Chai Open qPCR User Manual
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Chai Open qPCR User Manual

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Open qPCR User Manual
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Open qPCR User Manual
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Summary of Contents for Chai Open qPCR

  • Page 1 Open qPCR User Manual Research use only Open qPCR User Manual Revision A...
  • Page 2 DISCLAIMS ALL WARRANTIES, EITHER EXPRESS OR IMPLIED, REGARDING THIS MANUAL AND ANY INFORMATION CONTAINED HEREIN, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A SPECIFIC PURPOSE. IN NO EVENT SHALL CHAI BE LIABLE FOR ERRORS OR FOR INCIDENTAL OR CONSEQUENTIAL DAMAGES RELATING TO THE FURNISHING, USE, OR PERFORMANCE OF THIS DOCUMENT OR OF ANY INFORMATION CONTAINED HEREIN.

  • Page 3: Table Of Contents

    TABLE OF CONTENTS PREFACE Intended Use Safety Conventions Safety Warnings and Precautions Marks of Conformity Technical Support CHAPTER 1: The Open qPCR System Introduction Instrument Technical Specifications General Features Heating & Cooling Optics Supported Consumables CHAPTER 2: Installation Getting Started...
  • Page 4 CHAPTER 6: System Settings Manage Users Software Update Network Settings Diagnostics CHAPTER 7: Maintenance Cleaning and Disinfecting Open qPCR Return and Repair CHAPTER 8: Troubleshooting Amplification Curves Melt Curves Factory reset Appendix A - Glossary...

  • Page 5: Preface

    The Open qPCR User Manual serves to provide a comprehensive understanding of the setup, operation and maintenance of the Open qPCR system. The Open qPCR instrument is intended for Research Use Only (RUO). Results produced from the instrument are not for use in diagnostic procedures.

  • Page 6: Safety Warnings And Precautions

    The information in this manual is intended to supplement the normal safety requirements established in the user’s country. The Open qPCR instrument complies with safety standards IEC 61010-1, level of pollution 2, and overvoltage category II. General Safety The Open qPCR Instrument must be connected to a 3-wire grounded outlet with the correct voltage rating.

  • Page 7: Marks Of Conformity

    Follow all local, state/provincial, and/or national regulations for proper disposal of the instrument calibrators. Marks of Conformity Icon Definition This device complies with Part 15 of the FCC Rules. Operation is subject to the following two conditions: (1) this device may not cause harmful interference, and (2) this device must accept any interference received, including interference that may cause undesired operation.

  • Page 8: Technical Support

    Technical Support Customer support is provided for all technical and service issues related to the Open qPCR instrument. Support Site Please visit https://support.chaibio.com for support-related inquiries. You will find resources and tips for getting started as well as troubleshooting help on this site.

  • Page 9: Chapter 1: The Open Qpcr System

    5 °C/s, the system’s heating and cooling efficacy results in rapid cycling and leads to assay completion in as little as 20 minutes. Multiple Open qPCR units may be connected to one computer via Wi-Fi or Ethernet, allowing simultaneous analysis for as many samples as desired.

  • Page 10: General Features

    Relative humidity: 20 – 80% non-condensing Max altitude: 2,000 m Power Voltage: 100 – 240 V* Frequency: 50 – 60 Hz Average Power: 75 W** Peak Power: 350 W System Specifications Samples: 16 wells, 100 µL tubes Sample volume: 10 – 50 µL Sensitivity: 1 copy Dynamic range: 10 Dimensions:...

  • Page 11: Heating & Cooling

    Figure 1.2 1.4 Heating & Cooling The Open qPCR’s dual-Peltier design and heated lid provide for precise temperature control of the individual reactions. With one side of the Peltier modules attached to a heat sink, the opposite side can then be cooled and heated with respect to the heat sink (Figure 1.3).

  • Page 12: Optics

    Open qPCR’s fast ramp rate of up to 5 °C/s allows for rapid thermal cycling, resulting in quick turnaround times from start to finish. The instrument’s firmware controls the thermal cycling temperature by using a control loop to ensure rapid heating and to control temperature overshoot around the desired point.

  • Page 13: Supported Consumables

    554 nm 1.6 Supported Consumables The instrument utilizes 100 µL low-profile PCR tube strips. Due to Open qPCR’s unique architecture, both cap and tube sides must be optically clear and they must not auto-fluoresce. For optimal results, please use Chai’s validated DNAse and RNAse-free tubes.

