Qiagen PyroMark Q24 MDx User Manual
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Qiagen PyroMark Q24 MDx User Manual

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Version 1
January 2016
®
PyroMark
Q24 MDx User Manual
IVD
MAT
1063078EN
QIAGEN GmbH, QIAGEN Strasse 1, 40724 Hilden, GERMANY
R4
Sample & Assay Technologies

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Summary of Contents for Qiagen PyroMark Q24 MDx

  • Page 1 Version 1 January 2016 ® PyroMark Q24 MDx User Manual 1063078EN QIAGEN GmbH, QIAGEN Strasse 1, 40724 Hilden, GERMANY Sample & Assay Technologies...
  • Page 2 ® , Milli-Q ® (Merck KGaA); Sepharose ® (GE Healthcare). Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law. HB-0332-004 © 2011–2016 QIAGEN, all rights reserved.

  • Page 3: Table Of Contents

    Contents Contents Safety Information Proper use Electrical safety Biological safety Chemicals Mechanical hazards Heat hazard Consumables Symbols on the PyroMark Q24 MDx and PyroMark Q24 products Introduction About this user manual General information 2.2.1 Technical assistance 2.2.2 Policy statement 2.2.3...
  • Page 4 Contents Sample preparation with the PyroMark Q24 MDx Vacuum Workstation Analysis software Installation Procedures System delivery and installation Requirements Installation of the analysis software 4.3.1 Installing or upgrading PyroMark Q24 MDx Software 4.3.2 Uninstalling PyroMark Q24 MDx Software Operating Procedures Instrument administration 5.1.1...
  • Page 5 5.6.3 Checking the instrument 5-30 Backup of PyroMark Q24 files 5-30 Maintenance Checking the performance of the PyroMark Q24 MDx 6-1 Maintenance of the PyroMark Q24 MDx Instrument 6.2.1 Cleaning the instrument 6.2.2 Cleaning the heating block and light guides...
  • Page 6 Verification of correct installation and operation Glossary Appendix A Technical data Environmental conditions Waste Electrical and Electronic Equipment (WEEE) FCC declaration EC declaration of conformity Appendix B Assay design and validation Appendix C Liability clause Index Index-1 Contents-4 PyroMark Q24 MDx User Manual 01/2016...

  • Page 7: Safety Information

    Safety Information Safety Information Before using the PyroMark Q24 MDx, it is essential that you read this user manual carefully and pay particular attention to the safety information. The instructions and safety information in the user manual must be followed to ensure safe operation of the system and to maintain the system in a safe condition.

  • Page 8: Electrical Safety

    Safety Information WARNING Risk of personal injury and material damage The PyroMark Q24 MDx Instrument is too heavy to be lifted by one person. To avoid personal injury or damage to the instrument, do not lift the instrument alone. WARNING...

  • Page 9: Biological Safety

    * OSHA: Occupational Safety and Health Administration (United States of America). † ACGIH: American Conference of Government Industrial Hygienists (United States of America). ‡ COSHH: Control of Substances Hazardous to Health (United Kingdom). PyroMark Q24 MDx User Manual 01/2016...

  • Page 10: Chemicals

    COSHH: Control of Substances Hazardous to Health (United Kingdom). Mechanical hazards The lid of the PyroMark Q24 MDx Instrument must remain closed during operation of the instrument. An audible warning signal will alert you if the lid is opened when it is not safe.

  • Page 11: Heat Hazard

    To ensure proper ventilation, maintain a minimum clearance of 10 cm (3.94 in.) at the sides and rear of the PyroMark Q24 MDx Instrument. Slits and openings that ensure the ventilation of the PyroMark Q24 MDx Instrument must not be covered. Consumables CAUTION Unsupported consumables Do not connect or use any consumables, accessories, or external equipment other than that specified.

  • Page 12: Symbols On The Pyromark Q24 Mdx And Pyromark Q24 Products

    Safety Information Symbols on the PyroMark Q24 MDx and PyroMark Q24 products Symbol Location Language Description Type plate CE mark on the back of the instrument Type plate CSA listing mark for on the back Canada and the USA of the...
  • Page 13 Reagents, Material number solutions, cartridge, plate Reagents, Component solutions, cartridge, plate Reagents, Contains solutions, cartridge, plate Reagents, Number solutions, cartridge, plate Reagents, Use-by date solutions, cartridge, plate Reagents, Temperature limitation solutions, cartridge, plate PyroMark Q24 MDx User Manual 01/2016...
  • Page 14 Type plate Legal manufacturer on the back of the instrument and all other products Cartridge Keep away from (sun)light Cartridge Method number Cartridge Instrument parameters Inside the Warning, consult user instrument manual PyroMark Q24 MDx User Manual 01/2016...

