Home

PyroMark® Q96 ID User Manual

Contents

1. DNA dNTP DNA p PPi net ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5 phosphosulfate This drives the conversion of luciferin to oxyluciferin by luciferase generating visible light in amounts proportional to the amount of ATP Light is detected using charged coupled devices CCDs and seen as a peak in the Pyrogram Each light signal is proportional to the number of nucleotides incorporated Sulfurylase I light APS PPi ATP luciferin oxyluciferin Luciferase a ATP Light time nucleotide incorporation generates light seen as a peak in Pyrogram Apyrase a nucleotide degrading enzyme continuously degrades unincorporated nucleotides and ATP When degradation is complete another nucleotide is added dNTP MY dNDP dNMP phosphate ATP PES ADP AMP phosphate Nucleotides are added one at a time Note Deoxyadenosine alfa thio triphosphate dATPaS is used instead of natural deoxyadenosine triphosphate dATP since it is used efficiently by the DNA polymerase but not recognized by the luciferase 3 2 PyroMark Q96 ID User Manual 12 2012 General Description 9 Asthe process continues the complementary sequence is built up and the nucleotide sequence is determined from the peak in the Pyrogram nucleotide sequence G c A GG cc T gt nucleotide added PyroMark Q96 ID principle The PyroMark Q96 ID perfo
2. 2 GHz or higher RAM 1 GB Free hard disk space 200 MB Graphics card Supporting the resolution of the monitor Monitor 1280 x 800 pixels True Color 32 bit or higher Pointer device Mouse or similar Interfaces Ethernet ports or external USB Ethernet adaptor PyroMark Q96 ID User Manual 12 2012 A 3 Appendix A Waste Electrical and Electronic Equipment WEEE This section provides information about disposal of waste electrical and electronic equipment by users The crossed out wheeled bin symbol see below indicates that this product must not be disposed of with other waste it must be taken to an approved treatment facility or to a designated collection point for recycling according to applicable local laws and regulations The separate collection and recycling of waste electrical and electronic equipment at the time of disposal helps to conserve natural resources and ensures that the product is recycled in a manner that protects human health and the environment 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 replacement product is being supplied by QIAGEN free recycling of its WEEE marked electronic equipment is provided To recycle electronic equipment contact your local QIAGEN sales office for the required return form Once the form is submitted you will be contacted by QIAGEN either to request
3. Important The PyroMark Data Converter Tool is only compatible with Microsoft Windows XP Users also planning to upgrade to Windows 7 must convert their data into the new format before upgrading their operating system Runs which cannot be converted refer to 6 4 2 cannot be accessed with the new PyroMark Q96 Software 6 2 PyroMark Q96 ID User Manual 12 2012 PyroMark Q96 Software 6 4 1 Installing the PyroMark Data Converter Tool 1 The PyroMark Data Converter can be downloaded at www giagen com goto PyroMarkDataConverter at no cost In order to access this page and download the software you must register your instrument with a valid serial number see back of instrument at www giagen com PyroMarkRegistration 2 Install the PyroMark Data Converter software on the instrument operating computer containing the database of runs to be converted This computer must be running the Microsoft Windows XP operating system 6 4 2 Using the PyroMark Data Converter Tool The PyroMark Data Converter tool converts Pyrosequencing runs set up and completed with the old PyroMark Q96 ID Software into a file format that is compatible with the new PyroMark Q96 Software This tool copies the run data converts the data to the new format and exports the converted data to the location specified by the user Only sequence analysis SQA and genotyping SNP runs which have been executed and completed will be converted CoG assays generated wi
4. the vacuum switch is closed Off and the vacuum pump is switched off Disconnect the vacuum pump from the mains power Remove the tool from the tubing Loosen the four screws using an Allen key Pull out the old filter probes SS eS Gently insert new filter probes without pressing on the filter tips 7 Replace and fasten the four screws and reconnect the vacuum pump to the mains power Replacing the tubing If the tubing is broken or distorted replace it Be sure to observe all federal state and local environmental regulations for the disposal of laboratory waste The following items are required B New tubing contact QIAGEN Technical Services E Beaker 7 10 PyroMark Q96 ID User Manual 12 2012 Maintenance 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 Disconnect the vacuum pump from the mains power Remove the broken tubing at one end and empty any liquid waste into an empty beaker Disconnect the other end of the tubing and discard the tubing and any liquid waste Cut the new vacuum tubing into three pieces and assemble it Use the old tubing as a template for the size of the new tubing pieces Ensure that the tubing is connected to the pump s vacuum fitting Reconnect the vacuum pump to the mains power PyroMark Q96 ID User Manual 12 2012 7 11 Maintenance This page intentional
5. Kit 20 ul ofthe PCR product gives satisfactory Pyrosequencing results in most cases This volume should be adjusted to achieve single peak heights of at least 20 RLU in the Pyrogram Pyrosequencing analysis Use default settings in the software for all assay setups if not otherwise stated Assay setup AQ and CpG assays When creating an AQ or CpG assay the sequence to analyze should contain a sufficient number of bases to generate at least five nonvariable reference peaks For InDels a few reference peaks should be included before the variable position If the sequencing primer is placed immediately adjacent to the position to analyze include part of the sequence following the variable position in the Sequence to Analyze text box Ensure that the last base in the text box is not part of the variable position Blank dispensations are automatically generated by the software and serve as built in quality controls for the assay Never exclude the blank dispensations generated by the software 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 the dispensation order with PyroMark Q96 ID User Manual 12 2012 B 5 Appendix B B 6 one blank dispensation and have at least the same number of blank dispensations as the number of variable positions Pay attention to tips and war
6. PyroMark Denaturation Solution cat no 979007 PyroMark Wash Buffer concentrate cat no 979008 PyroMark Annealing Buffer cat no 979009 Assembly of the PyroMark Q96 Vacuum Workstation Assemble the PyroMark Q96 Vacuum Workstation as follows 1 Place the PyroMark Q96 Vacuum Workstation on a flat surface Connect vacuum tubing to the straight blue connector on the cap of the liquid waste container Attach the other end of this tubing to the vacuum pump Connect a second piece of vacuum tubing to the angled blue connector on the cap of the liquid waste container Attach the other end to the connector on the back of the PyroMark Q96 Vacuum Workstation With a third piece of tubing attach the Vacuum Prep Tool to the vacuum tubing connector on the top of the PyroMark Q96 Vacuum Workstation see image below Test the PyroMark Q96 Vacuum Workstation by switching on the vacuum pump and opening the switch on the front of the workstation If not vacuum is created turn off the vacuum pump and open the cap of the liquid waste container to release any built up air pressure Note The liquid waste container must be able to withstand absolute vacuum DNA amplification Amplify the DNA to be analyzed by PCR using one of the primers biotinylated See Appendix B for details on assay design and validation PyroMark Q96 ID User Manual 12 2012 5 7 Operating Procedures 5 4 3 Immobilization of the PCR product to beads Biotin
7. Wartungsarbeiten gem den Anweisungen in Abschnitt 7 dieses Handbuchs durch QIAGEN stellt alle Reparaturen in Rechnung die nachweislich auf eine inkorrekte Wartung zur ckzuf hren sind WARNUNG ACHTUNG A Verletzungsgefahr und Besch digung des Ger ts Der PyroMark Q96 ID ist sehr schwer und sollte nicht von einer Person angehoben werden Heben Sie das Ger t nicht allein an um eine Verletzung und oder Besch di gung des Ger ts zu vermeiden E 2 PyroMark Q96 ID User Manual 12 2012 Appendix E 1 2 WARNUNG A Schutz vor Stromschlag Ziehen Sie die Netzanschlusskabel aus den Steckdosen bevor Sie Wartungsarbeiten am Ger t vornehmen Gefahr durch Stromschlag Jede Unterbrechung des Schutzleiters Erdungs bzw Masseleiter im Ger t oder au erhalb des Ger ts und jede Abtrennung des Schutzleiters am Anschluss der Netzleitung erh ht die Gefahr eines Stromschlags Eine absichtliche Unterbrechung der Schutzleiter verbindung ist verboten Gef hrliche Spannung im Ger t Wenn das Ger t an die Stromversorgung angeschlossen ist sind die Anschlussstellen spannungsf hrend Durch das ffnen der Abdeckungen oder das Entfernen von Geh useteilen k nnen spannungsf hrende Komponenten freigelegt werden Um einen zufriedenstellenden und sicheren Betrieb des PyroMark Q96 ID zu gew hrleisten befolgen Sie bitte die nachstehenden Hinweise M Die Ger te Netzkabel m ssen an Wechselstro
8. are based on the sequence context as well as the results in the analyzed positions Deviations from this built in quality control are shown as warnings in the Well Information area 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 Q96 ID User Manual 12 2012 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 All materials replaced under this warranty will be warranted only for the duration of the original warranty period and in no case beyond the original expiration date of original warranty unless authorized in writing by an officer of the Company Read out devices interfacing devices and associated software will be warranted only for the period offered by the original manufacturer of these products Representations and warranties made by any person including representatives of QIAGEN which are inconsistent or in conflict with the conditions in this warranty shall not be binding upon the Company unless produced in writing and approved by an officer of QIAGEN PyroMark Q96 ID Us
9. at any time Environmental conditions Operating conditions Power 100 120 V 220 240 V 50 60 Hz Power consumption 280 VA max Overvoltage Il category Air temperature 18 28 C 64 82 F Relative humidity 30 90 relative humidity noncondensing Altitude Up to 2000 m 6500 ft Place of For indoor use only operation Draft free location not close to window Keep instrument out of direct sunlight Pollution level 2 Environmental 3K2 IEC 60721 3 3 class Transportation conditions Air temperature 25 C to 60 C 13 F to 140 F Relative humidity Max 7596 noncondensing PyroMark Q96 ID User Manual 12 2012 A 1 Appendix A Storage conditions Air temperature 10 C to 40 C 50 F to 104 F Relative humidity Max 75 noncondensing Mechanical data and hardware features PyroMark Q96 ID Dimensions closed Clearance space Mass Capacity Chemical resistance A 2 Width 520 mm 20 47 in Height 490 mm 19 29 in Depth 620 mm 24 41 in Width 540 mm 21 26 in Height 570 mm 22 44 in Depth 640 mm 25 20 in 52 kg 114 64 Ib Up to 96 samples per run Instrument covers pH 4 to pH 9 common detergents 70 ethanol Inside the instrument 70 ethanol PyroMark Q96 ID User Manual 12 2012 Appendix A PyroMark Q96 Software Operating system Microsoft Windows XP English version or Microsoft Windows 7 32 bit English version Processor Intel Core Duo processor
10. auf EN C Tick Mark Zeichen der f r Australien Ger ter ckseite Herstellerkennung N17965 6 Typenschild auf EN Markierung gem 20 der RoHS Richtlinie f r Ger ter ckseite China Beschr nkung der Verwendung bestimmter gef hrlicher Stoffe in Elektro und Elektronikger ten Typenschild auf EN Entspricht US der amerikanischen und S P Ger ter ckseite kanadischen c US Sicherheitsstandards PyroMark Q96 ID User Manual 12 2012 E 7 Appendix E This page intentionally left blank E 8 PyroMark Q96 ID User Manual 12 2012 Index Index A Analyzing the run 5 17 Annealing of sequencing primer to samples 5 13 Assay design and validation B 1 setup B 5 Assembly of the PyroMark Q96 Vacuum Workstation 5 7 D DNA amplification 5 7 F Filter probes function test 7 9 replacement 7 10 G General Description 3 1 features 3 5 Pyrosequencing principle 3 3 Glossary 9 7 Immobilization of the PCR product to beads 5 8 Informations de s curit D 1 Installation grounding requirements 4 2 moving the instrument 4 3 power requirements 4 2 site requirements 4 1 Intended use 2 3 L Liability clause C 1 M Maintenance changing the fuses 7 4 cleaning the cartridge 7 5 cleaning the heating block 7 3 cleaning the lens array 7 3 cleaning the vacuum workstation 7 8 routine 7 2 O Operating Procedures 5 1 finishing work 5 22 managing instrument parameters 5 4 post run pro
11. available in the PyroMark Q96 Software User Guide press the F1 key when in PyroMark Q96 Software Analysis modes PyroMark Q96 Software has four analysis modes AQ SNP CpG and SQA To toggle between the modes select AQ SNP CpG or SQA in the toolbar Note How the analysis is performed can be modified using the Analysis Setup tab 5 18 PyroMark Q96 ID User Manual 12 2012 Operating Procedures Overview tab Ser 5 eS Ce Z E Reciicate 1 Page 1 Regticane 7 Ra iicate Bagio Y Wegkcww 1 S2 1 In the Overview tab click the Zoom Tool to zoom into a quadrant of the 96 well plate Use the arrow around the zoom tool or click the desired quadrant of the plate to change which 24 wells are displayed 2 Click the Analyze Selected Wells button to analyze the selected wells with a valid analysis setup for the current analysis mode or click the Analyze all Wells icon to analyze all wells OH Analyze all wells QU Analyze the selected wells only 5 6 2 Viewing the analysis results By selecting an analyzed light blue well in the Overview tab the corresponding Pyrogram and theoretical histogram if an AQ SNP or CpG assay or compensated Pyrogram if an SQA assay are displayed in the Pyrogram pane The well information including analysis warnings is listed in the Well Information area If several wells are selected in the plate overview information f
