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: 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.
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 61010-1:2010 (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.
Preface 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.
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Contents Chapter 1 QX200™ Droplet Digital PCR System 1.1 Introduction 1.2 System Components 1.3 Droplet Digital PCR Workflow 1.4 System Setup and General Operation Instructions 1 1 2 4 4 Chapter 2 Using the QX200 Droplet Reader 5 Chapter 3 Using QuantaSoft Software 3.1 Setup 3.1.1 Using the Well Editor 3.1.2 Using the Experiment Editor 3.1.3 Using the Advanced Options 3.2 Run 3.3 Analyze 3.3.1 Viewing Channel Data (1D Amplitude) 3.3.2 Viewing Clustering Plots (2D Amplitude) 3.3.
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1 QX200™ Droplet Digital™ 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.
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.
Chapter 1 QX200 Droplet Digital PCR System Table 1.2. Additional materials required.
Chapter 1 QX200 Droplet Digital PCR System 1.3 Droplet Digital PCR Workflow Droplet Digital PCR involves the following steps (4.5–5.5 hours for the complete workflow): ■■ ■■ ■■ ■■ 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 1.
2 Using the QX200™ Droplet Reader 1. Power on the QX200 droplet reader using the switch at the back. Allow it to warm up for 30 min, then switch on the PC and launch QuantaSoft™ software. 2. Check the indicator lights on the front of the droplet reader (Table 2.1). The first two lights at left should be solid green, indicating power is on, there is sufficient oil in the designated oil reservoir, and there is <700 ml in the waste bottle.
Chapter 2 Using the QX200 Droplet Reader 3. Place the 96-well PCR plate into the plate holder: a. Place the 96-well PCR plate containing the amplified droplets into the base of the plate holder. Well A1 of the PCR plate must be in the top left position. b. Move the release tabs of the top of the plate holder into the “up” position and place the top on the PCR plate. Firmly press both release tabs down to secure the PCR plate in the holder.
Chapter 2 Using the QX200 Droplet Reader Correct Incorrect Correct and incorrect placement of the plate holder. Note the release tabs are in the “up” position in the incorrect placement at right. 5. In QuantaSoft software, click Setup in the left navigation bar to define your experiment (see Chapter 3), then click Run. The run indicator light (far right) flashes green to indicate droplet reading is in progress. 6. When droplet reading is complete, all four indicator lights are solid green.
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3 Using QuantaSoft™ Software QuantaSoft software (v. 1.4 and later) organizes and provides one-click access to the three main steps of droplet analysis in the left navigation bar, moving you through the entire workflow: ■■ Setup — enter information about the samples, assays, and experiments (see Section 3.1) ■■ Run — start the run and control the instrument, if needed (see Section 3.2) ■■ Analyze — compute nucleic acid concentration (see Sections 3.3 and 3.
Chapter 3 Using QuantaSoft Software 3.1 Setup File name Options for advanced data analysis and setup Load settings and data from previous experiments Open experiment editor Load or create a template (settings only) Instrument maintenance options (appear only when connected to the droplet reader or when in simulation mode) Define experiment settings Start the run View and analyze data Abort run or exit program Plate map QuantaSoft software Setup interface.
Chapter 3 Using QuantaSoft Software 3.1.1 Using the Well Editor Use the well editor to define the settings (samples, experiment type, and detection type) for the plate. Sample and experiment types are color-coded and can be customized for easy reference in the plate map. Reset — restore default settings Apply — apply settings without exiting well editor Cancel — close without saving changes OK — save changes and close well editor Well editor.
Chapter 3 Using QuantaSoft Software 4. Define Target 1 (channel 1, the FAM channel) and Target 2 (channel 2, the VIC or HEX channel). Assign each assay a Name and sample Type. Settings appear in the Applied Well Settings box as you enter them. When you are done, click Apply or OK to save the information. The settings appear in the well in the plate map. Tips: You can change the selected well using the plate map without exiting the well editor.
Chapter 3 Using QuantaSoft Software 3.1.3 Using the Advanced Options Click Options in the Setup window to see all advanced options for data collection and analysis. Reprocess data with a newer version of the software Export amplitude data for selected wells to a .csv file (one file for each well) Select wells by row (checked) or by column (unchecked) Set threshold display (color) Advanced options. 3.2 Run 1. Click Run in the left navigation bar to start the run. 2.
Fill Chapter in Chapter 3 Using Name QuantaSoft Here on Software Master Page (33_man_left header/footer) Channel 1 (FAM) fluorescence vs. event number Well data stream Channel 2 (VIC or HEX) fluorescence vs. event number Channel 1 histogram (events vs. amplitude) Channel 2 histogram (events vs. amplitude) Switch data views (charts to larger window) Analyzed wells Run indicator Sample name Experiment Conc. channel 1 unknown Conc. channel 2 unknown Interface during an active run.