  • Page 15: Introduction

    Thank you for choosing the PyroMark Q24 MDx. We are confident it will become an integral part of your laboratory. Before using the PyroMark Q24 MDx, it is essential that you read this user manual carefully and pay particular attention to the safety information (see Section 1).

  • Page 16: General Information

    (see back cover or visit www.qiagen.com). 2.2.2 Policy statement It is the policy of QIAGEN to improve products as new techniques and components become available. QIAGEN reserves the right to change specifications at any time. In an effort to produce useful and appropriate documentation, we appreciate your comments on this user manual.

  • Page 17: Intended Use Of Pyromark Q24 Mdx

    Introduction Intended use of PyroMark Q24 MDx PyroMark Q24 MDx is a system for detecting changes in specified variable positions in DNA that may have clinical relevance. The PyroMark Q24 MDx is intended for in vitro diagnostic use in Europe.
  • Page 18 Preventive Laboratory technicians Appropriately trained maintenance or equivalent and experienced personnel familiar with use of computers and automation in general Servicing and QIAGEN Field Service annual Specialists only preventive maintenance PyroMark Q24 MDx User Manual 01/2016...

  • Page 19: General Description

    PyroMark Q24 MDx Software: Software only  PyroMark Q24 MDx Vacuum Workstation: Vacuum  Workstation only PyroMark Q24 MDx System: All of the above, plus any  PyroMark Kits Pyrosequencing principle Pyrosequencing uses sequencing by synthesis for accurate and quantitative analysis of DNA sequences.
  • Page 20 (dATP) since it is used efficiently by the DNA polymerase, but not recognized by the luciferase. 9. As the process continues, the complementary sequence is built up and the nucleotide sequence is determined from the peak in the Pyrogram. PyroMark Q24 MDx User Manual 01/2016...

  • Page 21: Pyromark Q24 Mdx Principle

    Q24 Reagents and placed in the dispensing unit. 2. The USB stick containing the run file created using PyroMark Q24 MDx Software is inserted into the USB port at the front of the instrument. The run is then started by the user.

  • Page 22: Pyromark Q24 Mdx Instrument

    LED is lit when the cooling device is receiving power USB port (inactive) Light button for the coolant level window Window showing the coolant level Instrument power connector 24V Cooler power connector 12V PyroMark Q24 Instrument exterior PyroMark Q24 MDx User Manual 01/2016...

  • Page 23: Process Chamber

    If the room temperature is too high, the heating block is cooled by the PyroMark Q24 MDx Instrument Cooling Device (preinstalled). Data are collected from all the wells simultaneously by 24 CCDs underneath the heating block.

  • Page 24: Sample Preparation With The Pyromark Q24 Mdx Vacuum Workstation

    Instrument should be prepared according to the instructions in Section 5.3, using the PyroMark Q24 MDx Vacuum Workstation. Analysis software The PyroMark Q24 MDx is shipped with PyroMark Q24 MDx Software. The computer used for setup of runs and data analysis should have the following as minimum specifications: ®...

  • Page 25: Installation Procedures

    Installation Procedures Installation Procedures System delivery and installation The unpacking and installation of the PyroMark Q24 MDx is carried out by a certified QIAGEN Field Service Specialist. A person who is familiar with your laboratory and computer equipment should be present during the installation.
  • Page 26 115 V AC, 60 Hz, 25 VA  230 V AC, 50 Hz, 25 VA  Make sure that the voltage rating of the PyroMark Q24 MDx is compatible with the AC voltage available at the installation PyroMark Q24 MDx User Manual 01/2016...

  • Page 27: Installation Of The Analysis Software

    1. Ensure that the computer meets the minimum requirements; see Section 3.6. 2. Close any programs running on the computer. 3. Insert the PyroMark Q24 MDx Software CD into the CD drive. 4. In the CD menu, click “Install PyroMark Q24 MDx Software”.

