Gel Doc XR+ and ChemiDoc XRS+ Systems with Image Lab 2.0 sw User Manual Rev A

CREDITS 1. Image Lab 2.0 is based in part on the work of the Qwt project (http://qwt.sf.net). 2. Image Lab 2.0 is based in part on the work of the CImg project  (http://cimg.sourceforge.net/).  See license for detail at  http://www.cecill.info/licences/Licence_CeCILL-C_V1-en.html 3. Image Lab 2.0 is based in part on the work of the Independent JPEG Group (http://www.ijg.org/)

TRADEMARKS Windows, Windows XP, Windows Vista, Windows 7, Excel, Microsoft, and Power Point are trademarks of Microsoft Corporation. FireWire, Mac, and Mac OS X are trademarks of Apple Inc. Intel Core is a trademark of Intel Corporation. Hoechst is a trademark of Hoeschst Aktiengesellschaft, Mitsubishi is a trademark of Mitsubishi Corporation. SYBR Safe, SYBR Green, SYBR Gold, and SYPRO are trademarks of Invitrogen Corporation. Coomassie is a trademark of BASF Aktiengesellschaft. Cy is a trademark of GE Healthcare group companies. Fluor and Quasar are trademarks of Biosearch Technologies, Inc. Other brands or product names are trademarks of their respective holders.

LEGAL NOTICES Bio-Rad Laboratories, Inc. is licensed by Invitrogen Corporation to sell SYPRO products for research use only, under US patent 5,616,502.

Molecular Imager with Image Lab 2.0

Safety and Regulatory Compliance Important Safety Information Please read these instructions before attempting to operate the  Molecular Imager® Gel Doc™ XR+ or ChemiDoc™ XRS+ imaging systems. This instrument is suitable for research use only. It must be used, therefore, only by specialized personnel who know the health risks associated with the reagents that are normally used with this instrument. Use of the Gel Doc XR+ and ChemiDoc XRS+ systems involves UV illumination. Proper precautions must be taken to avoid eye and skin exposure to the UV radiation. This instrument is meant for use only by trained personnel who know the health risks associated with UV radiation normally used with this instrument. The acrylic shield provides some UV protection. However, it does not guarantee complete protection, and it is designed to shield only the person working in front of the system. WARNING! Use of the acrylic screen does not guarantee the user protection from UV radiation. The use of protective eyeglasses, mask, and/or gloves is strongly recommended.

Warranty The Gel Doc XR+ and ChemiDoc XRS+ imaging systems are warranted against defects in materials and workmanship for 1 year. If any defect occurs in the instrument during this warranty period, Bio-Rad Laboratories, Inc. will repair or replace the defective parts at its discretion without charge. The following defects, however, are specifically excluded: • Defects caused by improper operation • Repair or modification done by anyone other than Bio-Rad Laboratories, Inc. or their authorized agent • Use of spare parts supplied by anyone other than Bio-Rad Laboratories, Inc. • Damage caused by accident or misuse • Damage caused by disaster • Corrosion caused by improper solvents or samples

i

Molecular Imager with Image Lab 2.0 |

General Precautions • • • • • •

Please read the instruction manual carefully The instrument must be used only for the intended purpose of gel documentation in research laboratories The instrument must be connected to a grounded power source line and protected by a circuit breaker Do not pour liquids directly on or inside the instrument Switch off all the lights immediately after use Clean the transilluminator platen after use

Regulatory Notices The Gel Doc XR+ and ChemiDoc XRS+ imaging systems are designed and certified to meet EN61010, the internationally accepted electrical safety standards, EMC regulations, and TUV requirements. Certified products are safe to use when operated in accordance with this system instruction manual. This instrument should not be modified or altered in any way. Modification or alteration of this instrument will: • Void the manufacturer’s warranty • Void the regulatory certifications • Create a potential safety hazard WARNING! Bio-Rad Laboratories, Inc. 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, Inc., or an authorized agent.

Power Safety Information VOLTAGE SETTING INFORMATION The universal hood of the Gel Doc XR+ or ChemiDoc XRS+ imaging system has a power supply that automatically chooses the correct voltage for your country or region.

