QX200™ Droplet Generator Instruction Manual Catalog #186-4002
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 61010-1:2001 (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.
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Contents Chapter 1 QX200™ Droplet Generator 1.1 Introduction 1.2 QX200 Droplet Generator 1.3 Installation and General Operation 1 1 2 3 Chapter 2 Droplet Generation 2.1 Sample Preparation 2.2 Operation of the QX200 Droplet Generator 2.3 Preparation for PCR 2.4 Subsequent Steps 5 5 6 10 12 Chapter 3 Specifications and Maintenance 3.1 Specifications 3.
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1 QX200™ Droplet Generator 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 Generator 1.2 QX200 Droplet Generator The QX200 droplet generator uses microfluidics to combine oil and water (sample) to create the droplets required for ddPCR analysis. It generates droplets from up to eight samples at a time in about 2 minutes. Following reaction preparation using the appropriate ddPCR supermix, 20 μl each of up to eight prepared samples (or blanks) and droplet generator oil are transferred to the droplet generator (DG8™) cartridge.
Chapter 1 QX200 Droplet Generator Table 1.2. Additional materials required for droplet generation.
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2 Droplet Generation 2.1 Sample Preparation Prepare the PCR reaction by combining 2x PCR supermix, 20x primers and probe, and DNA sample. Mix by vortexing in short pulses; centrifuge briefly. ■■ ■■ ■■ ■■ ■■ ■■ The concentration of intact human genomic DNA should be <66 ng per 20 µl reaction. If using higher concentrations, digest DNA with a restriction endonuclease that does not cut target or reference amplicons Use one of the PCR supermixes recommended in Table 1.
Chapter 2 Droplet Generation 2.2 Operation of the QX200™ Droplet Generator The QX200 droplet generator prepares droplets for up to eight samples at a time. Droplet generation takes ~2 minutes for each set of eight samples (~30 minutes for a 96-well plate). ■■ ■■ All 8 sample wells in the DG8 droplet generator cartridge must contain sample (or 1x buffer control), and all 8 oil wells must contain droplet generator oil Do not load sample or oil into the DG8 cartridge unless it is inserted in the holder 1.
Chapter 2 Droplet Generation 2. Transfer 20 μl of each prepared sample to the sample wells (middle row) of the DG8 cartridge. Air bubbles can cover the bottom of the well and result in 2,500–7,000 fewer droplets and poor data quality. They are difficult to see. To avoid creating air bubbles, use the following pipetting technique, which also ensures samples wet the bottoms of the wells so they are wicked into the microchannels (necessary for proper droplet generation).
Chapter 2 Droplet Generation 4. Using a multichannel pipet, fill each oil well (bottom row) with 70 μl DG oil from the reagent trough. Filling the oil wells with droplet generator oil. 5. Hook the gasket over the cartridge holder using the holes on both sides. The gasket must be securely hooked on both ends of the holder; otherwise, pressure sufficient for droplet generation will not be achieved. Correct placement of the gasket over the cartridge holder. 6.
Chapter 2 Droplet Generation Press to open and close Status indicator lights QX200 droplet generator with DG8 cartridge in place. 8. When droplet generation is complete, all three indicator lights are solid green. Open the door by pressing the button, and remove the holder (with DG8 cartridge still in place) from the unit. Remove the disposable gasket from the holder and discard it.
Chapter 2 Droplet Generation 2.3 Preparation for PCR 1. Pipet 40 μl of the contents of the top wells (the droplets) into a single column of a 96-well PCR plate.
Chapter 2 Droplet Generation 2. Seal the PCR plate with foil immediately after transferring droplets to avoid evaporation. Use pierceable foil plate seals that are compatible with the PX1™ PCR plate sealer and the needles in the QX200 droplet reader (for example, catalog #181-4040). Follow the instructions in the PX1 PCR Plate Sealer Instruction Manual (bulletin 10023997). a. Set the plate sealer temperature to 180°C and time to 5 sec. b. Touch the arrow to open the PX1 tray door.
Chapter 2 Droplet Generation 2.4 Subsequent Steps Once the 96-well plate containing the droplets is sealed, place it into the thermal cycler for PCR amplification. Refer to the supermix product inserts for cycling conditions. When PCR amplification is complete, remove the 96-well plate from the thermal cycler and read the droplets using the QX200 droplet reader (follow the instructions in the QX200 Droplet Reader Instruction Manual, bulletin 10031906).
3 Specifications and Maintenance 3.1 Specifications 5 inches (127 mm) 11 inches (279 mm) 14 inches (356 mm) Weight 10 lb. (4.
Chapter 3 Specifications and Maintenance 3.2 Maintenance 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 186-3030 Catalog # 186-4001 Droplet Generator Oil for Probes, 2 x 7 ml 186-3005 Droplet Generator Oil for Probes, 10 x 7 ml 186-4005 Droplet Generator Oil for EvaGreen Dye, 2 x 7 ml 186-4006 Droplet Generator Oil for EvaGreen Dye, 10 x 7 ml 186-3031 Droplet Reader Oil, 1 x 1 L 186-3004 Droplet Reader Oil, 2 x 1 L 186-4002 186-4003 186-4007 Description QX200™ Droplet Digital™ PCR System, includes droplet generator, droplet reader, laptop c
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
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