  • Page 28: Uninstalling Pyromark Q24 Mdx Software

    Installation Procedures (see step 4) and click “Install PyroMark Q24 MDx Software”. 6. When the software has been successfully installed, click “Exit Setup” in the CD menu. 7. Please use Windows Update (www.update.microsoft.com) to check for any critical updates to the .NET Framework 3.5.

  • Page 29: Operating Procedures

    Operating Procedures Operating Procedures This section describes how to operate PyroMark Q24 MDx. Before proceeding, we recommend that you familiarize yourself with the features of PyroMark Q24 MDx Instrument by referring to Section 3.4. Instrument administration 5.1.1 Setting date and time...

  • Page 30: Copying Recently Saved Runs

    6. Remove the USB stick. 5.1.4 Copying log files If you need to send log files to QIAGEN Technical Services, copy files as follows: 1. When the instrument is not processing, insert one of the USB sticks supplied into the USB port at the front of the instrument.

  • Page 31: Extracting Damaged Runs

    2. Select the information you want to view and press “OK”. 5.1.7 Upgrading the instrument software If you have received a software upgrade from QIAGEN, upgrade the software as follows: 1. Save the upgrade files on one of the USB sticks supplied.

  • Page 32: Run An External Application

    5.2.1 Starting PyroMark Q24 MDx Software In the Windows “Start” menu, select “(All) Programs/PyroMark/PyroMark Q24”. The PyroMark Q24 MDx Software User Guide can be accessed at any time by pressing the “F1” key when in the software. 5.2.2 Setting up an assay 1.

  • Page 33: Setting Up A Run

    4. Add an assay to each well used, e.g., drag an assay from the shortcut browser to a well or a selection of wells. A well is colored according to the assay loaded into the well. PyroMark Q24 MDx User Manual 01/2016...
  • Page 34 ID (if the last part of the entered sample ID is a number). For more information, see the PyroMark Q24 MDx Software User Guide (press the “F1” key when in PyroMark Q24 MDx Software). PyroMark Q24 MDx User Manual 01/2016...

  • Page 35: Managing Instrument Methods

    “Save As”. 2. Enter a name for the new method and press “Enter”. 3. Change the method settings in the dialog box to match those posted on www.qiagen.com/Products/PyroMarkQ24MDx.aspx. 4. Click “Save”. PyroMark Q24 MDx User Manual 01/2016...

  • Page 36: Sample Preparation

    Note Note about the instrument method (optional). Sample preparation Samples to be analyzed using the PyroMark Q24 MDx Instrument should be prepared according to the instructions below. The following equipment and reagents are required for sample preparation. All reagents and solutions should be at room temperature (15–25°C) before starting.

  • Page 37: Pyromark Q24 Mdx Vacuum Workstation Function Test

    5.3.1 PyroMark Q24 MDx Vacuum Workstation function test Before using the PyroMark Q24 MDx Vacuum Workstation, check that the filter probes are working properly by performing the function test as follows: 1. Add 100 µl high-purity water to each well of a 24-well PCR plate.

  • Page 38: Dna Amplification

    5. Seal the PCR plate (or the strips) using strip caps. Ensure that no leakage is possible between the wells. 6. Agitate the PCR plate (or strips) constantly for at least 5–10 min using a mixer (1400 rpm). 5-10 PyroMark Q24 MDx User Manual 01/2016...

  • Page 39: Separation Of Dna Strands And Release Of Samples Into The Pyromark Q24 Plate

    Section 5.3.5 by placing it (without a plate) on a heating block at 80°C. Procedure 1. Ensure that the PyroMark Q24 MDx Vacuum Workstation has been assembled correctly and securely. PyroMark Q24 MDx User Manual 01/2016 5-11...
  • Page 40 Approximately 50 ml 1x Wash Buffer (3)  Approximately 50 ml high-purity water (4)  Approximately 70 ml high-purity water (5)  A suggested setup is shown in the following image. Refill the troughs to these levels whenever necessary. 5-12 PyroMark Q24 MDx User Manual 01/2016...
  • Page 41 Buffer. Add 25 µl of the solution to each well of a PyroMark Q24 Plate that is to be used. Note: Use one of the supplied PyroMark Q24 Plate Holders as support when preparing and moving the plate. PyroMark Q24 MDx User Manual 01/2016 5-13...
  • Page 42 15. Transfer the tool to the trough containing Wash Buffer. Flush the filter probes for 10 s. 16. Raise the tool to beyond 90° vertical for 5 s, to drain liquid from the filter probes (see following image). 5-14 PyroMark Q24 MDx User Manual 01/2016...
  • Page 43 25. At the end of a working day, liquid waste and any remaining solutions should be discarded and the PyroMark Q24 MDx Vacuum Workstation should be checked for dust and spillage, (see Section 5.6.2). PyroMark Q24 MDx User Manual 01/2016...