FUSES The universal hood of the Gel Doc XR+ or ChemiDoc XRS+ imaging system has two user-serviceable fuses, F1 and F2, which are located on the bottom rear panel and are a part of the power entry module. Refer to the Maintenance Chapter for fuse replacement instructions.

ii

Table of Contents  Safety and Regulatory Compliance... i

Chapter 1. Introduction... 1 Gel Doc™ XR+ Imaging System... 1 ChemiDoc XRS+ Imaging System... 2 System Components... 2 CCD Camera and Lenses... 2 Universal Hood... 3 Image Lab 2.0 Software... 3 Emission Filters... 3 Optional Accessories... 4 Printer... 4 Conversion Screens... 4 Computer Requirements... 4 Computer Specifications... 5

Chapter 2. Gel Doc™ XR+ Imager... 7 Gel Doc XR+ Imager Workflow... 7 Gel Doc XR+ Applications... 8 Gel Doc XR+ Technical Specifications... 9

Chapter 3. ChemiDoc™ XRS+ Imager... 11 ChemiDoc XRS+ Imager Workflow... 11 ChemiDoc XRS+ Applications... 12 ChemiDoc XRS+ Technical Specifications... 13

Chapter 4. System Calibration... 15

iii

Molecular Imager with Image Lab 2.0 | Table of Contents

Chapter 5. Image Lab Software Overview... 17 Software Overview... 18 Main Window... 18 Toolbar... 19 Results Data... 19 Display Toolbox... 19 Start Page... 20 Analysis Tool Box... 20 Status Bar... 21 Menu Items... 21

Chapter 6. Protocols... 27 Creating Custom Protocols... 27 Acquisition Settings... 28 Report Settings... 36 Choosing a Protocol... 36 Editing Protocols... 37 Position Gel... 38 Running a Protocol... 38 Application Tables... 39

Chapter 7. Viewing Images... 41 Results Overview... 41 Displaying Gel Images... 42 Display Gel Options... 42 Zoom Tools... 43 Fit in Window... 43 Image Transform... 44 Image Colors... 45 3-D Projection... 46 Image Info... 47 Displaying Data... 48 Lane and band definitions:... 48 Volume definitions:... 48 Analysis Table Options... 49 Lane Profile... 52 Standard Curve... 54 Report... 54

iv

Chapter 8. Analyzing Images... 55 Analysis Tool Box... 55 Auto Analysis... 55 Analysis Toolbox... 57 1. Image Tools... 58 2. Lane and Band Tools... 60 3. Molecular Weight Analysis Tools... 63 4. Quantity Tools... 64 5. Annotation Tools... 67 6. Volume Tools... 69

Chapter 9. Generating Reports... 73 Report... 73 Display Report General Tab... 74 Lane and Band Table Tab... 75 Volume Table Tab... 76 Print Report... 76 Print Report to .pdf File... 76 Adjust the Printer Settings... 76

Chapter 10. Exporting Results... 77 Exporting Gel Images... 77 File Menu Export... 78 Analysis Table Export... 79

Chapter 11. Maintenance... 81 UV Transilluminator Lamp and Starter Replacement... 81 Epi-Illumination Lamp Replacement... 82 Fuse Replacement... 83

Glossary... 85 Regression Method Calculations... 87

Appendix A: Troubleshooting... 89

v

Molecular Imager with Image Lab 2.0 | Table of Contents

Appendix B: Accessories... 91 Calibrating Accessories... 91 Installing Optional Accessories... 92 UV/White Light Conversion Screen... 92 Gel Alignment Template Kit... 94 White Light Transilluminator... 95 Orange Fluorescence Reference Plate... 96 Ordering Information... 97

Index... 101

vi

Molecular Imager with Image Lab 2.0

1

Introduction Welcome to Bio-Rad Molecular Imager systems! This manual describes how to use the Gel Doc XR+ and ChemiDoc XRS+ imaging systems. Gel Doc XR+ and ChemiDoc XRS+ gel documentation systems offer high performance and are easy to use. They both contain a charge coupled device (CCD) camera to capture images in real time, and allow you to accurately position your sample and generate optimized image data. The Gel Doc XR+ and ChemiDoc XRS+ systems use the same light-tight enclosure (the universal hood) which contains built-in UV and white light illumination, but different CCD cameras. Both systems feature dynamic flat fielding technology for superior image uniformity and accurate quantitation. Bio-Rad Image Lab™ software version 2.0 software controls image capture and optimization for your selected applications, analyzes results, and produces reports based on your desired output, all in a single workflow.