12. either be forward or reverse If designing primers with PyroMark ADSW the primer that needs to be biotinylated is indicated The biotinylated primer should be purified by HPLC or an equivalent procedure since free biotin will compete with the biotinylated PCR product for binding sites on the streptavidin coated Sepharose beads PyroMark Q96 ID User Manual 12 2012 B 1 Appendix B B 2 Amplicon length The optimal amplicon length for Pyrosequencing assays is between 80 and 200 bp although products up to 500 bp may work well Amplicons for CpG assays should ideally be shorter than 200 bp Sequencing primer 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 PCR setup PCR reactions of 25 ul are set up using the PyroMark PCR Kit Ensure that you follow the instructions provided in the PyroMark PCR Handbook Run the PCR at the optimal annealing temperature for 45 cycles Using fewer cycles may produce insufficient yield of template DNA and interfere with subsequent Pyrosequencing reactions due to the presence of unused biotinylated primer The PCR product should give one strong band with minimal excess of primers when analyzed on an agarose gel Starting template The yield and quality of PCR product is affected by both the quality and quantity of the nucleic acid starting template This is
13. in the toolbar to enter a run name and save the file in the desired folder 3 In the Instrument Parameters drop down list select the instrument parameters file that corresponds to the PyroMark Q96 Cartridge to be used 4 Optional Enter the Reagent ID Plate ID plate Bar code or a Run note 5 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 Alternatively right click a well or selection of wells and select Load Assay PyroMark Q96 ID User Manual 12 2012 5 3 Operating Procedures 5 3 3 6 Click id in the toolbar to save the run setup 7 Print a list of required volumes of reagents and the plate setup by selecting Pre Run Information from the Tools menu and when the report opens clicking 4 Further information There are several ways to set up a plate For example it is possible to import and paste a sample layout defined in a text file and drag copy and increment a sample ID if the last part of the entered sample ID is a number For more information see the PyroMark Q96 Software User Guide press the F1 key when in the PyroMark Q96 Software Note To base your run on a previous run right click the processed run file in the shortcut browser and select Copy and Rerun from the context menu Only the run setup not the run and analysis data will b
14. les autres produits EN Fabricant l gal PyroMark Q96 ID User Manual 12 2012 Appendix D Symbole Emplacement Langue Description l int rieur de EN Avertissement se l appareil reporter au manuel d utilisation Type de EN Num ro de s rie plaque l arri re de l instrument et de la boite Plaque EN Symbole DEEE signal tique X l arri re de l appareil zal Plaque EN Label FCC de la FC signal tique a Federal l arri re de Communications l appareil Commission des tats Unis Plaque EN Label C Tick pour signal tique l Australie identifiant l arri re de du fournisseur N17965 l appareil OS Plaque EN Label RoHS pour la 20 signal tique Chine restriction de l arri re de l utilisation de certaines l appareil substances dangereuses dans l quipement lectrique et lectronique Plaque EN Conforme aux normes signal tique de s curit des Etats P l arri re de Unis et du Canada c US l appareil PyroMark Q96 ID User Manual 12 2012 D 7 Appendix D This page intentionally left blank D 8 PyroMark Q96 ID User Manual 12 2012 Appendix E Appendix E 1 Sicherheitshinweise Vor der Inbetriebnahme des PyroMark Q96 ID sollten Sie dieses Handbuch sorgf ltig durchlesen beachten Sie insbesondere die Sicherheitshinweise Die Anweisungen und Sicherheitsinformationen in diesem Handbuch m ssen vom Anwender befolgt werden u
15. nationaler und regionaler Ebene eingehalten werden OSHA Occupational Safety and Health Administration Vereinigte Staaten von Amerika t ACGIH American Conference of Government Industrial Hygienists Vereinigte Staaten von Amerika COSHH Control of Substances Hazardous to Health Vereinigtes K nigreich E 4 PyroMark Q96 ID User Manual 12 2012 Appendix E 1 4 Gefahren durch mechanische Teile Der Deckel des PyroMark Q96 ID muss w hrend des Betriebs geschlossen sein WARNUNG Sich bewegende Ger teteile Um einen Kontakt mit sich bewegenden Teilen beim A Betrieb des PyroMark Q96 ID zu vermeiden darf das Ger t nur mit geschlossenem Deckel betrieben werden Entfernen Sie nicht die Abdeckplatten im Ger teinneren befinden sich keine Bauteile die vom Anwender gewartet werden m ssen Setzen Sie sich umgehend mit dem Technischen Service von QIAGEN in Verbindung falls einmal ein Problem mit dem PyroMark Q96 ID auftreten sollte WARNUNG Quetsch und Sto gefahr Die Dispensiereinheit der Prozesskammer Deckel A und oder Teile des Automationsmoduls k nnten jederzeit in Bewegung gesetzt werden WARNUNG Scharfe Nadeln Ber hren Sie nicht die scharfen Nadeln am Boden der A Reagenzienkartusche Gehen Sie vorsichtig mit den Nadeln um Kleine Partikel oder Fasern k nnen die Nadeln verstopfen PyroMark Q96 ID User Manual 12 2012 E 5 Appendix E 1 5 berhitzungsgefahr WARN
16. particularly true for amplification of long regions from DNA that has been fragmented by bisulfite treatment or extracted from paraffin embedded material Quality of starting template Since PCR consists of multiple rounds of enzymatic reactions it is more sensitive to impurities such as proteins phenol chloroform salts ethanol 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 PyroMark Q96 ID User Manual 12 2012 Appendix B These include the QlAprep system for rapid plasmid purification the QlAamp and DNeasy systems for rapid purification of genomic DNA and viral nucleic acids and the RNeasy system for RNA preparation from a variety of sources For more information about QlAprep QlAamp DNeasy and RNeasy products contact one of our Technical Service Departments see back cover or visit www giagen com Quality of starting template when performing CpG assays Critical parameters for a successful PCR using bisulfite treated DNA templates include complete bisulfite conversion and DNA fragments that are long enough for PCR EpiTect Plus Bisulfite Kit provides a fast and reliable procedure for efficient bisulfite conversion and a unique DNA Protect Buffer prevents DNA fragmentation during the bisulfite conversion reaction For more information about EpiTect products cont
17. pneumatic dispensation system should deliver an air pulse every 0 6 s the heating block should oscillate vibrate Close the process chamber lid and the instrument lid The Information light on the instrument will go out and the audible warning signal will cease Click on each well in Well overview The intensity level in the Pyrogram should be about 4000 for all wells Click Stop in the run progress bar Click Yes in the dialog box that appears The run will be stopped immediately and the data will be saved 6 8 PyroMark Q96 ID User Manual 12 2012 Maintenance 7 Maintenance The following maintenance procedures must be carried out to ensure reliable operation of the PyroMark Q96 ID M Regular performance checks M Cleaning of the instrument Following these procedures ensures that the PyroMark Q96 ID is free of dust and liquid spills Before undertaking maintenance procedures it is recommended that you familiarize yourself with the safety information in Section 1 Important Disconnect the instrument from mains power before cleaning Servicing The PyroMark Q96 ID is supplied with a warranty that lasts for 1 year beginning on the date of shipment The warranty includes all repairs due to mechanical breakdown Application development software upgrades accessories and disposable items are not included in the warranty QIAGEN offers comprehensive Service Support Agreements including Wa
18. removed without disconnecting the tubing If the vacuum workstation must be cleaned e g dust or spillage follow the instructions in Section 7 7 PyroMark Q96 ID User Manual 12 2012 5 25 Operating Procedures This page intentionally left blank 5 26 PyroMark Q96 ID User Manual 12 2012 PyroMark Q96 Software 6 1 6 1 1 6 1 2 6 1 3 6 2 PyroMark Q96 Software The operator s computer is configured for communication with the PyroMark Q96 ID Instrument System requirements Operator s and office computers See Section 3 9 for minimum specifications Printer All printers supported by the operator s computer are suitable QIAGEN recommends the use of a laser printer Data storage device A removable hard drive or similar should be used for backing up the result files This is required if the computer used to analyze the data is not connected to a LAN Installing PyroMark Q96 Software Note If the computer is connected to a network network policy settings may prevent you from completing this procedure For more information contact your system administrator 1 Close any programs running on the computer 2 Insert the PyroMark Q96 ID Application Software 2 5 CD into the CD drive 3 Inthe CD menu click Install PyroMark Q96 Software If the CD menu does not appear automatically m Select My Computer in the Windows Start menu m Right click the CD drive with the software CD
19. s responsibility to validate the performance of such product combination for any particular application The PyroMark Q96 ID System is intended for use by professional users such as technicians and physicians trained in molecular biological techniques and the operation of the PyroMark Q96 ID System PyroMark Q96 ID User Manual 12 2012 2 3 Introduction 2 3 1 Requirements for PyroMark Q96 ID users The table below covers the general level of competence and training necessary for transportation installation use maintenance and servicing of the PyroMark Q96 ID Servicing and QIAGEN Field Service annual preventive Specialists only maintenance Task Personnel Training and experience Delivery No special requirements No special requirements Installation QIAGEN Field Service Specialists only Routine use Laboratory technicians or Appropriately trained and running equivalent experienced personnel protocols familiar with use of computers and automation in general Preventive Laboratory technicians or Appropriately trained and maintenance equivalent experienced personnel familiar with use of computers and automation in general 2 4 PyroMark Q96 ID User Manual 12 2012 General Description 3 1 3 2 General Description The PyroMark Q96 ID is a multi application sequence analysis system which uses Pyrosequencing technology for the real time analysis of specific DNA sequences including single
20. serve as the operator s computer see Section 3 8 PyroMark Q96 ID User Manual 12 2012 3 5 General Description 3 5 3 6 3 7 Internal features of the PyroMark Q96 ID Internal view of the PyroMark Q96 ID EI Dispensing unit B Process chamber Process chamber The process chamber includes a Peltier heating block that maintains the correct temperature of the PyroMark Q96 Plate Low and its contents An integral mixer enables rapid mixing of sample and reagents in the PyroMark Q96 Plate Low Dispensing unit Reagents are manually added to the reagent cartridge which is then inserted into the dispensing unit on top of the process chamber lid During a run the dispensing unit is positioned over each well in the PyroMark Q96 Plate Low and reagents are dispensed in a zigzag fashion using a pneumatic system 3 6 PyroMark Q96 ID User Manual 12 2012 General Description 3 8 3 8 1 3 8 2 Controls and indicators Front panel The standby switch is located on the lower left hand side of the front panel Three indicator lights can be found in the middle of the front panel Top light Power green When illuminated this indicates the instrument has been connected to the mains electricity supply and is receiving power Middle light Busy yellow When this light is illuminated or flashing a run is in progress and the lid should not be opened Bottom light Information yellow If this light is ill
21. 24 6 PyroMark Q96 Software 6 1 6 1 System requirements 6 1 6 1 1 Operator s and office computers 6 1 6 1 2 Printer 6 1 6 1 3 Data storage device 6 1 6 2 Installing PyroMark Q96 Software 6 1 6 3 Uninstalling PyroMark Q96 Software 6 2 6 4 Upgrading from PyroMark Q96 ID Software 6 2 6 4 1 Installing the PyroMark Data Converter Tool 6 3 6 4 2 Using the PyroMark Data Converter Tool 6 3 6 5 Data backup 6 5 6 6 Installation testing 6 5 7 Maintenance 7 1 7 1 Shutdown procedure 7 2 7 2 Routine maintenance of the PyroMark Q96 ID 7 2 7 3 Cleaning the lens array 7 3 PyroMark Q96 ID User Manual 12 2012 Contents 3 Contents 7 4 Cleaning the heating block 7 3 7 5 Changing the fuses 7 4 7 6 Cleaning and testing the reagent cartridge 7 5 7 6 1 Standard cleaning procedure 7 5 7 6 2 Clogged cartridge cleaning procedure 7 6 7 6 3 Cartridge function check 7 6 7 7 Cleaning the PyroMark Q96 Vacuum Workstation 7 8 7 8 Testing and replacing the filter probes 7 9 7 8 1 Function test for filter probes 7 9 7 8 2 Replacing filter probes 7 10 7 9 Replacing the tubing 7 10 8 Troubleshooting 8 1 8 1 Unexpected results 8 1 8 2 Sequence errors 8 2 8 3 Peak errors 8 3 8 4 Instrument related errors 8 5 8 5 Vacuum workstation related errors 8 6 9 Glossary 9 1 Appendix A A 1 Technical data A 1 Environmental conditions A 1 Waste Electrical and Electronic Equipment WEEE A 4 FCC declaration A 5 Appendix B B 1 Assay design and validation B 1 Contents 4 PyroMar
22. 96 Vacuum Workstation check that the filter probes are working by performing the function test as follows 1 Add 100 ul high purity water to each well of a 96 well PCR plate Fill a trough with 70 ml high purity water Start the vacuum pump Apply vacuum to the vacuum prep tool by opening the vacuum switch Lower the filter probes into the trough Keep them in position for approximately 20 s Ensure that the water is transferred to the waste container i e that vacuum has been applied If this has not happened check the connections Lower the filter probes into the PCR plate and check that the water is aspirated evenly across all wells and that all wells are empty after a maximum of 10 s If the wells are not empty after 10 s repeat the procedure from step 1 If the function test fails twice replace the filter probes see below PyroMark Q96 ID User Manual 12 2012 7 9 Maintenance 7 8 2 7 9 Replacing filter probes Each filter probe can be replaced individually To ensure proper flow rate through the filter probes all probes should be replaced after preparation of approximately 100 plates Note Use gloves powder free to avoid contaminating the filter probes The following items are required M Powder free gloves M Allen key MB High purity water Milli Q 18 2 MQ x cm or equivalent M New filter probes cat no 979010 Procedure 1 Ensure that no vacuum is applied to the Vacuum Prep Tool i e