  • Page 44: Annealing Of Sequencing Primer To Samples

    30 times. If the reagent cartridge has not been used in 4 weeks or longer (e.g., it has been stored), clean the cartridge and check that it can be used for 5-16 PyroMark Q24 MDx User Manual 01/2016...

  • Page 45: Processing A Run On The Pyromark Q24 Mdx Instrument

    Processing a run on the PyroMark Q24 MDx Instrument The lid of the PyroMark Q24 MDx Instrument must remain closed during operation of the instrument. An audible warning signal will alert you if the lid is opened when it is not safe.

  • Page 46: Instrument Software

    2. Switch on the instrument. The power switch is located at the rear of the instrument (see image in Section 3.4). 5-18 PyroMark Q24 MDx User Manual 01/2016...

  • Page 47: Starting The Run

    To view the contents of a folder, select the folder and press “Select”. To go back to the previous view, press “Back”. 4. When the run file is selected, press “Select” to start the run. PyroMark Q24 MDx User Manual 01/2016 5-19...

  • Page 48: Monitoring The Run

    The enzyme mix and substrate mix are dispensed to all wells used. Then the nucleotides are dispensed to the wells according to their dispensation order, which is defined in the assay file. Stopped The run has been aborted. 5-20 PyroMark Q24 MDx User Manual 01/2016...

  • Page 49: After The Run

    8. Discard the plate. 9. If the reagent cartridge is to be reused, clean it according to the instructions below. 10. If this was the last run for the day, follow the instructions in Section 5.6. PyroMark Q24 MDx User Manual 01/2016 5-21...
  • Page 50 Hold the cartridge over a sink or beaker while pressing firmly on top of each compartment with a finger (wear powder-free gloves). A jet of water should come straight out of the tip of each needle. 5-22 PyroMark Q24 MDx User Manual 01/2016...
  • Page 51 7. When the reagent cartridge is dry, store the reagent cartridge in a PyroMark Q96 HS Tip Holder Box to protect it from dust and (sun)light. PyroMark Q24 MDx User Manual 01/2016 5-23...

  • Page 52: Analyzing The Run

    Analyzing the run Detailed instructions for analyzing the run are available in the PyroMark Q24 MDx Software User Guide (press the “F1” key when in PyroMark Q24 MDx Software). 1. Move the processed run file from the USB stick to a computer running PyroMark Q24 MDx Software.

  • Page 53: Viewing The Analysis Results

    : Shows the quality assessment of all variable positions in the well or of all the bases in the base-called sequence. : Shows the quality assessment at the end of the quality control window (SQA assays only). PyroMark Q24 MDx User Manual 01/2016 5-25...
  • Page 54 CpG assays with a light yellow background color. CpG analysis results The methylation percentages are displayed in Pyrogram, for example . The quality assessment is displayed by the background color of the result. 5-26 PyroMark Q24 MDx User Manual 01/2016...

  • Page 55: Analysis Reports

    “Reports” menu. For more information about the reports, see the “View, Print, and Save Analysis Reports” section of the PyroMark Q24 MDx Software User Guide (press the “F1” key when in PyroMark Q24 MDx Software). PyroMark Q24 MDx User Manual 01/2016...

  • Page 56: Finishing Work And Shutting Down

    “OK”. 2. When the message “It is now safe to turn off the instrument” appears, switch off the instrument. The power switch is located at the rear of the instrument. 5-28 PyroMark Q24 MDx User Manual 01/2016...