Gel Doc™ XR+ Imaging System Gel Doc XR+ imaging system is a high-resolution gel documentation system that allows fast, easy quantitation of gels and blots. Position your sample inside the imager and follow the onscreen steps to run a protocol with only one mouse click. You can customize your applications within an existing protocol or create a new protocol using many options presented by the Image Lab software. Features include: • Detection of dynamic ranges that cover > 3 orders of magnitude

1

Molecular Imager with Image Lab 2.0 | Introduction

• •

•

Dynamic flat fielding specific to fluorescent and colorimetric applications Smart, application-based protocol setup using Image Lab software, which assists you by presenting appropriate filter and illumination sources for each application Ability to upgrade to the ChemiDoc XRS+ system

ChemiDoc XRS+ Imaging System In addition to the gel documentation applications of Gel Doc XR+ presented above, the ChemiDoc XRS+ imaging system offers sensitive chemiluminescent detection. The system includes a supersensitive 16-bit CCD camera that is deeply cooled for faint sample detection and for accurate quantification of image data. Features include: • Imaging applications that require excellent sensitivity (chemiluminescent western blots) • Smart, application-based protocol setup using Image Lab software, which assists you by presenting appropriate filter and illumination sources for each application • Exceptional sensitivity and a dynamic range greater than 4 orders of magnitude • Flexibility to image chemiluminescent, fluorescent, and colorimetric samples with dynamic flat fielding specific to each application

System Components CCD Camera and Lenses Either the Gel Doc XR+ or the ChemiDoc XRS+ camera is placed on top of a light-tight enclosure (the universal hood) for capturing images. The camera comes with a motorized zoom lens (MZL) that allows a remote adjustment of the lens control functions (zoom, focus, and iris). A patent-pending software algorithm controls the MZL, giving the user automatic image focus, once an initial calibration is performed during system installation. See the Technical Specifications table, included in the chapter which discusses your camera, for complete specifications of each system. A +1 diopter lens is factory installed to allow the entire sample stage to be visible. This lens should always remain on the MZL assembly.

2

System Components

Universal Hood The universal hood is designed to capture fluorescent and chemiluminescent images without using a photographic darkroom. The enclosure has built-in white light epi-illumination and UV transillumination. For easy sample loading, the UV transilluminator is located in the drawer of the universal hood and can be accessed from the front of the enclosure. When not imaging, the lights in the darkroom enclosure turn off automatically. The universal hood has touchpad buttons to perform various functions; however, Image Lab software controls all of these functions remotely, removing any requirement for manual control of the lens and lights.  Running a protocol overrides any touchpad input.

Image Lab 2.0 Software Each system ships with a full version of Image Lab 2.0 software. In addition to controlling the imager systems, image capture, and optimization, Image Lab software can be used to annotate and document images, analyze molecular weights (or base pairs, when imaging nucleic acid gels), and to determine accurate quantitation and purity of samples. You can print all or a subset of your data in a report. Alternatively, you can export your data to other software, such as Microsoft Office programs, for further analysis or presentation options. See Chapters 5-10 for detailed instructions on operating the software.

Emission Filters The universal hood can hold two different emission filters for fluorescent applications. No filter is required to image chemiluminescent samples. A standard filter (catalog # 170-8081) is used for colorimetric (white light) applications and is included in the installation kit.

3

Molecular Imager with Image Lab 2.0 | Introduction

Optional Accessories Bio-Rad Laboratories, Inc. offers a selection of optional filters and illumination sources. See Appendix B for ordering information. For a complete listing of accessory filters, UV light sources, optional parts and replacement parts, please See “Appendix B: Accessories” on page 91.