23. Manual 12 2012 Operating Procedures Note If performing multiplex analysis of pooled PCR products use 5 10 ul of each product and reduce water volume to maintain the total volume The total volume should be 80 ul after addition of the master mix and PCR product 5 Seal the PCR plate using strip caps Ensure that no leakage is possible between the wells 6 Agitate the PCR plate constantly for at least 5 10 min using a mixer 1400 rpm Note Sepharose beads sediment quickly and capturing of beads must take place immediately once the agitation is complete If more than 1 minute has elapsed agitate the plate again before capturing the beads Note During immobilization prepare the Vacuum Workstation for the sample preparation 7 Proceed with strand separation see next section 5 4 4 Strand separation WARNING Hazardous chemicals w4 The Denaturation Solution used with the vacuum A workstation contains sodium hydroxide which is irritating to eyes and skin Always wear safety glasses gloves and a lab coat The responsible body e g laboratory manager must take the necessary precautions to ensure that the surrounding workplace is safe and that the instrument operators are not exposed to hazardous levels of toxic substances chemical or biological as defined in the applicable Safety Data Sheets SDSs or OSHA ACGIH or COSHH documents For more information visit www giagen com safety Venting for fumes and dispos
24. PyroMark Q96 ID User Manual December 2012 Power PyroMark Q96 ID n QIAGEN Trademarks QIAGEN DNeasy EpiTect PyroMark Pyrosequencing Pyrogram GlAamp QlAprep Q Solution RNeasy QIAGEN Group Adobe Reader Adobe Systems Incorporated Intel Core Intel Corporation Microsoft Windows Microsoft Corporation Milli Q Merck KGaA Sepharose GE Healthcare 2011 2012 QIAGEN all rights reserved Contents Contents 1 Safety Information 1 1 1 1 Proper use 1 1 1 2 Electrical safety 1 2 1 3 Chemical hazards 1 3 1 4 Mechanical hazards 1 3 1 5 Heat hazards 1 4 1 6 Symbols used on the PyroMark Q96 ID 1 4 2 Introduction 2 1 2 1 About this user manual 2 1 2 2 General information 2 2 2 2 1 Technical assistance 2 2 2 2 2 Policy statement 2 2 2 2 3 Version management 2 2 2 3 Intended use of the PyroMark Q96 ID 2 3 2 3 1 Requirements for PyroMark Q96 ID users 2 4 3 General Description 3 1 3 1 PyroMark Q96 ID definitions 3 1 3 2 Pyrosequencing principle 3 1 3 3 PyroMark Q96 ID principle 3 3 3 4 External features of the PyroMark Q96 ID 3 5 3 5 Internal features of the PyroMark Q96 ID 3 6 3 6 Process chamber 3 6 3 7 Dispensing unit 3 6 3 8 Controls and indicators 3 7 3 8 1 Front panel 3 7 PyroMark Q96 ID User Manual 12 2012 Contents 1 Contents 3 8 2 Rear panel 3 7 3 9 Computer and software 3 8 4 Installation Procedures 4 1 4 1 Instrument del
25. Q96 ID may cause personal injuries or damage to the instrument The PyroMark Q96 ID must only be operated by qualified personnel who have been appropriately trained Servicing of the PyroMark Q96 ID must only be performed by QIAGEN Field Service Specialists PyroMark Q96 ID User Manual 12 2012 1 1 Safety Information WARNING Risk of personal injury and material damage W2 CAUTION The PyroMark Q96 ID is too heavy to be lifted by one A person To avoid personal injury or damage to the instrument do not lift the instrument alone Perform the maintenance as described in Section 7 QIAGEN charges for repairs that are required due to incorrect maintenance 1 2 Electrical safety Disconnect the line power cord from the power outlet before servicing WARNING Electrical hazard W3 lead inside or outside the instrument or disconnection of the protective conductor terminal is likely to make the instrument dangerous Intentional interruption is prohibited A Any interruption of the protective conductor earth ground Lethal voltages inside the instrument When the instrument is connected to line power terminals may be live and opening covers or removing ports is likely to expose live parts To ensure satisfactory and safe operation of the PyroMark Q96 ID and PyroMark Q96 Vacuum Workstation follow the advice below M The line power cord must be connected to a line power outlet that has a pro
26. Q96 ID must remain closed during operation of the instrument WARNING Moving parts W5 To avoid contact with moving parts during operation of the A PyroMark Q96 ID the instrument must be operated with the lid closed Do not remove the cover panels since there are no user serviceable parts inside If there is a problem with the PyroMark Q96 ID contact QIAGEN Technical Services immediately WARNING Pinch and impact hazards w A The dispensing unit process chamber lid and robot may move at any time PyroMark Q96 ID User Manual 12 2012 1 3 Safety Information WARNING Sharp needles W7 Do not touch the sharp needles at the bottom of the A reagent cartridge Handle the needles with care Small particles and fibers may obstruct the needles 1 5 Heat hazards WARNING Hot surface we The external heating block can reach temperatures of up A to 80 C 176 F Avoid touching them when they are hot CAUTION Risk of overheating C1 A To ensure proper ventilation maintain a minimum clearance of 10 cm 4 in at the sides and rear of the PyroMark Q96 ID Slits and openings that ensure the ventilation of the PyroMark Q96 ID must not be covered 1 6 Symbols used on the PyroMark Q96 ID Symbol Location Language Description Type plate on EN CE mark C the back of the instrument All products EN Catalog or recorder number All products EN Temperature l
27. South techservice kr qiagen com Luxembourg techservice bnl8qiagen com Mexico techservice mx qiagen com The Netherlands techservice bnl qiagen com Norway techservice nordicGqiagen com Singapore techservice sg qiagen com Sweden techservice nordicGqiagen com Switzerland techservice ch qiagen com UK techservice uk qiagen com USA techservice us qiagen com 1074525 12 2012 QIAGEN Sample amp Assay Technologies
28. System M Intel Core Duo processor 2 GHz or higher MB 200 MB free hard drive capacity E 1 GB RAM M Ethernet ports or external USB Ethernet adaptor M Monitor with 1280 x 800 pixels True Color 32 bit M Graphics card supporting the resolution of the monitor M Pointer device mouse or similar 3 8 PyroMark Q96 ID User Manual 12 2012 Installation Procedures 4 2 Installation Procedures Instrument delivery and installation The unpacking and installation of the PyroMark Q96 ID is carried out by a certified QIAGEN Field Service Specialist A member of your group who is familiar with laboratory and computer equipment should be present during the installation The following items are delivered M PyroMark Q96 ID instrument M Operator s computer M PyroMark Q96 Vacuum Workstation must be ordered separately M PyroMark Q96 ID User Manual and other technical documentation in PDF format on accompanying CD ROM M PyroMark Q96 Software on accompanying CD ROM Requirements Site The PyroMark Q96 ID and PyroMark Q96 Vacuum Workstation must be located out of direct sunlight away from heat sources and away from sources of vibration and electrical interference Refer to Appendix A for the operating conditions temperature and humidity The site of installation should be free of excessive drafts excessive moisture excessive dust and not subject to large temperature fluctuations Refer to Appendix A for the weight and dimens
29. UNG Hei e Oberfl chen ws Der externe Heizblock kann Temperaturen von bis zu A 80 C erreichen Ber hren Sie diese Teile nicht wenn sie aufgeheizt sind ACHTUNG Uberhitzungsgefahr Vergewissern Sie sich dass ein Mindestabstand von 10 cm A zwischen Seitenw nden und R ckseite des PyroMark Q96 ID und der Wand eingehalten wird damit eine ausreichende Bel ftung des Ger ts gew hrleistet ist Die L ftungsschlitze und ffnungen die f r die Be und Entl ftung des PyroMark Q96 ID bestimmt sind d rfen nicht verdeckt werden 1 6 Symbole auf dem PyroMark Q96 ID Symbol Fundstelle Sprache Beschreibung Typenschild auf EN CE Kennzeichnung C der Ger ter ckseite Reagenzien EN Katalognummer REF L sungen Kartusche Platte Va Reagenzien EN Zuldssiger L sungen Temperaturbereich Kartusche Platte Typenschild auf EN Hersteller i S d der R ckseite des Gesetzes Ger ts und allen anderen Produkten Im Ger teinneren EN Warnhinweis lesen Sie das Handbuch ml A E 6 PyroMark Q96 ID User Manual 12 2012 Appendix E Symbol Fundstelle Sprache Beschreibung Typenschild auf EN Seriennummer Ger ter ckseite und Verpackungskarton Typenschild auf EN WEEE Markierung der Ger ter ckseite BE Typenschild auf EN FCC Kennzeichen C der der Federal Ger ter ckseite Communications Commission der Vereinigten Staaten Typenschild
30. act one of our Technical Service Departments see back cover or visit www qiagen com Quantity of starting template The annealing efficiency of a primer to the template is an important factor in PCR Owing to the thermodynamic nature of the reaction the primer template ratio strongly influences the specificity and efficiency of PCR and should be optimized empirically If too little template is used primers may not be able to find their complementary sequences Too much template may lead to an increase in mispriming events PCR optimization The PyroMark PCR Kit will produce satisfactory results in most cases However if a higher Mg concentration is required we recommend using the 25 mM MgCl provided in the kit PyroMark Q96 ID User Manual 12 2012 B 3 Appendix B The recommended annealing temperature is 60 C and 56 C for genomic DNA and bisulfite treated DNA respectively when using PyroMark ADSW 2 0 Addition of Q Solution provided with the PyroMark PCR Kit may improve PCR yield and specificity for difficult templates that for example have a high degree of secondary structure or templates that are GC rich For all PCR optimization tests analyze 5 ul of a 25ul PCR on an agarose gel and aim for one strong specific band with minimal excess of primers Please refer to the PyroMark PCR Handbook for further troubleshooting Equal amplification of both alleles in AQ and CpG assays Reliable results in quantif
31. al of wastes must be in accordance with all national state and local health and safety regulations and laws 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 Q96 ID User Manual 12 2012 5 9 Operating Procedures Procedure 1 Ensure that the PyroMark Q96 Vacuum Workstation has been assembled correctly and securely see Assembly of PyroMark Q96 Vacuum Workstation page 5 7 The mains plug should be easily accessible in case the vacuum pump needs to be disconnected quickly from the mains power Note Perform the function test to ensure that the filter probes are working properly see Section 7 8 1 All probes should be replaced after preparation of approximately 100 plates or when needed Note Check and empty the waste container if necessary see Section 5 7 5 Fill five separate troughs supplied with the PyroMark Q96 Vacuum Workstation as follows Approximately 110 ml ethanol 70 1 Approximately 90 ml Denaturation Solution 2 Approximately 110 ml Wash Buffer 3 Approximately 110 ml high purity water 4 Approximately 180 ml high purity water Parking Position P A suggested setup is shown on the next page Refill the troughs to these levels whenever necessary The total volume that ca
32. and select Explore m Double click the file autorun exe 4 Follow the instructions that appear in the Setup Wizard PyroMark Q96 ID User Manual 12 2012 6 1 PyroMark Q96 Software 6 3 6 4 Note If NET Framework 3 5 has to be installed installation is prompted by the Setup Wizard the installation must be restarted after the NET Framework installation has been completed i e open the CD menu see step 3 and click Install PyroMark Q96 Software 5 When the software has been successfully installed click Exit Setup in the CD menu 6 Please use Windows Update www update microsoft com to check for any critical updates to the NET Framework 3 5 Uninstalling PyroMark Q96 Software 1 Select Control Panel in the Windows Start menu 2 If using Microsoft Windows XP click Add or Remove Programs if using Microsoft Windows 7 click Programs and then Programs and Features 3 In the list of programs select PyroMark Q96 4 Click Remove XP or Uninstall 7 Upgrading from PyroMark Q96 ID Software Previous versions of PyroMark Q96 ID Software stored Pyrosequencing runs in an Oracle database Users upgrading to the new PyroMark Q96 Software must first use the PyroMark Data Converter Tool to convert this run database into a format that is compatible with the new software Note This procedure should only be followed by users upgrading from the old PyroMark Q96 ID Software
33. arting the PyroMark Q96 ID ensure that the mains power cable is correctly inserted into the mains power connector on the rear panel and connected to a grounded mains power socket Turn the power switch to I On All indicator lights turn on when power is connected After 1 3 minutes the Busy light will turn off and the Information light will start flashing The instrument is now ready for a run PyroMark Q96 ID User Manual 12 2012 5 1 Operating Procedures When a run is started the flashing Information light will turn off and the Busy light will illuminate Keep the lid closed during a run If the Information light illuminates during a run the operator s computer has lost contact with the instrument 5 2 2 Starting the PyroMark Q96 Software 1 Inthe Windows Start menu select All programs PyroMark PyroMark Q96 2 The PyroMark Q96 Software User Guide can be accessed at any time by pressing the F1 key when in the software 5 3 Preparing a run 5 3 1 Setting up an assay 1 In the shortcut browser right click the folder you want to place the assay in and select New Assay followed by the desired assay type AQ CpG SNP or SQA from the context menu Note To add a shortcut to a folder or drive click Add Folder Shortcut 2 Click id in the toolbar to enter a run name and save the file in the desired folder Note You cannot use the same name as another file saved in t
