis, and qbasePLUS software setup • CFX Manager software video tutorial Remove all packing materials and store them for future use. If any items are missing or damaged, contact your local Bio-Rad office.

System Requirements To operate the CFX96 or CFX384 system, use the following power sources and cables: • Input power. 100 - 240 VAC, 50 - 60 Hz • Indoor use. Ambient temperature of 15 - 31oC. Relative humidity maximum of 80%, noncondensing.

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System Installation

• USB cable. If the system is going to be controlled by a computer via a USB cable, the provided cable from Bio-Rad is sufficiently shielded for use. NOTE: For a full list of the safety and compliance requirements for this instrument, see “Safety and Regulatory Compliance” on page iii.

System Overview The CFX96 system or CFX384 system includes two components: • Optical reaction module. This module includes an optical system to collect fluorescent data and a thermal cycler block NOTE: The serial number of the CFX96 or CFX384 optical reaction module is located on the back. • C1000TM thermal cycler base. The C1000 base includes a user interface to control the system when running in stand-alone mode, the power button, and ports (both on the back panel) to connect to a computer

Indicator LED Open button

Front panel

Figure 1. Front view of the CFX96 system.

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CFX96 and CFX384 Systems Instruction Manual

When open, the CFX96 or CFX384 system includes the features shown in Figure 2.

Inner lid with heater plate

Block Close button

Figure 2. Inside view of the CFX96 system. WARNING! Avoid touching the inner lid or block: These surfaces can be hot. • Inner lid with heater plate. The heater lid maintains temperature on the top of the consumable to prevent sample evaporation. Avoid touching or otherwise contaminating the heater plate. Never poke anything through the holes; the optics shuttle system could be damaged • Block. Load samples in this block before the run • Close button. Press this button on the inside of the lid to close the motorized lid WARNING! Prevent contamination of the instrument by spills, and never run a reaction with an open or leaking sample lid. For information about general cleaning and maintenance of the instrument, see “Instrument Maintenance” (page 144). The back panel of the C1000 chassis includes these features (Figure 3): • Power switch. Press the power switch to turn on power to the system • Power input. Plug in the power cord here • Ethernet port. Connect an ethernet cable to email run logs and stand-alone data files • USB connections. Use these ports to connect the CFX96 system or CFX384 system to a computer or to connect an S1000TM thermal cycler

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System Installation

Power switch

Power input

Ethernet USB connections port

Figure 3. Back panel of C1000 thermal cycler. WARNING! Avoid contact with the back panel of the C1000 cycler during operation.

Setting up the system The CFX96 or CFX384 real-time PCR detection system should be installed on a clean, dry, level surface with sufficient cool airflow to run properly. The CFX96 system or CFX384 system can be run in two modes: stand-alone or software-controlled. If you are running the system under software-controlled mode, make sure there is sufficient space for a computer during setup. To insert the optical reaction module into the reaction module bay of the C1000 chassis, follow these instructions: 1. Place the C1000 chassis in a suitable location with the locking bar down. Remove any previously installed reaction modules.

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CFX96 and CFX384 Systems Instruction Manual

2. Lift the optical reaction module using the handle indents above the side air vents (Figure 4).

Figure 4. Lifting the optical reaction module into the C1000 chassis. 3. Position the module in the reaction module bay of the C1000 chassis, leaving about 2 cm of space in the front. When in the chassis bay, the optical module should be covering the Bio-Rad logo in front of the bay of the C1000 chassis. 4. Reach around and pull up the locking bar of the C1000 until it is flush with the sides of the module bay. This action moves the module forward, locking it into place (Figure 5).

Figure 5. Locking the optical module into place.

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System Installation

5. Check that the module is completely and evenly seated in the C1000 base. Check the space around the bottom of the module. There should be no extra space between the module and the base; the space should be even. 6. Plug the power cord into the back of the C1000 base (Figure 3) and into an appropriate three-pronged electrical outlet. 7. Press the power switch on the back panel of the C1000 thermal cycler to start the system. 8. Follow the instructions on the C1000 front panel to remove the red shipping screw from the inner heater lid. • Open the optical module lid by pressing the button below the Bio-Rad logo. • Turn the screw counterclockwise to remove it from the hole in the inner heated lid that corresponds to well A1 for the CFX96 or to the well adjacent to the left side of B1 for the CFX384. 9. Remove the shipping plate from the thermal cycler block. 10.Close the optical module lid by pressing the button positioned in front of the block. 11.Press the Screw Removed button to confirm the shipping screw has been removed. NOTE: If the shipping screw is not removed at this step, it will be detected by CFX Manager software. Follow instructions to remove the screw (page 18). TIP: The shipping screw must be in place when the module is shipped. Save this screw in a safe place for future shipping.

Installing CFX Manager Software CFX Manager software is run on a personal computer (PC) with either the Windows XP, Windows Vista, or Windows 7 operating system and is required to analyze real-time PCR data from the CFX96 system or CFX384 system. This software can also be used to control the CFX96 system or CFX384 system in software-controlled mode. Table 6 lists the computer system requirements for the software. Table 6. Computer requirements for CFX Manager software.

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System

Minimum

Recommended

Operating system

Windows XP Professional SP2 and Windows XP Professional SP2 and above or Windows 7. above, Windows Vista Home Premium or Windows 7 Home Premium and above.

Drive

CD-ROM drive

CD-RW drive

Hard drive

10 GB

20 GB

Processor speed

2.0 GHz

2.0 GHz

RAM

1 GB RAM (2 GB for Windows Vista)

2 GB RAM

Screen resolution

1024 x 768 with true-color mode

1280 x 1024 with true-color mode

USB

USB 2.0 Hi-Speed port

USB 2.0 Hi-Speed port

CFX96 and CFX384 Systems Instruction Manual

To install CFX Manager software: 1. The software must be installed on the computer by a user with administrative privileges. Make sure you are logged in with administrative privileges. 2. Place the CFX Manager software CD in the computer’s CD drive. 3. The software launch page should appear automatically. Double-click Install Software on the software launch page (Figure 6). TIP: Click the Documentation button to find searchable PDF copies of the instrument manuals and other documentation. 4. Follow the instructions on-screen to complete installation. When completed, the Bio-Rad CFX manager software icon will appear on the desktop of the computer. 5. If the launch page does not appear automatically, double-click (CD drive):Bio-Rad CFX, then open and follow instructions in the Readme.txt file. See “Installing the Software Manually” on page 146.

Figure 6. Software installation screen.

Installing the Drivers If the CFX96 system or CFX384 system is going to be run in software-controlled mode, drivers must be installed on the computer. Use only the supplied USB cable, which is sufficiently shielded to prevent data loss. To install the system drivers: 1. Connect the C1000 chassis to the computer by plugging a USB cable into the USB 2.0 A port located on the back of the chassis (Figure 3) and then connecting the cable to the USB 2.0 B port located on the computer. 2. If it is not already turned on, turn on the system using the power switch on the back of the C1000 chassis. Follow the instructions in the Found New Hardware Wizard that launches after the instrument is first detected by the computer. 3. On the first screen, select Yes, this time only to instruct the Windows operating system to connect to Windows Update to search for software. Click Next.

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