Printer For your convenience, Bio-Rad offers an optional USB printer for use with the Gel Doc XR+ and ChemiDoc XRS+ systems: the Mitsubishi P93DW Thermal Printer, (catalog #170-8089).

Conversion Screens WHITE LIGHT CONVERSION SCREEN The white light conversion screen (catalog #170-8001) is a phosphor screen that produces white light transillumination when placed on top of the UV transilluminator.

WHITE LIGHT TRANSILLUMINATOR An optional white light transilluminator (catalog #170-7950) may also be used with either system. The white light transilluminator plugs inside the universal hood and is controllable by the touchpad or the computer software.

XCITABLUE™ CONVERSION SCREEN The optional XcitaBlue screen kit (catalog # 170-8182) is a UV to blue light conversion screen which allows you to visualize DNA samples while protecting them from UV damage.

Computer Requirements The Image Lab 2.0 software runs under Windows XP Professional, Windows Vista, Windows 7, or Mac OS X. Images scanned at high resolution can be quite large. The amount of memory required for using the program is mainly determined by the size of the images you will scan and analyze. For this reason, we recommend that you archive images on a network file server or on removable storage media. Bio-Rad can also provide an appropriate computer to use with this system. Please contact your local  Bio-Rad representative for more details.

4

Computer Specifications

Computer Specifications Specifications

Minimum

Operating system

Windows XP SP3

Recommended Windows XP SP3 Professional

Windows 32-bit Vista SP2

Windows 32-bit Vista SP2 Ultimate

Windows 7

Windows 7 Professional

Mac OS X 10.5

Mac OS x 10.5

Processor

Pentium 4 or equivalent (Windows)  at 2.0 GHz

Core 2 Duo 2.0 GHz or higher

Hard disk space

20 GB

> 100 GB

Memory (RAM)

1,024 MB

> 1,024 MB

Ports for connecting instrument

1 free USB 2.0 port

1 free USB 2.0 port

Other software (optional)

Microsoft Excel 2000 or later (Windows)

Microsoft Excel 2000 or later (Windows)

Office 2008 or iWork software (Mac)

Office 2008 or iWork software (Mac)

5

Molecular Imager with Image Lab 2.0 | Introduction

6

Molecular Imager with Image Lab 2.0

2

Gel Doc™ XR+ Imager Refer to the Gel Doc XR+ Installation Guide (10016995) found in your  Gel Doc XR+ Installation Kit (catalog # 170-8199) for instructions to install your Image Lab software, and to assemble and calibrate your system. If you need to re-calibrate your system because you have acquired new accessories, refer to Chapter 4.

Gel Doc XR+ Imager Workflow Here are the basic steps to acquiring, analyzing, and archiving an image using the Gel Doc XR+ system and Image Lab 2.0 software: 1. Select an existing protocol, or customize a new one. 2. Position the sample to be imaged. 3. Run your selected protocol. 4. View the displayed results. 5. Optimize the analysis. 6. Generate your report. 7. Save or export the results.

7

Molecular Imager with Image Lab 2.0 | Gel Doc™ XR+ Imager

Gel Doc XR+ Applications The Gel Doc XR+ system is capable of running a wide variety of protocols. Those below are examples of the detection regents which may be used to image your samples. Contact Bio-Rad technical support to determine whether your application can be imaged on this instrument. See Chapter 6, Protocols, for complete instructions on designing protocols.

NUCLEIC ACID GELS • • • • • • •

Ethidium bromide SYBR® Green SYBR® Safe SYBR® Gold GelGreen™ GelRed™ Fast Blast™

PROTEIN GELS • • • • • • • • •

8

Coomassie Blue Copper stain Zinc stain Flamingo Oriole Silver stain Coomassie Fluor Orange SYPRO Ruby Krypton

Gel Doc XR+ Technical Specifications

Gel Doc XR+ Technical Specifications Applications Chemiluminescence

No

Fluorescence*

Yes

Colorimetry/densitometry

Yes

Gel documentation

Yes

Hardware Specifications Maximum sample size (cm) Maximum image area (cm)

28 x 36 25 x 26

Excitation source

Trans-UV and Epi-White are standard (302 nm included, with 254 and 365 nm available as options). Optional Trans White, self-powered or conversion screen. Optional XcitaBlue™ UV/blue conversion screen.