34. cedures 5 17 run 5 14 setting up a run 5 3 setting up an assay 5 2 starting the system 5 1 P PCR B 1 optimization B 3 setup B 2 Q Quality assessments B 8 R Reagent preparation 5 13 S Safety Information 1 1 chemical hazards 1 3 electrical safety 1 2 heat hazards 1 4 mechanical hazards 1 3 proper use 1 1 Sample preparation 5 6 PyroMark Q96 ID User Manual 12 2012 Index 1 Index Servicing 7 1 T Set up assay B 5 Technical assistance 2 2 PCR B 2 Technical data A 1 Sicherheitshinweise E 1 Troubleshooting 8 1 Software 6 1 Tubing data backup 6 5 replacement 7 10 installation 6 1 installation testing 6 5 V system requirements 6 1 upgrading software 6 2 Starting template B 2 Strand separation 5 9 W Waste disposal A 4 Ventilation 1 4 4 2 Index 2 PyroMark Q96 ID User Manual 12 2012 www qiagen com Australia techservice au giagen com Austria techservice at qiagen com Belgium techservice bnl qiagen com Brazil suportetecnico brasil amp qiagen com Canada techservice ca qiagen com China techservice cn giagen 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 giagen com India techservice india qiagen com Ireland techservice uk giagen com Italy techservice it qiagen com Japan techservice jp giagen com Korea
35. de l appareil ne sont pas expos s des niveaux dangereux de substances chimiques ou biologiques toxiques comme cela est d fini dans les fiches de donn es de s curit FDS ou dans les documents de l OSHA de ACGIH t ou du COSHH applicables Pour plus d informations voir le site www giagen com safety L evacuation des vapeurs et la mise au rebut des d chets doivent s effectuer conform ment toutes les r glementations et lois nationales r gionales et locales relatives la sant et la s curit OSHA Occupational Safety and Health Administration tats Unis d Am rique Administration pour la sant et la s curit du travail ACGIH American Conference of Government Industrial Hygienists tats Unis d Am rique Conf rence am ricaine des hygi nistes industriels gouvernementaux COSHH Control of Substances Hazardous to Health Royaume Uni Contr le des substances dangereuses pour la sant D 4 PyroMark Q96 ID User Manual 12 2012 Appendix D 1 4 Dangers m caniques Quand l appareil PyroMark Q 96 ID est en marche son couvercle doit rester ferm AVERTISSEMENT A Pieces mobiles Afin d viter le contact avec les pi ces en mouvement lorsque l appareil PyroMark Q96 ID est en marche son couvercle doit rester ferm Ne pas d monter les panneaux du capot Ils ne renferment aucune pi ce r parable par l utilisateur En cas de probl me avec l appareil Py
36. dge pack and take out a new cartridge The cartridge has to be handled with care in order to maintain accuracy 3 Remove the plastic needle cover from the underside of the reagent cartridge Save the cover for use when disposing of the reagent cartridge Do not use the plastic needle cover between runs since applying and removing the cover several times may damage the needles Note If re using a reagent cartridge ensure that it is cleaned appropriately see Section 7 6 If handled correctly a reagent cartridge can be used for up to 20 runs Note It is important that the reagents are pipetted into the correct reagent cartridge compartments 4 Place the reagent cartridge with the label facing towards you Pipet the recommended volume of substrate solution into the S compartment of the reagent cartridge 5 14 PyroMark Q96 ID User Manual 12 2012 Operating Procedures 5 6 Pipet the recommended volume of enzyme solution into the E compartment Pipet the recommended volume of each dNTP solution into the A C G and T compartments Note do not use filtered tips to pipet nucleotides PyroMark Q96 Cartridge from above with indicated compartments for enzymes substrate and nucleotides 5 5 Run 5 5 1 Loading the PyroMark Q96 Plate Low 1 En co RE Open the instrument lid Note The dispensing unit and process chamber will move if a run is accidentally started Open the process chamber lid Open
37. dispensed to the well A legend indicates which reagent corresponds to each color Mixer frequency temperature of the process chamber and pressure in the dispensing unit are displayed in real time in the Instrument Status Monitor Instrument Status Monitor Mixer Hz Temperature C Pressure mbar 35 0 28 0 650 0 35 2 28 0 649 6 PyroMark Q96 ID User Manual 12 2012 5 17 Operating Procedures A run progress bar displays the current state of a run e g preparing for run adding reagents run end of run connection lost and the time remaining to complete the run Run Progress Run Remaining time 57 Minutes Warnings during a run Errors may occur during a run which triggers warnings that will appear in the Warnings pane The warning is also documented in the Run Log of the Run Information If an error occurs during a run a dialog box that appears at the end of the run gives the user the option to review the Run Information Restoring a run If the connection to the instrument is lost during a run a message box will appear to inform the user Upon clicking OK the Run view will close but the instrument will continue to process the run The data collected will be stored on the instrument and the run file can be restored See the PyroMark Q96 Software User Guide for more information 5 6 Post run procedures 5 6 1 Analyzing the run Detailed instructions for analyzing the run are
38. e copied Managing instrument parameters Instrument parameters should be selected according to the reagent cartridge that will be used for the run The code number printed on the PyroMark Q96 Cartridge corresponds to specific instrument parameter settings provided at www giagen com Products PyroMarkQ 96ID aspx Note We recommend that only instrument parameters supplied by QIAGEN are used Important In order to access instrument parameter settings online you must register your instrument with a valid serial number see back of instrument at www giagen com Managing instrument parameters in PyroMark Q96 Software 1 Select Instrument Parameters from the Tools menu to open the instrument parameters dialog box PyroMark Q96 ID User Manual 12 2012 Operating Procedures Right click an existing instrument parameter file listed in the upper pane and select Duplicate from the context menu Enter a new file name e g Instrument parameters Code 0005 Go to the Web page cited above and open the file Managing instrument parameters for the PyroMark Q96 ID found under the User Support tab In the provided table find the dispensing pressures and pulse time settings that correspond to the Code number printed on the PyroMark Q96 Cartridge to be used Enter the dispensing pressures and pulse time into the corresponding fields of the Instrument Parameters dialog box Save the new instru
39. e top with high purity water 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 the needle 5 Ifa needle is blocked wet the needle tip with a drop of high purity water PyroMark Q96 ID User Manual 12 2012 7 5 Maintenance N OS Press several droplets through each needle Discard the high purity water Leave the cartridge to dry on its side on a lint free cloth Do not apply the cap Note Ensure that the needles are dry on the outside before using them Store the cartridge in the PyroMark Q96 HS Tip Holder Box between uses 7 6 2 Clogged cartridge cleaning procedure If reagent solutions have been left in the cartridge a more thorough cleaning process might be needed to clear the needles Procedure 1 Discard any remaining solutions 2 Rinse the cartridge several times with high purity water 3 Fill the compartments with high purity water and immerse the cartridge in a beaker with high purity water covering the needles Leave the cartridge in the beaker for 1 hour 4 Proceed with the standard cleaning procedure in section 7 6 1 beginning with step 4 7 6 3 Cartridge function check To check if a cartridge can be used for analysis a simple function test can be performed 1 Fill the compartments completely with high purity water Take care not to let the water run ove
40. een Below the expected range M Green Within the expected range M Dark green Above the expected range SQA analysis results The base called sequence is displayed in the Overview tab above the Pyrogram of the selected well The bases in the base called sequence and the peaks in the compensated Pyrogram in the lower window of the Pyrogram pane are colored according to their quality assessments Analysis reports To generate a report select the desired report from the Reports menu For more information about reports see the View Print and Save Analysis Reports section of the PyroMark Q96 Software User Guide press the F1 key when in the PyroMark Q96 Software Note To view reports generated in PDF format a PDF reader must be installed on the computer Adobe Reader can be downloaded at www adobe com 5 22 PyroMark Q96 ID User Manual 12 2012 Operating Procedures 5 7 Finishing work 5 7 1 After a run is finished Remove and dispose of the reagent cartridge 1 Open the instrument lid 2 Open the dispensing unit cover 3 Remove the reagent cartridge 4 Close the dispensing unit cover Ensure that the latch snaps into its locked position Note If the reagent cartridge is to be reused clean it directly after use as described in Section 7 6 Sharp needles Do not touch the sharp needles at the bottom of the reagent cartridge Handle the needles with care Small particles a
41. en the heating block and correctly the instrument clean the lens array see Section 7 3 If the problem persists the contact pins may need replacing Contact QIAGEN Technical Services 8 5 Vacuum workstation related errors Comments and suggestions a No vacuum is Turn off the vacuum pump and open the cap to received the waste container to release any pressure Close the cap and start the pump again Empty the waste container if full Ensure that the tubing is connected correctly and that there is no leakage b Vacuum lost during Ensure thot the tubing is connected correctly and sample preparation there is no leakage c Filter probes not Ensure filter probes are working properly see working properly Section 7 8 1 d Liquid left in some Replace the corresponding filter probes see wells in the Section 7 8 2 immobilization plate e White remains If more than 1 min has elapsed since the plate or Sepharose beads strips was agitated agitate again for 1 min in the before capturing the beads immobilization plate 8 6 PyroMark Q96 ID User Manual 12 2012 Glossary 9 Term Biotin Dispensation order Drop off Enzyme Histogram Homopolymer Instrument parameters Glossary Description A molecule that can bind very strongly to streptavidin PCR primers can be biotinylated to enable the resulting PCR product to bind to streptavidin coated Sepharose beads Defines the nucleotides and the order in which they sh
42. enior Director Quality and Compliance Automated Systems PyroMark Q96 ID User Manual 12 2012 A 7 Appendix A This page intentionally left blank A 8 PyroMark Q96 ID User Manual 12 2012 Appendix B Appendix B Assay design and validation Assay design Pyrosequencing assays can be designed using the latest version of PyroMark Assay Design Software ADSW The program automatically generates primer sets that include both 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 PCR For PCR amplification we recommend using the PyroMark PCR Kit Cat no 978703 which is specifically optimized for Pyrosequencing analysis and enables highly specific and unbiased amplification of template DNA for various Pyrosequencing applications such as mutation detection SNP analysis methylation analysis and de novo sequencing The convenient master mix format enables specific amplification of various starting materials such as genomic DNA from a variety of sources as well as bisulfite converted DNA using only one protocol PCR primers One of the primers must be biotin labeled to enable immobilization of the PCR amplicons to streptavidin coated beads Streptavidin Sepharose High Performance GE Healthcare for the preparation of a single stranded DNA template The orientation of the assay can
43. er Manual 12 2012 C 1 Appendix C This page intentionally left blank C 2 PyroMark Q96 ID User Manual 12 2012 Appendix D Appendix D 1 Informations de s curit Avant d utiliser le PyroMark Q96 ID il est imp ratif de lire attentivement ce manuel et de porter une attention particuli re aux informations de s curit Afin de garantir un fonctionnement du syst me en toute s curit et de maintenir le systeme en bon tat de marche il est imp ratif de suivre les instructions et les informations de s curit fournies dans le pr sent manuel d utilisation Les types d informations de s curit suivants sont fournis tout au long du manuel AVERTISSEMENT A Le terme AVERTISSEMENT signale des situations risquant d entra ner des accidents corporels dont l utilisateur ou d autres personnes pourraient tre victime Les d tails concernant ces circonstances sont donn s dans un encadr identique celui ci ATTENTION A Le terme ATTENTION signale des situations risquant d entra ner des d t riorations de l appareil ou de tout autre mat riel Les d tails concernant ces circonstances sont donn s dans un encadr identique celui ci Les conseils donn s dans ce manuel ont pour but de venir compl ter les exigences de s curit habituelles en vigueur dans le pays de l utilisateur et non de s y substituer PyroMark Q96 ID User Manual 12 2012 D 1 Appendix D 1 1 Utilisa
44. es l int rieur de l quipement Lorsque l quipement est reli l alimentation les bornes peuvent tre sous tension et l ouverture de capots ou le retrait de pi ces risque d exposer des l ments sous tension Afin que le PyroMark Q96 ID fonctionne de mani re satisfaisante et en toute s curit suivre les conseils suivants M Les cordons d alimentation de l appareil doivent tre branch s sur des prises d alimentation munies d un conducteur de protection terre masse M Les fiches d alimentation reli es au secteur doivent tre facilement accessibles s il est n cessaire de d brancher rapidement l quipement du secteur M Utiliser uniquement les fiches et cordons d alimentation fournis avec le syst me E Si vous remplacez les fusibles ne les remplacer qu avec des fusibles du m me type et quivalent en voltage comme indiqu sur le fusible PyroMark Q96 ID User Manual 12 2012 D 3 Appendix D 1 3 Produits chimiques AVERTISSEMENT A Produits chimiques dangereux La solution de d naturation utilis e avec le poste de travail sous vide contient de l hydroxyde de sodium qui est irritant pour les yeux et la peau Toujours porter des lunettes de protection des gants et une blouse de laboratoire La personne responsable par exemple le directeur du laboratoire doit prendre les pr cautions n cessaires afin de garantir que le lieu de travail environnant est s r et que les op rateurs