Chapter 3 Using QuantaSoft Software Results table Well selector Graphical data display Y-axis display options Copy graph to clipboard or print Data analysis interface. Data from a CNV analysis are shown.
Chapter 3 Using QuantaSoft Software Channel 1 vs. channel 2 clustering plot Channel data Plot of ratio of unknown:reference (a/b or a/[a+b]) Concentration data Plot of measured copy numbers Plot of # droplet events counted Graphical data display options. A concentration plot from a CNV analysis is shown, with display options across the top. 3.3.1 Viewing Channel Data (1D Amplitude) Click 1D Amplitude to visualize the data collected from each channel of selected wells.
Chapter 3 Using QuantaSoft Software Channel selector Channel 1 fluorescence amplitude vs. event number Channel 1 (FAM) histogram of events vs. amplitude Reset automatic threshold settings Channel 2 fluorescence amplitude vs. event number Threshold adjustment tool (single well) Channel 2 (VIC or HEX) histogram of events vs. amplitude Threshold settings Y-axis log scale toggle Viewing channel data for a single well. Processed data from both channels of a single well are shown.
Chapter 3 Using QuantaSoft Software 3.3.2 Viewing Clustering Plots (2D Amplitude) Click 2D Amplitude to view the channel 1 vs. channel 2 clustering plot and enable options for manually or automatically adjusting the thresholds used in assigning positives and negatives for each detection channel.
Chapter 3 Using QuantaSoft Software 3.3.3 Viewing Concentration Data (Concentration) Concentration data for each target appear in the wells in the plate map and are tabulated in the results table. Click Concentration to visualize data in concentration plots. Use the radio buttons to select the channels displayed. Error bars reflect total error or Poisson 95% confidence limits. These data can be exported for analysis in other spreadsheet applications (for example, Microsoft Excel).
Chapter 3 Using QuantaSoft Fill in Chapter Name Here on Software Master Page (33_man_left header/footer) 3.3.4 Viewing Copy Number Data (Copy Number) Click Copy Number to view copy number for selected wells/samples. Toggle well, name, or label display Viewing copy number data. Hover over data points to reveal well identity, concentration, and Poisson confidence limits. Solid data points (not shown) indicate merged data; open data points (shown) indicate data from single wells. 3.3.
UsingHere QuantaSoft Software 34_man_left header/footer Fill inChapter Chapter3Name on Master Page 3.3.6 Viewing Events Click Events to view the number of droplet events counted for selected wells/samples. Use the radio buttons to select the channels displayed. View positive, negative, or total droplet counts, or any combination of these. Select channel(s) to display Select events to display Toggle well, name, or label display Viewing event data. Data from absolute quantification are shown. 3.3.
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4 Specifications and Chapter Title Here Maintenance 4.1 Specifications 11.5 inches (292 mm) 26 inches (660 mm) 20.5 inches (521 mm) Weight 56.6 lb. (26 kg) Size (W x D x H) 26 x 20.5 x 11.
Chapter 4 Specifications Maintenance Fill in Chapter Name Hereand on Master Page 4.2 Maintenance 4.2.1 General Maintenance Procedures Surfaces of the instrument may require general cleaning. Use deionized/distilled water for general wipe down with a slightly dampened cloth. For decontamination, 10% bleach followed by 70% ethanol and/or deionized/ distilled water may be used. Do not use acetone or tap water. Inspect equipment regularly for damaged external components or wiring. Do not use if damaged.
Appendix A Ordering Information QX200™ ddPCR™ System 186-3005 Catalog # 186-4001 Droplet Generation Oil for Probes, 10 x 7 ml 186-4005 Droplet Generation Oil for EvaGreen, 2 x 7 ml 186-4006 Droplet Generation Oil for EvaGreen, 10 x 7 ml 186-3004 ddPCR droplet Reader Oil, 2 x 1 L 186-4002 Description QX200™ Droplet Digital™ PCR System, includes droplet generator, droplet reader, laptop computer, software, associated component consumables QX200 Droplet Generator, includes droplet generator, 1 box o
Appendix A Ordering Information 186-3021 186-3022 One-Step RT-ddPCR Kit for Probes, 2 ml (2 x 1 ml), 200 x 20 µl reactions, 2x RT-ddPCR mix, includes 1 manganese acetate tube One-Step RT-ddPCR Kit for Probes, 5 ml (5 x 1 ml), 500 x 20 µl reactions, 2x RT-ddPCR mix, includes 2 manganese acetate tubes 186-4033 QX200™ ddPCR™ EvaGreen® Supermix, 2 ml (2 x 1 ml), 200 x 20 µl reactions 186-4034 QX200 ddPCR EvaGreen Supermix, 5 ml (5 x 1 ml), 500 x 20 µl reactions 186-4035 QX200 ddPCR EvaGreen Supermix, 2
10031906 Rev D US/EG 1014 Sig 1213
