Automated Droplet Generator Instruction Manual Catalog #186-4101
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.
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: ■■ ■■ ■■ ■■ I EC 61010-1:2010 (3rd ed.), EN61010-1:2010 (3rd ed). Electrical Equipment for Measurement, Control, and Laboratory Use — Part 1: General requirements N 61326-1:2006 (Class A). Electrical equipment for measurement, control, and laboratory use.
Instrument Safety Warnings Alteration of this instrument voids the warranty and safety certification and creates a potential safety hazard. This instrument is intended for laboratory use only. Bio-Rad Laboratories 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 or an authorized agent.
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 ■■ ■■ 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 ■■ Change gloves
Table of Contents Chapter 1. QX200™ AutoDG™ Droplet Digital™ PCR System . . . . . . . . . . 1 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 System Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Installation and General Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 System Setup and General Operation Instructions . . . . . . . . . . . . .
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1 QX200™ AutoDG™ Droplet Digital™ PCR System 1.1 Introduction Bio-Rad’s QX200 AutoDG Droplet Digital PCR (ddPCR™) system combines water-oil emulsion droplet technology with microfluidics to perform accurate and precise digital PCR. The system consists of two instruments — an Automated Droplet Generator and a Droplet Reader — and associated consumables.
QX200 AutoDG Droplet Digital PCR System The ddPCR System lets you: ■■ ■■ ■■ ■■ ■■ ■■ Achieve absolute quantification without the use of a standard curve Detect rare DNA target copies with unmatched sensitivity Determine copy number variation with unrivaled accuracy Measure gene expression levels with precision Design scalable assays for high sensitivity or high throughput Expand applications using flexible ddPCR chemistry — EvaGreen or probes Applications and uses include: ■■ ■■ ■■ ■■ ■■ Copy number va
1.2 System Components The system’s guided quick load allows users to get started faster. The large, color touch screen provides simple setup and plate tracking. Select the desired number of columns across a 96-well plate, load Droplet Generation Oil and only the consumables you need as prompted by the system, and start the run. A countdown timer will display the time remaining so users can maximize their time in the lab. When the plate is ready, users are notified and a time-elapsed counter begins.
QX200 AutoDG Droplet Digital PCR System Table 2. Additional materials required for Droplet Digital PCR.
1.4 System Setup and General Operation Instructions 3. P erform PCR — remove the 96-well PCR plate containing droplets from the Automated Droplet Generator, seal the plate with foil, and perform PCR to end point (~40 cycles) using a PX1™ Plate Sealer and C1000 Touch™ Thermal Cycler (see Table 2). 4. R ead droplets — load the plate into the QX200 (or QX100) Droplet Reader and start your run.
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2 Using the Automated Droplet Generator 2.1 Powering On the Automated Droplet Generator The Automated Droplet Generator (AutoDG™) is designed to remain powered on in order to preserve positive airflow inside of the instrument and track consumable use. The instrument stays in an idle state when not being used. Please see Section 2.2 for instructions on starting a run from idle mode. 1.
Using the Automated Droplet Generator 2. If the instrument deck is empty, the indicator lights on the deck of the Automated Droplet Generator should be off, indicating that no consumables are present. The corresponding areas of the touch screen will be gray. Fig. 2. Home screen, no consumables loaded. If consumables were present in the instrument when power was lost or disconnected, an error message will appear prompting you to check and reset the consumables if necessary.
2.2 Starting a Run from Idle Mode on the AutoDG 2.2 Starting a Run on the AutoDG Instrument 1. Bring the Automated Droplet Generator out of idle mode by touching the screen. Please wait while the instrument performs a self-check. 2. Check the indicator lights on the deck of the AutoDG Instrument nd the consumable icons on the touch screen. Table 3 explains the deck lighting and touch screen icons and the instrument status they indicate. Table 3.
Using the Automated Droplet Generator A B Fig. 4. A, Home screen; B, Configure Sample Plate screen. A B C Fig. 5. Configure Sample Plate screen with A, full plate of samples selected; B, half plate of samples selected; C, alternating columns of samples selected. 4. Based on the number of columns selected in the previous step, the consumable icons on the screen will begin to blink yellow to indicate where new consumables need to be loaded into the instrument.
2.2 Starting a Run from Idle Mode on the AutoDG Fig. 6. The Automated Droplet Generator keeps track of the consumables so long as it is connected to a power supply. Based on the number of columns configured, the system will prompt for only the number of consumables that need to be loaded to complete the run. In this example, a full plate has been selected. 5. Open the door on the AutoDG Instrument by lifting up on the handle at the front of the instrument.