45. etting moisture inside the instrument M The cleaning liquid must be applied to the cloth only The following items are required M Ethanol 70 B Clean nonabrasive lint free cloths Procedure 1 Shut down the instrument see Section 7 1 2 Open the instrument lid 3 Clean the area around the dispensing unit and process chamber using a lint free cloth moistened with 70 ethanol 4 Open the process chamber lid Clean the area around the heating block 7 2 PyroMark Q96 ID User Manual 12 2012 Maintenance 7 3 Cleaning the lens array The following items are required for cleaning the lens array beneath the heating block M Ethanol 70 M Clean nonabrasive lint free cloths Procedure 1 Shut down the instrument see Section 7 1 2 Open the instrument lid 3 Loosen the bayonet screw behind the heating block by turning it Ya turn counter clockwise until a click is heard 4 Lift out the heating block Avoid damaging the contact pins on the underside of the block 5 Carefully clean the lens array with a cloth moistened with 7096 ethanol 6 Letthe lens array dry before remounting the heating block 7 Place the heating block on top of the lens array Ensure that the rear part of the heating block is properly seated over the contact pins Do not force the heating block into place 8 Turn the bayonet screw 4 turn clockwise 7 4 Cleaning the heating block The following items are required for cleaning t
46. ew tab summarizes quality assessments Select the Quality option in the upper right corner to display the quality assessment of all variable positions in a selected well or of all bases in the base called sequence SQA assays also include quality assessment at the end of the quality control window Quality levels are indicated with different colors Blue Passed Yellow Check Red Failed White Not analyzed Either analysis is not supported by the software e g analyzing single nucleotide polymorphisms in CoG mode or the variable position has been deselected by the user AQ SNP and CpG assays only AQ SNP and CpG analysis results Allele frequencies AQ analysis genotypes SNP analysis and methylation percentages CpG analysis are displayed at PyroMark Q96 ID User Manual 12 2012 5 21 Operating Procedures 5 6 3 the top of the Pyrogram The quality assessment is indicated by the background color of the result Variable regions are highlighted with a blue gray background color In CpG analysis bisulfite treatment controls are highlighted in the Pyrogram with a light yellow background color Select the Ranges option in the upper right corner of the Overview tab to show methylation bars in the plate overview These bars indicate the methylation level for each CpG site in a selected well as compared to the expected level Expected ranged can be set under the Analysis Setup tab M Light gr
47. f the peak levels in difficult assays Ensure that the initial DNA sample is pure or that the assay is capable of specifically amplifying and or sequencing only one target sequence in the sample The assay may otherwise generate a mixed sequence that cannot be analyzed PyroMark Q96 ID User Manual 12 2012 Appendix B Pay attention to tips and warnings indicated by the red icon and make suitable modifications when the icon appears Validation of a new assay Controls All new assays have to be validated by the user Use a reference DNA sample when testing a new assay and ensure that appropriate analysis parameters in the PyroMark Q96 Software are used Interactions between primers or loops formed on single stranded DNA can serve as priming sites for base incorporation by DNA polymerase The following controls should be included when an assay is analyzed for the first time W PCR without template DNA This will show if the primers interact to give a background signal in Pyrosequencing reactions M PCR with template DNA but with no sequencing primer This will show if the template can loop back on itself and give a background signal in Pyrosequencing reactions M Sequencing primer without any PCR product This will show if the sequencing primer can form duplexes or hairpins and give background signal in Pyrosequencing reactions M Biotinylated primer without any PCR product This will show if the biotinylated primer can form dup
48. follow up information for scheduling collection of the electronic waste or to provide you with an individual quote PyroMark Q96 ID User Manual 12 2012 Appendix A FCC declaration The United States Federal Communications Commission USFCC in 47 CRF 15 105 declared that the users of this product must be informed of the following facts and circumstances This device complies with part 15 ofthe FCC 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 This Class B digital apparatus complies with Canadian ICES 003 The following statement applies to the products covered in this manual unless otherwise specified herein The statement for other products will appear in the accompanying documentation Note This equipment has been tested and found to comply with the limits for a Class B digital device pursuant to part 15 ofthe FCC Rules These limits are designed to provide reasonable protection against harmful interference in a residential installation This equipment generates uses and can radiate radio frequency energy and if not installed and used in accordance with the instructions may cause harmful interference to radio communications However there is no guarantee that interference will not occur in a particular installation If this equipment d
49. g sequencing primer added Wrong sequence to analyze entered Wrong dispensation order chosen 8 3 Peak errors a Wide peaks Comments and suggestions Assay specific problem due to secondary structure Contaminated or nonhomogeneous samples Follow sample preparation instructions carefully see Section 5 4 Reagents incorrectly stored or dissolved Too much or too little template Too much template DNA Check amount of DNA template used Reaction volume in wells was too high Use lower volume PyroMark Q96 ID User Manual 12 2012 8 3 Glossary b c d Nonspecific peaks Low signal High baseline noise Comments and suggestions Sequence signals from the self annealed sequencing primer and or biotinylated PCR primer and DNA template or nonspecific annealing of the sequencing primer Run DNA template sequencing primer and biotinylated PCR primer in individual wells If significant peaks appear consider redesigning the PCR and or sequencing primer s Contaminated nucleotides Background of contaminating sequence from PCR Consider redesigning sequencing primer Contact QIAGEN Technical Services if this problem persists Too little template Reagents incorrectly stored or dissolved Too much salt in the sample Follow sample preparation instructions carefully see Section 5 4 Dirty lens array Clean the lens array see Section 7 3 Contaminated nucleotides or background of conta
50. he heating block M Ethanol 70 M Cotton swabs Procedure 1 Shut down the instrument see Section 7 1 2 Open the instrument lid 3 Clean each well hole in turn with a cotton swab moistened with 70 ethanol 4 Allow the heating block to dry before closing the instrument lid PyroMark Q96 ID User Manual 12 2012 7 3 Maintenance 7 5 Changing the fuses The fuses of the PyroMark Q96 ID should be checked if the instrument power indicator is not illuminated even though the power cable is connected to the mains power outlet and the standby switch is on Important Ensure that the PyroMark Q96 ID is disconnected from the mains power before removing fuses from the instrument Have a qualified technician e g facilities staff ensure that the mains power outlet is live The following item is required M Small flat head screwdriver Procedure 1 Turn off the standby switch at the front of the instrument and disconnect the mains power cable 2 If necessary carefully turn the instrument so that the rear panel can be easily accessed 3 Use a small screwdriver to release the securing lugs on each side of the fuse holder 4 Pull out the fuse holder using your fingers The fuse holder contains two fuses type F 6 3 A H 250 V Check both fuses and replace as necessary Note Replace fuses only with fuses of the specified type and current rating 5 Insert the fuse holder it is keyed and can only be replaced
51. he plate or strips was agitated agitate again for 1 min before capturing the beads Apply vacuum to the tool by opening the vacuum switch Carefully lower the filter probes into the PCR plate to capture the beads containing immobilized template Hold the probes in place for 15 s Take care when picking up the tool Ensure that all liquid is aspirated from the wells and that all beads have been captured onto the filter probe tips Note If the wells still contain liquid or white beads remain the filter probes may need replacing see Section 7 8 2 Transfer the tool to the trough containing 70 ethanol trough 1 Flush the filter probes for 5 s Transfer the tool to the trough containing Denaturation Solution trough 2 Flush the filter probes for 5 s Transfer the tool to the trough containing Wash Buffer trough 3 Flush the filter probes for 10 s Raise the tool to beyond 90 vertical for 5 s to drain liquid from the filter probes see image PyroMark Q96 ID User Manual 12 2012 Operating Procedures 17 While holding the tool over the PyroMark Q96 Plate Low close the vacuum switch on the worktable Off 18 Release the beads in the plate containing 0 4 uM sequencing primer in 40 ul Annealing Buffer by shaking the tool gently from side to side in the plate Allow the filter probes to rest on the bottom of the wells 19 Transfer the tool to the trough containing high purity water trough 4 and agitate the
52. he same folder 3 If creating an AQ CpG or SNP assay type or paste the sequence to analyze into the Sequence to Analyze text box and then click the Generate Dispensation Order button If creating a CpG assay enter the sequence after the bisulfite treatment If entering an SQA assay enter the dispensation order 4 Click id in the toolbar Note Before running your samples validate your assay using a reference DNA sample Note When using QIAGEN kits use the settings stated in the kit handbooks 5 2 PyroMark Q96 ID User Manual 12 2012 Operating Procedures 5 3 2 Optional If desired enter a note about the assay and set up the variable positions in the Variable Positions tab If creating a CpG assay it is recommended that bisulfite treatment controls are added In the sequence before bisulfite treatment check if the suggested bisulfite controls are Cs converted to Ts read as Gs and As in a reverse assay and are suitable for controls or not To add a control left click a bold orange T or A in the histogram preferable at the beginning of the sequence e Al Lu SS HR Ho LL If applicable during the assay validation edit the analysis parameters Setting up a run 1 In the shortcut browser right click the folder you want to place the run file in and select New Run from the context menu Note To add a shortcut to a folder in the shortcut browser click Add Folder Shortcut 2 Click id
53. ication assays depend on equal amplification of both alleles and this must be carefully tested To ensure equal amplification in a CpG assay unmethylated DNA can be mixed with increasing proportions of completely methylated DNA We recommend using EpiTect Control DNAs Cat no 59695 which provide bisulfite treated completely methylated and unmethylated DNA in ready to use solutions Regression analysis of the frequency of one allele measured in the PyroMark Q96 ID as a function of the input expected allele should give an R value greater than 0 9 For an AQ assay the allelic variants including the variable position can be mixed at different ratios similar to the procedure for a CpG assay If the variable position in an AQ assay is a SNP the easiest way to test for equal amplification is to compare the peak heights from a heterozygote If the SNP is represented by single base incorporations e g AAC TGG the two alleles C and T peaks should give peaks of equal height An InDel heterozygote should give 50 deletion PyroMark Q96 ID User Manual 12 2012 Appendix B Sample preparation Use 5 20 ul of a 25 ul PCR for immobilization of PCR amplicons to streptavidin coated sepharose beads Streptavidin Sepharose High Performance GE Healthcare according to the instructions in Section 5 4 3 This will be equivalent to approximately 1 4 picomoles of PCR product depending on the PCR efficiency Note When using the PyroMark PCR
54. imitation Type plate on EN Legal manufacturer the back of the instrument and all other products 1 4 PyroMark Q96 ID User Manual 12 2012 Location Type plate on the back of the instrument Language EN Safety Information Description Warning consult user manual Type plate on EN Serial number the back of the instrument and box Type plate on EN Waste Electrical and the back of Electronic Equipment the instrument WEEE EEE FE Type plate on EN FCC mark of the United the back of States Federal the instrument Communications Commission Type plate on EN C Tick mark for Australia the back of supplier identification the instrument N17965 N15128 63 Type plate on EN RoHS mark for China the 237 the back of restriction of the use of the instrument certain hazardous substances in electrical and electronic equipment Type plate on EN CSA listing mark for SP the back of Canada and the USA C s the instrument PyroMark Q96 ID User Manual 12 2012 1 5 Safety Information This page intentionally left blank 1 6 PyroMark Q96 ID User Manual 12 2012 Introduction 2 1 Introduction Thank you for choosing the PyroMark Q96 ID We are confident it will become an integral part of your laboratory Before using the PyroMark Q96 ID it is essential that you read this user manual carefully and pay particular attention to the safety information The instructions and