  • Page 14: Chapter 2: Installation

    Do not touch the power switch or power cord with wet hands. When the Open qPCR system is not in use, ensure that the instrument lid is closed and secured in place. This avoids any dust or debris, which may interfere with the system performance.
  • Page 15 IP address over the Internet. Option A - Connecting to Open qPCR via USB On the computer being used to connect to the Open qPCR, open a browser and navigate to the following link: http://beagleboard.org/getting-started#step2...

  • Page 16: Setting A Static Ip Address

    IP address to access the software. 2.3 Setting a Static IP Address The Open qPCR system must be connected by Ethernet in order to set a static IP address. If applicable, consult with your IT department before proceeding.

  • Page 17: Logging In And Creating An Account

    Figure 2.2 3) Proceed to set the appropriate static IP address and select Save Changes once complete. 4) Once the changes are saved, the screen will automatically refresh to the home page. The new IP address will display on both the current web browser and the instrument’s LCD screen.

  • Page 18: Chapter 3: Calibration

    CHAPTER 3 – Calibration 3.1 When to Calibrate the Open qPCR The Open qPCR needs to be calibrated before its initial use. Calibration must also be run each time any of the following events occur: The lid height is adjusted...

  • Page 19: Open Qpcr Single Channel Calibration

    Always make sure that your tubes are dry externally and capped tightly before inserting them into the Open qPCR. Failure to do so may cause liquids to vaporize and create deposits on the internal components of the instrument. 3.3 Open qPCR Single Channel System Calibration The Open qPCR single channel system requires calibration with the Fluorescein Calibrator kit.
  • Page 20 Preparing for Calibration The Fluorescein solution is light sensitive. Keep aliquoted solution protected from light. If you are using calibrators that have previously been stored at 4 °C, you do not need to equilibrate the solution to room temperature before running the calibration process. You may use the calibrator solution directly from the fridge.
  • Page 21 Figure 3.3 4. The screen will prompt you to prepare 4 new 8-well PCR tubes (Figure 3.4). Gather the previously prepared strip solutions and select Begin. Figure 3.4 5. Insert two water strips into the instrument. Press down on the tubes gently once they are inserted.
  • Page 22 6. Close the lid, making sure that the latch clicks and select Next. You will see the screen below during the lid heating and data collection process. Figure 3.6 7. Allow the instrument to read fluorescence for 2-3 minutes before proceeding to the next step.

  • Page 23: Open Qpcr Dual Channel Calibration

    3.4 Open qPCR Dual Channel System Calibration The Open qPCR dual channel system requires calibration with the FAM + HEX Calibrator Set. The calibrators are provided in lyophilized format. Prior to calibrating the Open qPCR, ensure that the lyophilized calibrators are reconstituted thoroughly (~ 45 minutes).
  • Page 24 Do not freeze the calibrator once reconstituted. Materials Required for Calibration FAM/HEX calibrator solution Distilled or deionized water 200 µL pipette 200 µL pipette filter tips Chai PCR Tube & Cap Strips, 8-Well Strips, Optically Clear, 100 µL PCR tube rack...
  • Page 25 Mini centrifuge Reconstitution Procedures Please note that the lyophilized calibrators may be difficult to see. 1. Before opening the calibrator tubes, use a mini centrifuge to spin down the lyophilized HEX and FAM vials for 10 seconds. It is important to gather all materials at the bottom of the tube.
  • Page 26 5. Prepare the water strips by aliquoting 25 µL/tube of water into 2 new PCR tube strips. Cap strip tubes tightly and spin down. Calibration Procedures Please note that the calibration process must be completed in one sitting. The heat block and lid temperature rises above 95 ° C. To avoid injury, do not make direct contact with the heat block, lid, or tubes immediately after a run.
  • Page 27 Figure 3.13 5. Insert two water strips into the instrument. Press down on the tubes gently once they are inserted. Adjust the lid height per the instructions in section Adjusting the Open qPCR Lid Height. Figure 3.14 6. Close the lid, making sure that the latch clicks and select Next. You will see the screen below during the lid heating process.
  • Page 28 7. Allow the instrument to read fluorescence for 2-3 minutes before proceeding to the next step. Figure 3.16 8. Once the reading completes, insert the FAM strips (Figure 3.17). Figure 3.17 9. Repeat steps 6 - 7 for both the FAM and HEX strips. 10.
  • Page 29 Figure 3.18 11. If calibration fails, you will see a screen displaying Calibration Failed (Figure 3.19). Repeat the calibration process in such instances. If failure persists on repeat, contact Technical Support. Figure 3.19 It is not recommended to reuse the calibration dyes because photo-bleaching occurs at different levels from well to well.