  • Page 57: Emptying The Waste Container And Troughs

    2. Discard any solutions left in the troughs. 3. Rinse the troughs with high-purity water, or replace them, if necessary. 4. Empty the waste container. Note: The cap can be removed without disconnecting the tubing. PyroMark Q24 MDx User Manual 01/2016 5-29...

  • Page 58: Checking The Instrument

    If it is not, contact QIAGEN Technical Services. Light button Backup of PyroMark Q24 files The data generated by PyroMark Q24 MDx Software is stored on the computer as files with the following suffixes: *.pyrorun (run files)  *.pyrosetup (assay files).

  • Page 59: Maintenance

    Maintenance Maintenance The following maintenance procedures must be carried out to ensure reliable operation of the PyroMark Q24 MDx: Regular performance checks  Cleaning of the instrument  Following these procedures ensures that the PyroMark Q24 MDx Instrument is free of dust and liquid spills.

  • Page 60: Maintenance Of The Pyromark Q24 Mdx Instrument

    Maintenance Maintenance of the PyroMark Q24 MDx Instrument 6.2.1 Cleaning the instrument If the instrument has been contaminated by dust and spillage, clean it according to the instructions below. Important points before starting: Avoid harsh cleaners and chemicals, and getting ...

  • Page 61: Cleaning The Heating Block And Light Guides

    70% ethanol (see image, next page). 8. Close the plate-holding frame and the instrument lid and reconnect the instrument to the mains power. PyroMark Q24 MDx User Manual 01/2016...
  • Page 62 Maintenance CAUTION Light guide maintenance Use lint free tissues to clean the space between the heating block and the light guide block inside the instrument. Do not use paper tissues. PyroMark Q24 MDx User Manual 01/2016...

  • Page 63: Maintenance Of The Pyromark Q24 Mdx Vacuum Workstation

    Maintenance Maintenance of the PyroMark Q24 MDx Vacuum Workstation WARNING Hazardous chemicals [W6] The Denaturation Solution used with the vacuum workstation contains sodium hydroxide, which is irritating to eyes and skin. Always wear safety glasses, gloves, and a lab coat.

  • Page 64: Testing And Replacing The Filter Probes

    The following items are required: Powder-free gloves  2 mm Allen key (supplied with the system)  High-purity water (Milli-Q 18.2 MΩ x cm or equivalent)  New filter probes (QIAGEN)  PyroMark Q24 MDx User Manual 01/2016...

  • Page 65: Replacing The Rubber Seal

    The rubber seal needs to be replaced  If the rubber seal needs to be replaced, the following items are required: Powder-free gloves,  2 mm Allen key (supplied with the system)  New rubber seal (QIAGEN)  PyroMark Q24 MDx User Manual 01/2016...

  • Page 66: Replacing The Tubing

    The following items are required: New tubing (QIAGEN)  Beaker  Procedure 1. Ensure that no vacuum is applied to the vacuum prep tool, i.e., the vacuum switch is closed (Off), and the vacuum pump is switched off. PyroMark Q24 MDx User Manual 01/2016...

  • Page 67: Replacing The Waste Filter

    5. Push the tubing onto the fittings of the new filter. 6. If necessary, empty the waste container. Note: The cap can be removed without disconnecting the tubing. 7. Reconnect the vacuum pump to the mains power. PyroMark Q24 MDx User Manual 01/2016...
  • Page 68 Maintenance This page intentionally left blank 6-10 PyroMark Q24 MDx User Manual 01/2016...

  • Page 69: Troubleshooting

    Troubleshooting Troubleshooting If you need to contact QIAGEN Technical Services about an error, note down the steps leading to the error and any information given in any dialog boxes. This will help the QIAGEN Field Service Specialist in solving the problem.

  • Page 70: Analysis-Related Errors

    Troubleshooting runs, please contact QIAGEN Technical Services. If requested to send an Environment Data file: Select “Export Environment Data” from the “Tools”  menu Select the destination folder for the data file from the  “Save in” drop-down list Enter the filename in the “File name” text box and ...
  • Page 71 Incorrect sequence Correct the sequence to analyze and, if necessary, to analyze rerun the samples. Background peaks Follow the recommendations in Appendix B the in Pyrogram first time an assay is run. Redesign the assay. PyroMark Q24 MDx User Manual 01/2016...