Illumination control

3 modes Trans-UV, Trans White, Epi-White

Detector CCD resolution (H x V)

CCD 1,392 x 1,040

Pixel size (H x V in microns)

4.65 x 4.65

Cooling system

Not applicable

Camera cooling temperature

Not available

Filter holder

3 positions (2 for emission filters, 1 for using no filter)

Emission filters

1 included (standard), 3 optional

Dynamic range

>3.0 orders of magnitude

Pixel density (gray levels)

4,096

Dynamic flat fielding

Application-specific, for all applications

Instrument size (cm)

36 (L) x 60 (W) x 96 (H)

Instrument weight (kg)

32

Operating Ranges Operating voltage

110/115/230 V AC nominal

Operating temperature

10–28°C (21°C recommended)

Operating humidity

<70% non-condensing

Automation Capabilities Workflow automated selection

Application driven, user selected or recalled by a protocol

Workflow automated execution

Controlled by a protocol via application specific setup for image area, illumination source, filter, analysis and reporting

Workflow reproducibility

100% repeatability via recallable protocols; from image capture to quantitative analysis and reports

Autofocus (patent pending)

Pre-calibrated focus for any zoom setting or sample height

Image flat fielding (patent pending) Autoexposure

Dynamic; pre-calibrated and optimized per application 2 user-defined modes; (intense or faint bands)

*Using the optional XcitaBlue kit (catalog #170-8182) is highly recommended if performing SYBR® Safe DNA applications, because the UV to blue conversion screen allows you to visualize DNA samples while protecting from UV damage.

9

Molecular Imager with Image Lab 2.0 | Gel Doc™ XR+ Imager

10

Molecular Imager with Image Lab 2.0

3

ChemiDoc™ XRS+ Imager Refer to the ChemiDoc XRS+ Installation Guide (10006999) found in your ChemiDoc Installation Kit (catalog # 170-8299) for instructions to install your Image Lab software, and to assemble and calibrate your ChemiDoc XRS+ system. If you need to re-calibrate your system because you have acquired new accessories, refer to Chapter 4.

ChemiDoc XRS+ Imager Workflow Here are the basic steps to acquiring, analyzing and archiving an image using the ChemiDoc XRS+ system and Image Lab software: 1. Select an existing protocol, or customize a new one. 2. Position the sample to be imaged. 3. Run your selected protocol. 4. View the displayed results. 5. Optimize the analysis. 6. Generate your report. 7. Save or export the results. When running chemiluminescent blots, you can use a signal accumulation mode, which takes a series of exposures within a period of time. This allows you to choose an image with optimal signal. Refer to Chapter 6 for complete instructions.

11

Molecular Imager with Image Lab 2.0 | ChemiDoc™ XRS+ Imager

ChemiDoc XRS+ Applications The ChemiDoc XRS+ system is capable of running protocols to image all the detection reagents from the Gel Doc XR+ list, plus the others added to this list. These are only examples of the detection regents which may be used to image your samples, not a complete list. Contact Bio-Rad technical support to determine whether your application can be imaged on this instrument. See Chapter 6, Protocols, for complete instructions on designing protocols.

NUCLEIC ACID GELS •

Ethidium bromide

•

GelGreen™

•

SYBR® Green

•

GelRed™

•

SYBR® Safe

•

Fast Blast™ stain

•

SYBR® Gold

PROTEIN GELS •

Coomassie Blue

•

SYPRO Ruby

•

Copper stain

•

Silver stain

•

Zinc stain

•

Coomassie Fluor Orange

•

Flamingo

•

Krypton

•

Oriole

BLOTS

12

•

Chemiluminescent

•

Qdots 625

•

Colorimetric

•

CY2

•

Qdots 525

•

Alexa 488

•

Qdots 565

•

DyLight 488