  • Page 30: Chapter 4: Assay Setup

    CHAPTER 4 – Assay Setup 4.1 General PCR Considerations It is important to minimize sample contamination and PCR product carryover when setting up a new experiment. Please take note of the following precautions: Wear a clean lab coat, clean gloves, and eye protection when preparing samples for PCR amplification.
  • Page 31 Figure 4.3 TEMPERATURE DETAILS Temperature details are displayed on the bottom left of the page. The Temperature, Ramp Speed, and Hold Duration can be manually adjusted by clicking directly on the values. Ramp ° speeds can be set between a range of 0.00001 and 5 C/s.
  • Page 32 ADD STEP On the right side of the page, there is an option to Add Step (Figure 4.6). Designate the respective sections to add the additional step(s). To do so, click on the arrows at either side of the screen or click directly on the appropriate stage in the orange section; the editable step will be bordered in black (Figure 4.7).
  • Page 33 Melt Curve Stage A melt curve is typically performed immediately after the amplification protocol in one continuous sitting. The purpose is to determine specificity of the PCR reaction and to check whether nonspecific amplification products, such as primer dimers, were formed. Refer to Section 1.4 Melt Curve Analysis for more details.
  • Page 34 Figure 4.10 DEFINE CYCLE NUMBER To set the number of cycles for a certain stage, click directly on the underlined default 40x Cycles shown in Figure 4.11 and type in the value. Figure 4.11 EDIT STAGES To delete or reposition a step, click on Edit Stages located on the right-hand side. To delete a step, select the Ä...

  • Page 35: Starting & Cancelling Runs

    ESTIMATED TIME TO COMPLETE To view the estimated time until test completion before starting a run, click anywhere on the right panel of the protocol. Once clicked, the Est. Time to Complete will be displayed on the page layout’s palette on the bottom portion. Figure 4.14 4.3 Starting &...

  • Page 36: Chapter 5: Result Analysis

    CHAPTER 5 – Result Analysis 5.1 Amplification Curve Depending on the protocol specifications, run results may be viewed as either an amplification curve, melt curve, or thermal profile. The default setting displays the amplification curve on the result screen (Figure 5.1). The plot may be viewed in either log or linear format. During a run, amplification curves are generated and can be monitored in real-time.
  • Page 37 Figure 5.3 Cq Calling: The software defaults to the Cy0 method for Cq calling as it is recommended. You may choose to select cpD2 for data analysis as the characteristics of the data itself can affect how Cq methods perform. Parameters: The parameters section is set to default at the values shown in Figure 5.3.
  • Page 38 The algorithm performs baseline subtraction on background subtracted values. Background subtracted values are obtained after the algorithm adjusts for well-to-well variations and deconvolution (for dual channels only) on the amplification data. Baseline subtraction is turned on by default. This can be turned off by toggling the Baseline Subtraction option on the top left of the amplification plot.

  • Page 39: Melt Curve

    Channels may also be turned on or off by toggling between the options (Figure 5.8). Figure 5.8 Single Channel Model For the single channel Open qPCR, amplification curves may only be viewed in either Log or Linear format. There is no option for differentiating between channels since there is only one channel.
  • Page 40 Melt curve analysis may also be used for genotyping purposes based on the differences in melt temperature between alleles. There are two types of curves used in the melt analysis application: normalized and derivative. Normalized Plot: This displays the various temperature increments on the x-axis and the normalized fluorescence signal intensity on the y-axis.

  • Page 41: Thermal Profile

    5.3 Thermal Profile To access the thermal profile, click on the arrow next to Amplification Curve at the top left of the page for a specific experiment. Select Thermal Profile (Figure 5.11). Figure 5.11 The thermal profile graph displays time on the x-axis and heat block temperature on the y-axis. You can manually hover over the graph to view the heat block and lid temperature at each time point.

  • Page 42: Data Export

    Back to Home: Returns to the home screen Experimental Properties: Displays the lid temperature throughout the experiment View Protocol: Displays the initial protocol setup page for the experiment View Result: Displays the results screen Export: Downloads the experiment file for further data analysis. Delete: Permanently deletes the experiment run from the database 5.5 Data Export Data can be exported via a .csv file.