55. in one way and push it into place using your fingers 6 Reposition the instrument reconnect the power cable and turn the standby switch on 7 Ifthe instrument still does not work please contact QIAGEN Technical Services 7 4 PyroMark Q96 ID User Manual 12 2012 Maintenance 7 6 Cleaning and testing the reagent cartridge If the reagent cartridge is to be reused it must be cleaned directly after use It is recommended that the reagent cartridge is used a maximum of 20 times WARNING A Sharp needles W7 Do not touch the sharp needles at the bottom of the reagent cartridge Handle the needles with care Small particles and fibers may obstruct the needles 7 6 1 Note Be sure to observe all federal state and local environmental regulations for the disposal of laboratory waste The following items are required M Powder free gloves E High purity water Milli Q 18 2 MQ x cm or equivalent M Clean lint free cloths There are two possible cleaning procedures A standard cleaning procedure Section 7 6 1 and a cleaning procedure for clogged cartridges Section 7 6 2 Ensure that a cleaned cartridge is usable by performing a cartridge function check Section 7 6 3 Standard cleaning procedure Procedure 1 Discard any remaining solutions 2 Rinse the cartridge four times with high purity water 3 Spray the outside of the needles with high purity water 4 Fill the compartments to th
56. instrument is now ready to receive instructions from the operator s computer 4 Start the PyroMark Q96 Software from the Start menu 5 In the shortcut browser right click the folder in which to place the installation test run file and select New Run from the context menu Ak PyroMark Q96 25 1 AQ Run Ar M File Tools Instrument Reports Window E Uzal gia Egg Shortcuts Ej Example Run SNP1 ES SQA1 2 2 QCpG Files TJ QCpC Example Run Setup E QCpG Assay Explore Refresh bes New Assay gt D New Run Remove Shortcut Properties 6 Click id in the toolbar to enter a run name and save the file in the desired folder 7 Add an assay to all wells e g select all wells and drag an assay from the shortcut browser to the selection 6 6 PyroMark Q96 ID User Manual 12 2012 PyroMark Q96 Software Note If there are no existing assays in the shortcut browser refer to section 5 3 1 to set up a new one 8 In the Instrument Parameters drop down list select the instrument parameters file that corresponds to the PyroMark Q96 Cartridge to be used 9 Click id to save 10 Open the instrument lid 11 Open the process chamber lid 12 Place an empty PyroMark Q96 Plate Low on the heating block 13 Close the process chamber lid 14 Open the dispensing unit cover and insert an empty reagent cartridge 15 Close the dispensing unit cover and press the latch down into its locked po
57. ions of the PyroMark Q96 ID and PyroMark Q96 Vacuum Workstation Ensure that the workbench is level dry clean vibration proof and has additional space for accessories Approximately 80 cm 31 5 in clearance above the workbench is required to accommodate the PyroMark Q96 ID with the lid open Allow at least 10 cm 4 in of free space behind the instrument for cabling PyroMark Q96 ID User Manual 12 2012 4 1 Installation Procedures The PyroMark Q96 ID must be placed within approximately 1 5 m 59 in of a properly grounded earthed AC power outlet The power line to the instrument should be voltage regulated and surge protected Note It is recommend to plug the instrument directly into its own wall socket and not to share the socket with other lab equipment CAUTION A Risk of overheating C1 To ensure proper ventilation maintain a minimum clearance of 10 cm at the sides and rear of the PyroMark Q96 ID Slits and openings that ensure the ventilation of the PyroMark Q96 ID must not be covered Power requirements The PyroMark Q96 ID operates at M 100 240 V AC 50 60 Hz 1 1 0 45 A Depending on the unit purchased the PyroMark Q96 Vacuum Workstation operates at M 100 VAC 50 60 Hz 1 7 1 4 A M 115VAC 60 Hz 1 5 A M 230 VAC 50 Hz 0 6 A Ensure that the voltage rating of the PyroMark Q96 ID and PyroMark Q96 Vacuum Workstation is compatible with the AC voltage available at the installation
58. ique to confirm there is only one specific band PyroMark Q96 ID User Manual 12 2012 8 1 Glossary Comments and suggestions Insufficient sample amounts Follow sample preparation instructions carefully see Section 5 4 Wells marked in the software do not correspond to the sample placement in the PyroMark Q96 Plate Low Check the plate was loaded according to the information under the Sample tab in the Run Setup dialog Insufficient enzyme or substrate for all the wells Check the reagent cartridge was filled according to the Volume Information under the Run Info tab Sequence errors Comments and suggestions Ensure thot all nucleotide chambers in the reagent cartridge contain nucleotides Check the reagent cartridge for blockages and rinse or replace it as necessary Check the sample and sample preparation Contaminated samples will lead to an unusually high consumption of substrate at substrate addition giving a high presequencing signal and no light produced at nucleotide dispensation b Results only in some wells of the plate 8 2 a Some peaks appear but the sequence stops 8 2 PyroMark Q96 ID User Manual 12 2012 Glossary b Faulty or poor sequence Comments and suggestions Contaminated or nonhomogeneous samples Follow sample preparation instructions carefully see Section 5 4 Reagents incorrectly stored or dissolved Too much or too little template Wron
59. ivery and installation 4 1 4 2 Requirements 4 1 4 3 Moving the PyroMark Q96 ID 4 3 4 4 Reinstalling the PyroMark Q96 ID Stand alone 4 4 4 5 Reinstalling the PyroMark Q96 ID Network installation 4 4 4 6 Connecting mains power 4 5 4 7 Setting up network properties 4 5 4 7 1 Used IP addresses for instrument and computer 4 5 5 Operating Procedures 5 1 5 1 Required items 5 1 5 2 Starting the system 5 1 5 2 1 Starting the PyroMark Q96 ID 5 1 5 2 2 Starting the PyroMark Q96 Software 5 2 5 3 Preparing a run 5 2 5 3 1 Setting up an assay 5 2 5 3 2 Setting up a run 5 3 5 3 3 Managing instrument parameters 5 4 5 4 Sample preparation 5 6 5 4 1 Assembly of the PyroMark Q96 Vacuum Workstation 5 7 5 4 2 DNA amplification 5 7 5 4 3 Immobilization of the PCR product to beads 5 8 5 4 4 Strand separation 5 9 5 4 5 Annealing of sequencing primer to samples 5 14 5 4 6 Reagent preparation 5 14 5 5 Run 5 15 5 5 1 Loading the PyroMark Q96 Plate Low 5 15 Contents 2 PyroMark Q96 ID User Manual 12 2012 Contents 5 5 2 Loading the reagent cartridge 5 16 5 5 3 Starting the run 5 17 5 5 4 Monitoring the run 5 17 5 6 Post run procedures 5 18 5 6 1 Analyzing the run 5 18 5 6 2 Viewing the analysis results 5 19 5 6 3 Analysis reports 5 22 5 7 Finishing work 5 23 5 7 1 After a run is finished 5 23 5 7 2 Leaving the instrument on 5 23 5 7 3 Exiting PyroMark Q96 Software 5 24 5 7 4 Cleaning the instrument 5 24 5 7 5 Emptying the waste container and troughs 5
60. k Q96 ID User Manual 12 2012 Contents Appendix C C 1 Liability clause C 1 Appendix D D 1 Informations de s curit D 1 Appendix E E 1 Sicherheitshinweise E 1 Index Index 1 PyroMark Q96 ID User Manual 12 2012 Contents 5 Contents This page intentionally left blank Contents 6 PyroMark Q96 ID User Manual 12 2012 Safety Information Safety Information Before using the PyroMark Q96 ID 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 instrument and to maintain the instrument in a safe condition The following types of safety information appear throughout this manual WARNING The term WARNING is used to inform you about situations that could result in personal injury to you or other A persons Details about these circumstances are given in a box like this one CAUTION The term CAUTION is used to inform you about situations A 1 1 WARNING CAUTION A that could result in damage to the instrument or other equipment Details about these circumstances are given in a box like this one The advice given in this manual is intended to supplement not supersede the normal safety requirements prevailing in the user s country Proper use Risk of personal injury and material damage W1 Improper use of the PyroMark
61. lexes or hairpins and give background signal in Pyrosequencing reactions M Sequencing primer and biotinylated primer together without 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 a peak after any nucleotide addition PyroMark Q96 ID User Manual 12 2012 B 7 Appendix B B 8 Quality assessment The user will be warned if something in the assay may reduce the quality of the result given by the analysis software The ultimate goal for a well optimized assay is that all variable positions in an AQ or CpG assay or the sequence in the quality control window of a SQA assay have the quality assessment Passed when using default or more stringent analysis parameters Such results will be shown as blue in the quality bar in the well after analysis Results of lower quality are indicated as Check yellow or Failed red together with error messages Analysis results For samples and positive controls aim for Sufficient signal intensities Aim for a single peak height of at least 20 RLU No background in blank dispensations No background in variable positions AQ and CpG Expected reference sequence pattern AQ and CpG All positions AQ and CpG and quality control window SQA with quality assessment Passed The quality assessments for AQ and CpG assays
62. ll as to the researchers at QIAGEN We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques For technical assistance and more information please see our Technical Support Center at www qiagen com goto TechSupportCenter or call one of the QIAGEN Technical Service Departments or local distributors 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 Please contact QIAGEN Technical Services 2 2 3 Version management This document is the PyroMark Q96 ID User Manual version 4 0 2 2 PyroMark Q96 ID User Manual 12 2012 Introduction 2 3 Intended use of the PyroMark Q96 ID The PyroMark Q96 ID System detects changes in specified variable positions in or base calling of DNA prepared from biological samples The PyroMark Q96 ID Instrument and PyroMark Q96 Vacuum Workstation are intended to be used only in combination with QIAGEN kits indicated for use with the PyroMark Q96 ID Instrument for the applications described in the kit handbooks If the PyroMark Q96 ID Instrument and PyroMark Q96 Vacuum Workstation are used with kits other than QIAGEN kits it is the user
63. ly left blank 7 12 PyroMark Q96 ID User Manual 12 2012 Glossary 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 Instrument Service Specialist in solving the problem When calling QIAGEN Technical Service about errors please have the following information ready Instrument serial number type and version Date of last maintenance performed Error code if applicable Time point when the error occurred for the first time Frequency of error occurrence i e intermittent or persistent error Photo of error if possible Take the following action before contacting QIAGEN Technical Services 1 Check important parameters in the Run Overview in the Instrument Status tab to assess if the system was working properly during the run 2 Consult the Troubleshooting sections below 8 1 Unexpected results Comments and suggestions a No result in any Check the reagent cartridge and ensure that it is wells of the plate correctly filled with reagents and or nucleotides Check that the needles are not damaged or obstructed and that the reagent cartridge cover is closed Insufficient sample or incorrectly conditioned sample e g too much dsDNA or salt present Follow sample preparation instructions carefully see Section 5 4 Check the PCR samples using a gel techn
64. m Steckdosen mit Schutzleiter Erdungs Masseleiter angeschlossen werden M Sorgen Sie daf r dass die Netzstecker jederzeit frei zug nglich sind falls das Ger t einmal schnell vom Stromnetz getrennt werden muss M Benutzen Sie nur Netzanschl sse und kabel die mit dem Ger t geliefert werden M Beim Austausch der Netzsicherung ersetzen Sie diese nur durch eine desselben Typs und der Stromst rke die auf dem Etikett Typenschild angegeben ist PyroMark Q96 ID User Manual 12 2012 E 3 Appendix E 1 3 Chemikalien WARNUNG A Gef hrliche Chemikalien Die Denaturierungsl sung die zusammen mit der Vakuum Arbeitsstation verwendet wird enth lt Natriumhydroxid das auf Augen und Haut reizend wirkt Tragen Sie immer eine Schutzbrille Laborhandschuhe und einen Laborkittel Die verantwortliche Person z B der Laborleiter muss alle erforderlichen Vorsichtsma nahmen treffen um sicherzustellen dass die unmittelbare Umgebung des Arbeitsplatzes sicher ist Auch d rfen die Grenzwerte in Bezug auf infekti se Erreger die in den entsprechenden Sicherheitsdatenbl ttern SDS oder den Vorschriften der OSHA ACGIH oder COSHH festgelegt sind nicht berschritten werden Weitere Informationen finden Sie unter www giagen com safety Beim Betrieb eines Abzugs und bei der Entsorgung von Abfallstoffen m ssen alle Bestimmungen und Gesetze zu Gesundheitsschutz und Sicherheit am Arbeitsplatz auf bernationaler
65. m einen sicheren Betrieb des PyroMark Q96 ID zu gew hrleisten und das Ger t in einem sicheren Zustand zu erhalten In diesem Handbuch werden die folgenden beiden Kategorien von Sicherheitshinweisen verwendet WARNUNG Der Begriff WARNUNG WARNING weist Sie auf Situationen hin in denen eine Verletzungsgefahr f r Sie selbst oder andere Personen besteht N here Einzelheiten ber diese Situationen werden in einem Textfeld wie diesem beschrieben ACHTUNG Der Begriff ACHTUNG CAUTION weist Sie auf Situationen hin in denen das Ger t oder andere Ger te besch digt werden k nnten N here Einzelheiten ber diese Situationen werden in einem Textfeld wie diesem beschrieben Die in diesem Handbuch enthaltenen Hinweise stellen eine Erg nzung und keinen Ersatz der blichen Sicherheits anforderungen dar die im jeweiligen Land gelten PyroMark Q96 ID User Manual 12 2012 E 1 Appendix E 1 1 Sachgem e Handhabung WARNUNG Verletzungsgefahr und Besch digung des Ger ts ACHTUNG Die unsachgem e Bedienung des PyroMark Q96 ID A kann zu einer Verletzung des Benutzers oder zur Besch digung des Ger ts f hren Die Bedienung des PyroMark Q96 ID darf nur durch qualifiziertes entsprechend geschultes Personal erfolgen Die Instandhaltung des PyroMark Q96 ID darf nur durch einen Servicespezialisten des QIAGEN Au endiensts durchgef hrt werden F hren Sie alle