Using the Automated Droplet Generator A B Fig. 7. A, DG32 Cartridge holders on the AutoDG deck in the “Load” state, indicated by yellow lights; B, green lights indicate the DG32 Cartridge has been correctly loaded and is ready. Fig. 8. DG32 consumables have been correctly loaded. The Ready status appears on the DG32 icons on the screen. 7. To load the AutoDG Pipet Tips along the center row of the instrument: a.
2.2 Starting a Run from Idle Mode on the AutoDG Fig. 9. Pipet tip boxes have been correctly loaded. The Ready status appears on the Pipet Tip icons on the screen. 8. To load the 96-well PCR plate containing your prepared ddPCR reactions into the front row of the instrument: a. The sample plate can be sealed with a PX1™ Plate Sealer and heat-sealing foil in advance of loading into the AutoDG Instrument. Each well should contain 22 µl of your prepared ddPCR reaction.
Using the Automated Droplet Generator The cooling block should be placed in a –20°C freezer for at least 2 hours before configuring a run on the AutoDG Instrument and inserting the Droplet Plate assembly into the instrument. The block goes from a pink color at room temperature to a dark purple color when properly cooled. 9. To load the Droplet Plate assembly: a. Remove the cooling block from the freezer and place into the front right plate holder, labeled on the screen as Droplet Plate.
2.2 Starting a Run from Idle Mode on the AutoDG ? Why do I need a cooling block? The AutoDG Instrument uses a cooling method to prevent droplet evaporation, much like the Droplet Generator requires you to cover the wells once droplets are transferred. 10. To load Automated Droplet Generation Oil into the instrument: a. Remove the cap from the bottle of Automated Droplet Generation Oil and twist the bottle into the tower of the oil delivery system at the front left corner of the instrument. b.
Using the Automated Droplet Generator Automated Droplet Generation Oil for EvaGreen should be used only in combination with the QX200™ ddPCR system and ddPCR EvaGreen Supermix. The use of other supermixes can lead to poor results and damage to the QX200 Droplet Reader. The QX100™ ddPCR System does not support EvaGreen chemistry; running this chemistry will damage the instrument. 11.
2.2 Starting a Run from Idle Mode on the AutoDG 12. Once you have confirmed the plate setup, touch the Start Run button to begin droplet generation. The door will automatically close at the beginning of the run and must remain closed during the run. Opening the door before the Droplets Ready message appears will cause the instrument to terminate the run. If the selected columns on the confirmation screen do not match the location of samples in your sample plate, touch Back to change.
Using the Automated Droplet Generator If for any reason you need to stop the run, touch the Abort button on the countdown timer screen. A confirmation window will appear; touch Yes to abort the run or No to return to the countdown timer. Please note that stopping the Automated Droplet Generator during a run can result in loss of the current column of samples being processed. A B Fig. 18. A, touching the Abort button will bring up a confirmation screen; B, system display with time since run aborted. 14.
2.2 Starting a Run from Idle Mode on the AutoDG If any errors were encountered during the run, an error notification will appear below the count-up timer. Touch the Errors encountered button to bring up the run logs and identify the error. Fig. 20. Droplet generation is complete. Please wait to remove the oil bottle until the orange message disappears from the screen. 15. Remove the droplet plate containing ddPCR droplets and seal within 30 min of droplet generation completing.
Using the Automated Droplet Generator Begin thermal cycling (PCR) within 30 min of sealing the plate, or store the plate at 4°C for up to 4 hr prior to thermal cycling. Refer to the supermix product inserts for cycling conditions. Fig. 21. PX1 PCR Plate Sealer (left) and a sealed 96-well plate (right). 2.3 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.
2.4 Advanced Loading and Switching of Automated Droplet Generation Oils 2.4.1 Loading a Bottle of Oil before Configuring a Plate To save time at the beginning of a run, a bottle of Automated Droplet Generation Oil can be loaded into the instrument, and the oil delivery system primed, at any point. When there is no bottle of oil loaded into the instrument, the Oil Level display on the screen will appear gray to indicate it is empty. The Oil Type display on the screen will not display an oil type. Fig. 22.
Using the Automated Droplet Generator 5. The instrument will check that there is oil in the bottle. If you did not load a bottle (or insert the bottle completely) as described in step 2, you will be prompted to load a bottle of the oil type that you selected in the previous screen. Once correctly loaded, the AutoDG Instrument will begin the flush and prime routine. A B C Fig. 24.
2.4 Advanced Loading and Switching of Automated Droplet Generation Oils A B Fig. 26. A, home screen displaying a full bottle of AutoDG Oil for Probes loaded; B, a partial bottle of AutoDG Oil for Probes loaded. 2.4.2 Changing between AutoDG Oil Types Changing between Automated Droplet Generation Oils can be performed at any time, and the oil delivery system primed. The flush and prime when changing chemistries is important for good ddPCR results.