  • Page 43: Changing An Experiment Name

    temperature_log.csv contains data for the lid and heat block temperatures per cycling period. 5.6 Changing an Experiment Name To change the name of your experiment post-run, select the icon on the top left of the results screen. Proceed to click on the experiment name (Figure 5.15) to delete before typing in the new name.

  • Page 44: Chapter 6: System Settings

    (Figure 6.3). Figure 6.3 USB: If the Open qPCR is connected via USB, select Download Image. This will download as a zip file. Do not open the zip file once downloaded. Select Browse & Install to upload the zip file.

  • Page 45: Network Settings

    Technical Support. 6.3 Network Settings You may connect your Open qPCR instrument to any available network via Ethernet or Wi-Fi. Ensure that your computer is connected to the same network as the instrument. You have the option to adjust your Ethernet settings as necessary under the Network Settings tab.

  • Page 46: Diagnostics

    6.4 Diagnostics The diagnostics section checks the thermal and optical integrity of the system. To perform either test, click on the respective Run Now tab. Instructions will be displayed on the screen once you begin. Run times for each diagnostic is approximately 5 minutes. Figure 6.6 Thermal Performance The thermal performance diagnostic tests the lid heating as well as the heating and cooling...
  • Page 47 Figure 6.8 (Dual Channel) Figure 6.9 (Single Channel)

  • Page 48: Chapter 7: Maintenance

    70% ethanol solution (dilute with water) to disinfect the surface or the wells. Complete with a water cleanse. Bleach is corrosive to the Open qPCR body. It is important that you follow the 10% bleach solution immediately (no longer than 30 seconds) with a 70% ethanol solution.

  • Page 49: Open Qpcr Return And Repair

    7.2 Open qPCR Return and Repair If the instrument needs to be returned for service, it must be disinfected prior to shipment. For direct customers of Chai, please contact Chai Technical Support for a copy of the Decontamination Certificate to initiate the return process.

  • Page 50: Chapter 8: Troubleshooting

    CHAPTER 8 – Troubleshooting 8.1 Amplification Curves Amplification Curves Problem Cause Solution Increase enzyme a. Low enzyme concentration or use a concentration different enzyme Inconsistent data where replicate b. Low target copy samples do not Increase sample number below the LOD for display similar concentration the assay...
  • Page 51 Amplification Curves Problem Cause Solution d. Instrument not Calibrate the instrument calibrated No amplification Ensure that the channel e. Dye detector assigned used to detect the to incorrect channel fluorophore matches correctly a. Master mix preparation Confirm master mix error calculations b.
  • Page 52 Amplification Curves Problem Cause Solution Design specificity: Confirm there are no mismatches between target and primer/probe sequences by performing a BLAST search to confirm specificity or target and assay sequences. Also confirm that the primer/probe does not span a SNP site a.
  • Page 53 Air bubbles present or Spin down tubes for 10 droplets adhered to tube seconds using a walls microcentrifuge Jagged curves Use Chai's validated PCR- strip tubes. The instrument c. Incorrect tube type requires 100 µL optically used clear, non-auto fluorescent tubes a.
  • Page 54 Amplification Curves Problem Cause Solution a. Reagent or genomic Repeat protocol with fresh contamination reagents Amplification in No Template Control (NTC) b. Primer-dimer Use a master mix with good formation hot start activity Reset baseline to 3 cycles Looping Data a.

  • Page 55: Melt Curves

    To perform the factory image reset: 1. Turn off your Open qPCR. Disconnect any USB cables. 2. Using a toothpick, press the reset button through the small hole in the rear of the machine.
  • Page 56 3. While holding down the reset button, power on the Open qPCR. Continue to hold the reset button down for 30 seconds. 4. Release the reset button. Keep the machine powered on for about 30 minutes. After 30 minutes, the device will reboot, and the Open qPCR logo will be displayed on the LCD.

  • Page 57: Appendix A - Glossary

    Amplification Plot – Displays a plot of the amplification cycles (X axis) versus fluorescence units (Y axis) Assay – Pertaining to the Open qPCR platform, this is a test that contains a PCR master mix and specific primers/probes to amplify a target sequence Background Fluorescence –...
  • Page 58 Singleplex PCR – This type of PCR contains a single primer set in the tube/well where only target or endogenous control can be amplified at a time. VIC – A fluorescent dye with l (absorption) at 538 nm and l (emission) at 554 nm...
  • Page 59 Corporate Headquarters 990 Richard Ave, Suite 110 Santa Clara, CA 95050 Toll-Free: +1(800) 642-4002 International: +1(650) 779-5577 www.chaibio.com...

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