QX100™ Droplet Reader and QuantaSoft™ Software Instruction Manual Catalog #186-3001, 186-3003
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.
Table of Contents Chapter 1 QX100™ 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 2 4 Chapter 2 Using the QX100™ 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.
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.
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.
vi | QX100 Droplet Reader and QuantaSoft Software Instruction Manual
1 QX100™ Droplet Digital™ PCR System 1.1 Introduction The QX100 Droplet Digital PCR (ddPCR™) system performs digital PCR with unrivaled accuracy and precision. The system consists of two instruments, the QX100 droplet generator and the QX100 droplet reader, and their associated consumables (see Section 1.2). The QX100 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 QX100 droplet reader.
Chapter 1 QX100 Droplet Digital PCR System 1.2 System Components The system consists of two instruments and associated software, consumables, and reagents (Table 1.
Chapter 1 QX100 Droplet Digital PCR System Table 1.1. QX100 Droplet Digital system components. Items shipped with the QX100 ddPCR system (catalog #186-3001). Catalog # refers to replacement items (quantities may be different).
Chapter 1 QX100 Droplet Digital PCR System The QX100 ddPCR system is compatible with hydrolysis probe (TaqMan) chemistry and can detect up to two probes at a time (FAM and VIC, or FAM and HEX), using a dye deconvolution matrix in the software to ensure target specificity. The system is not compatible with SYBR® Green or EvaGreen® chemistry; using these chemistries or other unapproved Bio-Rad supermixes may harm the instrument and voids the warranty. 1.
2 Using the QX100™ Droplet Reader 1. Power on the QX100 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 QX100 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 QX100 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.
Chapter 2 Using the QX100 Droplet Reader 8 | QX100 Droplet Reader and QuantaSoft Software Instruction Manual
3 Using QuantaSoft™ Software QuantaSoft software 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 Section 3.
Chapter 3 Using QuantaSoft Software 3.1 Setup Filename Options for advanced data analysis and setup Load settings and data from previous experiments Load or create a template (settings only) Open experiment editor Instrument maintenance options (appear only when connected to the droplet reader) 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 Tips: You can change the selected well using the plate map without exiting the well editor. To append sample or assay names with numbers incrementally through selected wells, select the Auto Inc check box next to Name. Use standard Windows keyboard shortcuts for copying, pasting, and deleting selections (for example, use Ctrl+c to copy a selection, Ctrl+z to undo an operation, etc.). 3.1.
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 chart options (threshold display) Advanced options. 3.2 Run 1. Click Run in the left navigation bar to start the run.
Chapter 3 Using QuantaSoft Software 3.3 Analyze In the Setup window, load a plate (filename.qlp), then click Analyze to open and analyze the data.
Chapter 3 Using QuantaSoft Software Channel 1 vs. channel 2 clustering plot Channel data Plot of measured copy numbers Plot ratio of unknown:reference (a/b or a/[a+b]) Plot of # droplet events counted Concentration data Graphical data display options. A clustering 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 Y-axis display options Channel 2 (VIC or HEX) histogram of events vs. amplitude Channel 2 fluorescence amplitude vs. event number Y-axis log scale toggle Threshold settings Copy graph to clipboard or print Threshold adjustment tool (single well) 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. Patterns observed in these plots can also indicate interactions between PCR products in the reactions.
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 Software 3.3.5 Viewing Ratio Data (Ratio) Click Ratio to view ratio data for selected wells/samples. Use the radio buttons to select a plot of the Ratio (unkown:reference) or Fractional Abundance (% of sample), and select Inverse to apply the inverse of either. Select ratio or abundance plot, inverse ratios Toggle well, name, or label display Viewing ratio data. Data from absolute quantification are shown.
Chapter 3 Using QuantaSoft Software 20 | QX100 Droplet Reader and QuantaSoft Software Instruction Manual
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 lbs. (26 kg) Size (W x D x H) 660 x 521 x 292 cm (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 QX100™ ddPCR™ System ddPCR Reagents Catalog # 186-3001 Description QX100™ Droplet Digital™ PCR System, includes droplet generator, droplet reader, laptop computer, software, associated component consumables 186-3010 ddPCR Supermix for Probes, 5 x 1 ml tubes 2x supermix, for use in sample preparation for droplet generator in the QX100 Droplet Digital PCR system 186-3026 ddPCR Supermix for Probes, 2 x 1 ml tubes QX100 Droplet Generator, includes droplet generator, 1 box
Appendix A Ordering Information Thermal Cyclers 185-1196 C1000 Touch™ Thermal Cycler with 96-Well Fast Reaction Module, includes C1000 Touch thermal cycler chassis, 96-well fast reaction module, USB flash drive 186-1096 T100™ Thermal Cycler, includes 96-well thermal cycler, power cord, tube support ring 24 | QX100 Droplet Reader and QuantaSoft Software Instruction Manual
QX100 Droplet Reader and QuantaSoft Software Instruction Manual | 25
Bio-Rad Laboratories, Inc. Life Science Group 10026321 Rev A AUS/EG 10026321 Rev US/EG Web site www.bio-rad.