Using the Automated Droplet Generator A B Fig. 27. An example of switching oil types. A, oil selection display with Probes selected; B, Change Oil button activated by touching the EvaGreen droplet. 6. The instrument will check that a new bottle has been properly loaded and that there is oil in the bottle. If you did not load a bottle (or insert the bottle completely) as described in step 3, you will be prompted to load a bottle of the oil type that you selected in the previous screen.
2.5 Accessing Run Logs Fig. 28. AutoDG Instrument run log displaying runs completed or terminated. Fig. 29. Example of Run Details for a run terminated by the system due to a pipet tip box lid being left on when placed in the instrument. The run details are displayed on the left of the screen and the error details on the right.
Using the Automated Droplet Generator To export the run log: 1. Touch the Run Log button on the home screen. 2. Remove the AutoDG Instrument’s side panel to the right of the touch screen. Slide the magnetic panel down to release, then pull straight out to remove the panel. 3. Insert a USB key into the USB port on the side of the instrument. 4. Touch the Export All button at the bottom left of the screen. 5. Wait while the system exports the run logs to the USB key. 6.
3 Specifications and Maintenance 3.1 Specifications 35 inches (89 cm) open 26 inches (66 cm) 22 inches (56 cm) 26 inches (66 cm) Weight 100 lb (45.
Specifications and Maintenance 3.2 Maintenance 3.2.1 General Care 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 on all surfaces except the door. Do not use acetone or tap water. Inspect equipment regularly for damaged external components or wiring. Do not use if damaged.
3.2 Maintenance 3.2.3 Cleaning the Oil Waste Reservoir It is recommended that you clean the oil waste reservoir on a quarterly basis, or more frequently if switching Droplet Generation Oil chemistries (probe/EvaGreen) frequently. The oil waste reservoir collects Droplet Generation Oil during the flush and prime routine; it is unused oil that has not come into contact with the ddPCR™ reaction. To access the oil waste reservoir: 1. Touch the Settings button on the home screen. 2.
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4 Automated Droplet Generator Troubleshooting The Automated Droplet Generator provides onscreen troubleshooting should an error occur. Guidance will be provided as to how to best resolve the error, or the instrument will advise that Bio-Rad Technical Support be contacted. 4.1 Restarting a Run after an Error If the AutoDG™ Instrument encounters an obstruction during the run, it will stop the run and display an error message with the area of the deck that needs attention. 1.
Automated Droplet Generator Troubleshooting Fig. 31. Example of error message displayed when the lid is left on the box of tips placed in the left tip box holder. The run stops and an error message indicating the position of the error displays on the touch screen. 4.2 Restarting a Run after a System Error If the AutoDG Instrument encounters a system error during the run, it will stop the run and display an error message requiring power cycling. 1. Unplug the AutoDG Instrument from the power supply. 2.
4.3 Exporting Logs for Technical Support 3. The AutoDG Instrument will not be able to recall the status of all consumables and will require manual confirmation (see Section 2.1). 4. Reset the unused consumables and touch the Configure Sample Plate icon on the home screen. 5. Select the columns of samples remaining to be processed on the screen and touch OK. 6. Touch the Start Droplet Generation icon to restart the run. 4.
Automated Droplet Generator Troubleshooting 4.4 Additional Troubleshooting Use the following table to help you troubleshoot issues with your AutoDG Instrument. If you have additional questions or if the recommended action does not resolve the problem, please contact Bio-Rad Technical Support. Table 4. Additional troubleshooting for the AutoDG Instrument.
Ordering Information QX200™ AutoDG™ ddPCR™ System Catalog # Description 186-4100 QX200 AutoDG Droplet Digital PCR System, includes Automated Droplet Generator, QX200 Droplet Reader, laptop computer, QuantaSoft Software 186-4101 Automated Droplet Generator, includes Automated Droplet Generator, power cord, and accessories 186-4003 QX200 Droplet Reader, includes droplet reader, ddPCR manual, plate holders, USB cable, power cord 186-4108 DG32 Automated Droplet Generator Cartridges, 1 pkg of 30, enoug
Ordering Information ddPCR Reagents (continued) Catalog # Description 186-3028 ddPCR Supermix for Probes, 50 ml (10 x 5 ml), 2x supermix 186-4033 QX200 ddPCR EvaGreen Supermix, 2 ml (2 x 1 ml), 2x supermix 186-4034 QX200 ddPCR EvaGreen Supermix, 5 ml (5 x 1 ml), 2x supermix 186-4035 QX200 ddPCR EvaGreen Supermix, 25 ml (5 x 5 ml), 2x supermix 186-4036 QX200 ddPCR EvaGreen Supermix, 50 ml (10 x 5 ml), 2x supermix 186-3021 One-Step RT-ddPCR Kit for Probes, 2 ml (2 x 1 ml), 2x supermix 186-3022
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