  • Page 72: Analysis Software-Related Errors

    Minus shift Ensure that homopolymers are followed by an extra dispensation. Analysis software-related errors For errors related to the analysis software, see the Troubleshooting Guide section of the PyroMark Q24 MDx Software User Guide. PyroMark Q24 MDx User Manual 01/2016...

  • Page 73: Instrument-Related Errors

    Ensure your upgrade installation files are located folders/files found in a folder called “Upgrade” at the root of the on USB memory USB stick. Note: For all other instrument error messages, please contact QIAGEN Technical Services. PyroMark Q24 MDx User Manual 01/2016...

  • Page 74: Vacuum Workstation-Related Errors

    The used USB stick is damaged or not compatible USB stick with the system. It is recommended that USB sticks supplied by QIAGEN are used. c) USB stick can not be Broken USB contact, contact QIAGEN Technical inserted Services. Vacuum workstation-related errors Comments and suggestions...

  • Page 75: Verification Of Correct Installation And Operation

    (measured as %C), single bases of all four nucleotides, and homopolymers of two and three bases. For information on how to use the PyroMark Control Oligo, see the PyroMark Q24 Validation Oligo Handbook, supplied with the product. PyroMark Q24 MDx User Manual 01/2016...
  • Page 76 Troubleshooting This page left intentionally blank PyroMark Q24 MDx User Manual 01/2016...

  • Page 77: Glossary

    For example, the sequence "TCCAGAA" can be analyzed with the dispensation order "TCAGA". Dispensation Defines the nucleotides and the order in which they order should be dispensed in Pyrosequencing runs. PyroMark Q24 MDx User Manual 01/2016...
  • Page 78 InDels Insertion and/or deletions. Instrument A method that describes physical settings for the methods instrument, such as mixer speed, block temperature, and pulse time settings. PyroMark Q24 MDx User Manual 01/2016...
  • Page 79 Pyrogram The graph resulting from a sequencing reaction performed using Pyrosequencing technology. Incorporated nucleotides are shown as peaks in the Pyrogram. PyroMark Q24 MDx User Manual 01/2016...
  • Page 80 Minus shift: A small proportion of the template sequences that fails to incorporate a nucleotide will be sequenced subsequent to the rest of template. PyroMark Q24 MDx User Manual 01/2016...
  • Page 81 Variable A region in the sequence that varies at one or more position variable bases. In PyroMark Q24 MDx Software, the variable positions are highlighted with a blue-gray background color in the histogram and Pyrogram. PyroMark Q24 MDx User Manual 01/2016...
  • Page 82 Glossary This page intentionally left blank PyroMark Q24 MDx User Manual 01/2016...

  • Page 83: Technical Data

    Appendix B Appendix A Technical data QIAGEN reserves the right to change specifications at any time. Environmental conditions Operating conditions: PyroMark Q24 MDx Instrument Input power 100–240 VAC, 50–60 Hz Maximum 160 VA Input power consumption Instrument rating 24 V DC, 40 W...
  • Page 84 Height: 700 mm (27.56 in.) Depth: 600 mm (23.62 in.) Mass 28 kg (61.74 lb.) Capacity Up to 24 samples per run Chemical pH 4 to pH 9, common detergents, 0.5 M sodium resistance hydroxide, 70% ethanol PyroMark Q24 MDx User Manual 01/2016...
  • Page 85 Appendix B Operating conditions: PyroMark Q24 MDx Vacuum Workstation Power 100 V AC, 50/60 Hz or 115 V AC, 60 Hz or 230 V AC, 50 Hz Power consumption: Maximum 25 VA Overvoltage category Air temperature 15–32ºC (59–90ºF) Relative humidity 20–90% Altitude Up to 2000 m (6500 ft.)
  • Page 86 Intel Pentium IV, 3 GHz (or higher) 1 GB 100 MB Free hard disk space Graphics card Supporting the resolution of the monitor Monitor 1280 x 1024 pixels Pointer device Mouse or similar Interfaces USB port and CD-ROM PyroMark Q24 MDx User Manual 01/2016...

  • Page 87: Waste Electrical And Electronic Equipment (Weee

    Recycling can be provided by QIAGEN upon request at additional cost. In the European Union, in accordance with the specific WEEE recycling requirements and where a...

  • Page 88: Fcc Declaration

    Reorient or relocate the receiving antenna.  PyroMark Q24 MDx User Manual 01/2016...