66. mains power as per Section 4 6 Reinstalling the PyroMark Q96 ID Network installation The operator s computer is configured with a built in Ethernet port to connect to the PyroMark Q96 ID and an external USB adaptor to connect to a Local Area Network LAN The main advantages of connecting the PyroMark Q96 ID to a LAN are M Easier access to run dota files M Assays can be set up and analyzed on office computers M Data such as SNP entries can be shared between different users M Access to LAN printing resources PyroMark Q96 ID User Manual 12 2012 Installation Procedures Install a LAN system as follows 1 Connect one end of the cross connected Ethernet network cable provided to the NET connector on the rear panel of the PyroMark Q96 ID Connect the other end of the network cable to the built in LAN outlet on the operator s computer Connect the second Ethernet network cable between the Ethernet USB adaptor of operator s computer and the nearest LAN connection point Connect mains power as per Section 4 6 Start up the operator s computer and assign a unique IP address to the second network adapter for communication with the LAN 4 6 Connecting mains power 1 Ensure the standby switch on the front of the PyroMark Q96 ID is in position press the lower part of the switch Connect the mains cable to the inlet on the rear of the PyroMark Q96 ID Connect the mains cable to a grounded mains p
67. ment parameter file When creating a new Run Setup select this new instrument parameter file from the list of instrument parameter files PyroMark Q96 ID User Manual 12 2012 5 5 Operating Procedures 5 4 Instrument parameters In the Instrument Parameters dialog box the following parameters are available Reagent pressure Pressure millibar for dispensation of the enzyme mix and substrate mix Enzyme pulse time Dispensation time milliseconds for the enzyme mix Substrate pulse time Dispensation time milliseconds for the substrate mix Nucleotide pressure Pressure millibar from the dispensation of nucleotides Nucleotide pulse Dispensation time time milliseconds for nucleotides Note Note about the instrument method optional Sample preparation Equipment and reagents to be supplied by the user PyroMark Q96 Vacuum Workstation PyroMark Q96 Plate Low cat no 979002 PyroMark 96 Sample Prep Thermoplate Low supplied with the PyroMark Q96 ID Mixer for immobilization to beads Heating block capable of attaining 80 C 96 well PCR plate and adhesive foil or strip caps Biotinylated PCR template Streptavidin Sepharose HP GE Healthcare cat no 17 5113 01 Sequencing primer High purity water Milli Q 18 2 MQ x cm or equivalent Ethanol 70 5 6 PyroMark Q96 ID User Manual 12 2012 Operating Procedures 5 4 1 5 4 2 PyroMark Binding Buffer cat no 979006
68. minating sequence from PCR Contact QIAGEN Technical Services if this persists PyroMark Q96 ID User Manual 12 2012 Glossary Comments and suggestions e Occasional peaks Cross talk or dispensation errors Avoid placing between assays with high signals close to assays with low dispensations signals Check and replace the reagent cartridge if necessary Light leaking into the instrument Do not place the instrument in strong sunlight Contact QIAGEN Technical Services for camera calibration 8 4 Instrument related errors Comments and suggestions a No contact between Check mains power supply to instrument and the operator s ensure the Power light is illuminated computer and the PyroMark Q96 ID Check the network cables are properly connected at the rear of the instrument and the wall outlets If contact was broken during a run a restore of the run may be necessary No match between IP numbers for the instrument and the operator s computer or the IP number conflicts with other equipment on the network Contact your system administrator b All lights on the Instrument software problem Contact QIAGEN PyroMark Q96 ID Technical Services remain on 5 minutes after it has been switched on PyroMark Q96 ID User Manual 12 2012 8 5 Glossary Comments and suggestions c Heating block Remove and reinsert the heating block see temperature control Section 7 4 networking If there is liquid betwe
69. n be added to each trough is 180 ml There is a mark on each trough indicating 90 ml PyroMark Q96 ID User Manual 12 2012 Operating Procedures 90 ml 180 ml Denaturation high purity Solution water E 110 ml 4 high purity E A water 110 ml pm m 110 ml 70 1 3 Wash ethanol Buffer PCR plate PyroMark plate Switch on the vacuum pump Apply vacuum to the tool by opening the vacuum switch Wash the filter probes by lowering the probes into high purity water Parking Position Flush the probes with 180 ml high purity water Ensure that the water is being transferred to the waste container If it is not ensure the tubing is connected correctly and is not broken Broken tubing should be replaced see Section 7 9 Ensure that the tubing is connected properly and there is no leakage Refill Parking Position with 180 ml high purity water Close the vacuum switch on the worktable Off and place the tool in the Parking Position Add 40 ul of 0 4 UM sequencing primer in Annealing Buffer to each well of the PyroMark Q96 Plate Low that is to be used PyroMark Q96 ID User Manual 12 2012 5 11 Operating Procedures 9 10 11 12 13 14 15 16 After shaking place the PCR plate and PyroMark Q96 Plate Low on the worktable Ensure that both plates are in the same orientation as when samples were loaded Note Sepharose beads sediment quickly If more than 1 min has elapsed since t
70. nd fibers may obstruct the needles Note To avoid injury replace the plastic needle cover on the reagent cartridge prior to disposal Note Be sure to observe all federal state and local environmental regulations for the disposal of laboratory waste Remove and dispose of the PyroMark Q96 Plate Low 1 Open the process chamber lid 2 Open the plate holding frame and remove the PyroMark Q96 Plate Low from the heating block 3 Close the plate holding frame and the process chamber lid Note Be sure to observe all federal state and local environmental regulations for the disposal of laboratory waste 5 7 2 Leaving the instrument on To ensure stable operating conditions the PyroMark Q96 ID should remain on between runs PyroMark Q96 ID User Manual 12 2012 5 23 Operating Procedures Shut down the instrument only if it will not be used again within two weeks See Section 7 1 for shutdown instructions 5 7 3 Exiting PyroMark Q96 Software Exit the PyroMark Q96 Software by choosing Exit from the File drop down menu 5 7 4 Cleaning the instrument Remove any spillages around the dispensing unit and process chamber with a clean lint free cloth See Sections 7 2 through 7 6 for detailed cleaning instructions 5 75 Emptying the waste container and troughs WARNING Hazardous chemicals W4 A The Denaturation Solution used with the vacuum workstation contains sodium hydroxide which is irritating
71. nings indicated by the red icon and make suitable modifications when the icon appears Bisulfite treatment controls in CpG assays When creating a CpG assay inclusion of bisulfite treatment controls is recommended Cytosines not followed by a Guanine indicated as orange Ts in PyroMark ADSW should be fully converted to Thymine during bisulfite treatment and can therefore be used as controls for the reaction When creating a CpG assay the software indicates possible dispensations as controls for the bisulfite treatment reaction The original sequence before bisulfite treatment can be entered in the assay and used to see if the suggested controls are Cs converted to Ts read as Gs and As in a reverse assay and suitable as controls or not The preferable controls are those dispensations that are located at the beginning of the sequence and or represent single base incorporations SQA assays Based on experience sequencing large numbers of templates the dispensation order n CTGA gives on average the best de novo sequencing quality Individual templates may however give better results with other dispensation orders SQA assays set up to differentiate multiple sequences should not be designed to distinguish variants based on accurate sequencing of homopolymers In addition it may be useful to have a few known bases at the beginning of the sequence preferably single peaks These can be used as reference peaks to aid the setting o
72. nucleotide polymorphisms SNPs mutation and methylation analysis The instrument can analyze 96 samples simultaneously An easy to use protocol is used to prepare samples Enclosed in the light proof instrument housing a light detection system with cooled charged coupled device CCD camera detects light emitted from reactions in the wells of the PyroMark Q96 Plate Low The instrument includes an embedded computer module with hard disk and network interface PyroMark Q96 ID definitions PyroMark Q96 ID Instrument Instrument only PyroMark Q96 Software Software only PyroMark Q96 Vacuum Workstation Vacuum Workstation only PyroMark Q96 ID Instrument software and installation PyroMark Q96 ID System All of the above plus any PyroMark kits Pyrosequencing principle Pyrosequencing uses sequencing by synthesis for accurate and quantitative analysis of DNA sequences 1 A sequencing primer is hybridized to a single stranded PCR amplified DNA template 2 The template is incubated with enzymes and substrates 3 The first of four nucleotides is added to the reaction If the nucleotide is complementary to the base in the template strand it will be incorporated into the DNA strand by the DNA polymerase PyroMark Q96 ID User Manual 12 2012 3 1 General Description 4 Each incorporation event is accompanied by release of pyrophosphate PPi in an equimolar quantity to the amount of nucleotide incorporated Polymerase
73. o dalabore orci Step 2 Select Data PEELE Es 4 Click LJ and select or create a new directory in which to save the converted data The path to the selected directory will be shown in the text field 5 Click Convert to convert and export all selected data to the specified directory The progress of the export will be displayed in the progress bar Data backup Good data management requires that data backups are made on a regular basis Note QIAGEN is not responsible for the setup and administration of the user s backup routine Installation testing Stand alone systems and LAN systems should be tested after installation using the test method below This test will check the main functions of the hardware and software If the test functions correctly the PyroMark Q96 ID can be used for running samples PyroMark Q96 ID User Manual 12 2012 6 5 PyroMark Q96 Software 1 Start the operator s computer Microsoft Windows starts and displays the welcome screen 2 Press Ctrl Ali Delete and log on as the Administrator No password is required The Windows desktop will appear 3 Start the PyroMark Q96 ID using the standby switch on the front panel All lights on the front panel will illuminate The instrument software is loaded onto the PyroMark Q96 ID This takes 1 3 min The yellow Busy light on the instrument will then go out and the yellow Information light will start to flash The
74. oes cause harmful interference to radio or television reception which can be determined by turning the equipment off and on the user is encouraged to try to correct the interference by one or more of the following measures M Reorient or relocate the receiving antenna M Increase the separation between the equipment and receiver M Connect the equipment into an outlet on a circuit different from that to which the receiver is connected M Consult the dealer or an experienced radio TV technician for help PyroMark Q96 ID User Manual 12 2012 A 5 Appendix A QIAGEN s FCC Compliance Tests were conducted using QIAGEN supported peripheral devices and shielded cables such as those you receive with your system Changes or modifications not expressly approved by QIAGEN could void the user s authority to operate the equipment C A 6 PyroMark Q96 ID User Manual 12 2012 Appendix A Declaration of Conformity Name and address of the company QIAGEN GmbH QIAGEN Strasse 1 40724 Hilden Germany We herewith declare that the product PyroMark Q96 ID meets all applicable requirements of the following European Directives Low Voltage Directive LVD 2006 95 EC Electromagnetic Compatibility Directive EMC 2004 108 EC and the relevant harmonized standards IEC 61010 1 2001 IEC 61010 2 081 2001 A1 2003 IEC 61326 1 2006 Accessories PyroMark Q96 Vacuum Workstation Hombrechtikon March 4 2011 NO x X Pit Muggli S
75. or COSHH documents For more information visit www qiagen com safety Venting for fumes and disposal of wastes must be in accordance with all national state and local health and safety regulations and laws 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 If the Vacuum Workstation needs to be cleaned to remove dust and spillage follow the instructions below The following items are required Powder free gloves High purity water Milli Q 18 2 MQ x cm or equivalent A mild detergent if necessary Clean lint free cloths 7 8 PyroMark Q96 ID User Manual 12 2012 Maintenance 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 2 Disconnect the vacuum pump from the mains power 3 Clean the worktable and the tool except for the filter probes using a clean lint free cloth moistened with water or a mild detergent Note Do not touch the tips of the filter probes 4 Wipe the worktable and the tool except for the filter probes dry using a clean lint free cloth 5 Reconnect the vacuum pump to the mains power 7 8 Testing and replacing the filter probes 7 8 1 Function test for filter probes Before using the PyroMark Q
76. or the well with the orange selection frame is shown PyroMark Q96 ID User Manual 12 2012 5 19 Operating Procedures TCT T CGGCACCA GBA GCT GA GCCAA TT CA TGGA CCAGAA CA ACCCGCT GT CGGGGT TGACCCACAA nt SATA Pyrograms from 2 or more wells can be viewed simultaneously by opening one or more additional Pyrograms of selected wells in the lower area of the Pyrogram pane Get an overview of the results The following well information can be viewed in the plate overview in the Overview tab Select to show the assay name y LI e AQ and SNP assays Select to show the sample ID EN Select to show the well note Select to show the quality bar The quality bar shows the quality assessment of all variable positions in the well or of all the bases in the base called sequence CpG assays Select to show the mean methylation percentage of all CpG sites in the well 5 20 PyroMark Q96 ID User Manual 12 2012 Operating Procedures ms Assy Select to show the methylation bar The re methylation bar shows the methylation r Semple ID level for each CpG site in the well E Note ea Quality om Ranges SQA assays Q Select to show the quality assessment at the end of the quality control window None The default number of bases included is Assy 20 Sample ID Note Quality Q Window Quality assessments The plate overview in the Overvi