  • Page 89: Ec Declaration Of Conformity

    Consult the dealer or an experienced radio/T.V.  technician for help. QIAGEN GmbH Germany is not responsible for any radio television interference caused by unauthorized modifications of this equipment or the substitution or attachment of connection cables and equipment other than those specified by QIAGEN GmbH, Germany.

  • Page 90: Assay Design And Validation

    PCR and sequencing primers. Each primer set is given a quality score based on several parameters that are specific for Pyrosequencing analysis. Ensure you use the correct assay type in PyroMark ADSW. We recommend the use of QIAGEN IVD-labeled assays for Pyrosequencing, which include all necessary optimized primers.
  • Page 91 Design sequencing primers using PyroMark ADSW. When designing an InDel assay, it is highly recommended that the sequencing primer is located a few bases before the variable position. We recommend the use of QIAGEN IVD-labeled assays for Pyrosequencing, which include all necessary optimized primers.
  • Page 92 EDTA, and other chemical solvents than single-step enzyme-catalyzed processes. QIAGEN offers a complete range of nucleic acid preparation systems, ensuring the highest-quality templates for PCR. These include the QIAprep ®...
  • Page 93 DNA in ready-to-use solutions. Regression analysis of the frequency of one allele measured in the PyroMark Q24 MDx as a function of the input (expected) allele, should give an R value greater than 0.9.
  • Page 94 Never exclude the blank dispensations as they function as excellent indicators of unspecific nucleotide incorporation. When manually generating a dispensation order, include an appropriate number of blank dispensations. If possible, start B-12 PyroMark Q24 MDx User Manual 01/2016...
  • Page 95 Thymine during bisulfite treatment and can therefore be used as controls for the reaction. When creating a CpG assay in PyroMark Q24 MDx Software, the software indicates possible dispensations as controls for the bisulfite treatment reaction. The original sequence (before...
  • Page 96 PCR product. This will show if the sequencing primer and the biotinylated primer can form duplexes and give background signal in Pyrosequencing reactions. Pyrograms from these controls should not show any significant peak after any nucleotide addition. B-14 PyroMark Q24 MDx User Manual 01/2016...
  • Page 97 The analysis results for SQA assays are based on the appearance of the peaks in Pyrogram, related to peak height levels estimated by the software. The inclusion of known bases in SQA assays can improve the estimation of peak height level. PyroMark Q24 MDx User Manual 01/2016 B-15...
  • Page 98 Appendix B This page intentionally left blank B-16 PyroMark Q24 MDx User Manual 01/2016...

  • Page 99: Liability Clause

    Appendix C Appendix C Liability clause QIAGEN shall be released from all obligations under its warranty in the event repairs or modifications are made by persons other than its own personnel, except in cases where the Company has given its written consent to perform such repairs or modifications.
  • Page 100 Appendix C This page intentionally left blank PyroMark Q24 MDx User Manual 01/2016...
  • Page 101 5-2 rubber seal, 6-7 recently saved runs, 5-2 tubing, 6-8 unsaved runs, 5-1 waste filter, 6-9 abort, 5-21 analysis, 5-24 Glossary, 8-1 finishing and shutting down, 5-28 monitoring, 5-20 processing, 5-17 PyroMark Q24 MDx User Manual 01/2016 Index-1...
  • Page 102 Ventilation, 1-5, 4-2 Starting template, 9 Verify proper installation and operation, View analysis results, 5-25 Technical assistance, 2-2 Technical data, 1 Test Warnings, 1-1 filter probes, 5-9 Waste disposal, 5 reagent cartridge, 5-22 Index-2 PyroMark Q24 MDx User Manual 01/2016...
  • Page 104 Australia techservice-au@qiagen.com  Austria techservice-at@qiagen.com  Belgium techservice-bnl@qiagen.com  Brazil suportetecnico.brasil@qiagen.com  Canada techservice-ca@qiagen.com  China techservice-cn@qiagen.com  Denmark techservice-nordic@qiagen.com  Finland techservice-nordic@qiagen.com  France techservice-fr@qiagen.com  Germany techservice-de@qiagen.com  Hong Kong techservice-hk@qiagen.com  India techservice-india@qiagen.com  Ireland techservice-uk@qiagen.com ...

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