77. ould be dispensed in Pyrosequencing runs continual decrease in signal height normally seen in the Pyrogram A protein or RNA working as a catalyst to enhance the speed of a biochemical reaction without altering it In Pyrosequencing technology a mixture of Klenow polymerase sulfurylase luciferase and apyrase is used in the sequencing reaction The theoretical representation of the expected Pyrosequencing peak pattern A stretch of identical bases in DNA In Pyrosequencing technology a stretch of more than two identical bases is regarded as a homopolymer A method that describes physical settings for the instrument such as mixer frequency block temperature and pulse time settings PyroMark Q96 ID User Manual 12 2012 9 1 Glossary Term IUPAC Out of phase Polymorphism Presequencing signal Description International Union of Pure and Applied Chemistry An organization providing recommendations on organic and biochemical nomenclature symbols terminology etc IUPAC codes A Adenine K TorG C Cytosine W TorA G Guanine S CorG T Thymine B C T or G not A R Purine A or G D A T or G not C Y Pyrimidine C or T H A T or C not G M CorA V A C orG not T N Any base A C G or T When one of the alleles is sequenced ahead of the other Genetic variations broadly encompassing any of the many types of variations in DNA sequences that are found within a given pop
78. ower outlet The PyroMark Q96 ID will automatically adjust to the correct voltage 4 7 Setting up network properties 4 7 1 Used IP addresses for instrument and computer The instrument IP address and the host IP address operator s computer are fixed and do not need to be changed if the PyroMark Q96 ID is connected to a LAN E Operator s computer IP address 192 168 255 200 M PyroMark Q96 ID IP address 192 168 255 201 PyroMark Q96 ID User Manual 12 2012 4 5 Installation Procedures This page intentionally left blank 4 6 PyroMark Q96 ID User Manual 12 2012 Operating Procedures 5 1 5 2 5 2 1 Operating Procedures This section describes how to operate the PyroMark Q96 ID Before proceeding it is recommended that you familiarize yourself with the features of the PyroMark Q96 ID by referring to Sections 3 4 and 3 5 Required items The following items are needed to perform a run Operator s computer loaded with PyroMark Q96 Software PyroMark Q96 Vacuum Workstation Sample preparation buffers PyroMark Gold Q96 Reagents PyroMark Q96 ID Cartridge PyroMark Q96 Plate Low High purity water Milli Q 18 2 MQ x cm or equivalent Powder free gloves Lint free tissues Starting the system The PyroMark Q96 ID requires a warm up time of 90 minutes for the CCD camera output to stabilize To save time switch on the instrument before starting to prepare samples Starting the PyroMark Q96 ID Before st
79. r the edge and wet the needles on the outside 2 Press firmly on top of each compartment in turn with a finger use powder free gloves A small jet of water should come out from the tip of each needle If no jet is seen fill the compartment with more water and try again 3 Check that the jet of water is straight in the direction of the needle If not fill with water again and press firmly 7 6 PyroMark Q96 ID User Manual 12 2012 Maintenance 4 Ifthe test fails discard the cartridge 5 After a cartridge has passed the function test allow it to dry completely at ambient temperature 15 25 C before using Cleaning the PyroMark Q96 Cartridge Al Filling the compartments with high purity water 3 Placing pressure on compartments Cartridge immersed in a beaker with high purity water PyroMark Q96 ID User Manual 12 2012 7 7 Maintenance 7 7 Cleaning the PyroMark Q96 Vacuum Workstation WARNING Hazardous chemicals w4 The Denaturation Solution used with the vacuum A workstation contains sodium hydroxide which is irritating to eyes and skin Always wear safety glasses gloves and a lab coat The responsible body e g laboratory manager must take the necessary precautions to ensure that the surrounding workplace is safe and that the instrument operators are not exposed to hazardous levels of toxic substances chemical or biological as defined in the applicable Safety Data Sheets SDSs or OSHA ACGIH
80. rms DNA sequencing using Pyrosequencing technology 1 After setting up the run in the PyroMark Q96 Software the PyroMark Q96 Plate Low containing DNA samples is placed on the heating block in the instrument and the reagent cartridge containing enzymes substrate and nucleotides is placed in the dispensing unit The run is then started when the user clicks the Run button in the Run Setup dialog box The operator s computer sends all information required for the analysis to the instrument Note During a run data are continuously stored on the operator s computer and on the computer embedded in the instrument If the communication between the instrument and the operator s computer is interrupted during the run data from the run will be automatically restored once communication has been re established The temperature of the heating block and chamber rises to preset levels The mixer speed adjusts to the preset level The pressure in the pneumatic dispensing system rises to the preset level PyroMark Q96 ID User Manual 12 2012 3 3 General Description 11 12 13 14 15 Once the temperatures and pressure reach the preset levels this may take several minutes the predispensation and dispensation of the reagents begins Enzyme and substrate mixtures are predispensed into the rectangular sink well at the right hand edge of the PyroMark Q96 Plate Low to ensure that the dispensation capillarie
81. roMark Q96 ID contacter imm diatement les Services techniques de QIAGEN AVERTISSEMENT A Risque de pincement et d impact L unit de distribution le couvercle de la chambre de r action et l instrument peuvent bouger tout moment AVERTISSEMENT A Aiguilles pointues Ne pas toucher les aiguilles pointues situ es au fond de la cartouche de r actifs Manipuler les aiguilles avec pr caution De petites particules ou des fibres peuvent boucher les aiguilles PyroMark Q96 ID User Manual 12 2012 D 5 Appendix D 1 5 Dangers lies a la chaleur AVERTISSEMENT A Surface chaude Le bloc chauffant peut atteindre des temperatures jusqu 80 C 176 F Eviter de le toucher quand il est chaud ATTENTION A Risque de surchauffe Afin de garantir une bonne ventilation laisser un d gagement d au moins 10 cm sur les c t s et l arri re de l appareil PyroMark Q96 ID Les fentes et les ouvertures qui assurent la ventilation de l appareil ne doivent pas tre obstru es 1 6 Symbole ce REF Emplacement Plaque signal tique l arri re de l appareil R actifs solutions cartouche plaque Langue EN EN Symboles sur le PyroMark Q96 ID Description Marquage CE R f rence R actifs solutions cartouche plaque EN Limitations de temp rature Plaque signal tique l arri re de l appareil et de tous
82. rranty Extensions Full Cover Support Agreements and instrument application training including on site installation and annual preventative maintenance Service Support Agreements maximize productivity and ensure high performance from your instrument In addition service histories are fully documented and all parts are certified and guaranteed Contact your local QIAGEN Field Service Specialist or your local distributor for more information about flexible Service Support Agreements from QIAGEN PyroMark Q96 ID User Manual 12 2012 7 1 Maintenance 7 1 Shutdown procedure Before carrying out maintenance ensure the PyroMark Q96 ID is shutdown using the following procedure 1 Choose Instrument from the main menu 2 Choose Shut Down Instrument 3 Click Yes to confirm shutdown of the instrument Error messages will appear if the selected instrument is not connected or the selected instrument is busy 4 All indicators on the front panel illuminate and the shutdown procedure begins Shutdown is complete when the message Now it is safe to turn off instrument is displayed 5 Turn off the standby switch on the front of PyroMark Q96 ID and check that the Power indicator lamp is turned off 7 2 Routine maintenance of the PyroMark Q96 ID The PyroMark Q96 ID should be checked and cleaned regularly t0 remove dust and spillages Important points before starting M Avoid harsh cleaners and chemicals and g
83. s are flushed and filled with solution The reagent cartridge is positioned over each well in turn and the enzyme mixture is dispensed The process is then repeated for the substrate mixture Note Dispensation of all reagents into the PyroMark Q96 Plate Low is in a zigzag fashion The mixing system vibrating process chamber is operational throughout the sequencing run The dispensation pressure is increased The nucleotides are predispensed into the rectangular sink well at the right hand edge of the PyroMark Q96 Plate Low The reagent cartridge is positioned over each well in turn and the programmed nucleotide is dispensed The CCD camera collects data simultaneously from all wells throughout the process The CCD camera is cooled to approximately 0 C to optimize performance and reduce background noise In wells where there is a positive reaction with the added nucleotide light is emitted giving rise to a peak in the Pyrogram A period of 65 seconds cycle time is allowed between the addition of each nucleotide to ensure that all enzymatic reactions are completed The programmed sequence of nucleotide addition continues until the end of the program has been reached PyroMark Q96 ID User Manual 12 2012 General Description 3 4 External features of the PyroMark Q96 ID The PyroMark Q96 ID EI Instrument lid El Standby switch E Indicator LED Note The PyroMark Q96 ID is delivered with a laptop to
84. safety information in the user manual must be followed to ensure safe operation of the instrument and to maintain the instrument in a safe condition A French and German translation of the safety information can be found in Appendices D and E respectively About this user manual This user manual provides information about the PyroMark Q96 ID in the following sections 1 Safety Information Introduction General Description Installation Procedures Operating Procedures PyroMark Q96 Software Maintenance Troubleshooting ON ED Glossary Appendices The appendices include Technical data Environmental conditions WEEE recycling requirements Assay design and validation Warranty terms French and German translations of safety information PyroMark Q96 ID User Manual 12 2012 2 1 Introduction 2 2 General information 2 2 1 Technical assistance At QIAGEN we pride ourselves on the quality and availability of our technical support Our Technical Services Departments are staffed by experienced scientists with extensive practical and theoretical expertise in sample and assay technologies and the use of QIAGEN products If you have any questions or experience any difficulties regarding the PyroMark Q96 ID or QIAGEN products in general do not hesitate to contact us QIAGEN customers are a major source of information regarding advanced or specialized uses of our products This information is helpful to other scientists as we
85. site Mains supply voltage fluctuations are not to exceed 10 of nominal supply voltages Grounding requirements To protect operating personnel the PyroMark Q96 ID must be correctly grounded earthed The instrument is equipped with a 3 conductor AC power cord To preserve this protection feature do not operate the instrument from an AC power outlet that has no ground earth connection 4 2 PyroMark Q96 ID User Manual 12 2012 Installation Procedures 4 3 Moving the PyroMark Q96 ID WARNING A Risk of personal injury and material damage w2 The PyroMark Q96 ID is too heavy to be lifted by one person To avoid personal injury or damage to the instrument do not lift the instrument alone If you need to transport the PyroMark Q96 ID package the instrument as follows 1 When the instrument is not processing shut down PyroMark Q96 Software and exit from Microsoft Windows Switch off the operator s computer and PyroMark Q96 ID and disconnect the mains power cable from the instrument To move the instrument to a new location within the same laboratory go to step 4 To move the instrument to another site go to step 7 Place the instrument onto a trolley and move to the new location Note The instrument weighs 52 kg Two persons are required Position the PyroMark Q96 ID on the bench See Section 4 2 for site requirements Follow the reinstallation procedures in Section 4 4 for
86. sition 16 Close the instrument lid 17 Check that the instrument is connected by selecting Preferences from the Tools menu Note If the instrument name does not appear click Find Instrument to have the software detect the connected PyroMark Q96 ID Instrument Upon detecting the instrument the software will enter the instrument serial number and URL Assign a name to the instrument and click Save PyroMark Q96 ID User Manual 12 2012 6 7 PyroMark Q96 Software 18 19 20 21 22 23 24 25 Start the run by clicking and check that the run starts The flashing Information light will go out and the Busy light will illuminate The Run view becomes active and the run progress bar displays the current state of the run The chamber and heating block temperatures and pressure will stabilize to preset values When these values are reached dispensation of the enzyme beige and substrate purple begins When dispensation of enzyme and substrate is complete the pressure increases to the nucleotide pressure value defined in the Instrument Parameters The run progress bar displays Run and dispensation of the nucleotides begins Function test Open the instrument lid There will be an audible warning signal and the yellow Information light will illuminate Open the process chamber lid The X Y table should move at a rate of about 1 well every 0 6 s the
87. stand alone installation or Section 4 5 for network installation To move the instrument to another site First mount the transport protection block under the instrument lid Pack the PyroMark Q96 ID and other components using the original packaging supplied with the instrument if available Note The total weight of the package including the instrument is 80 kg The instrument alone weighs 52 kg Use suitable lifting equipment when moving the package PyroMark Q96 ID User Manual 12 2012 4 3 Installation Procedures 4 4 4 5 9 Atthe new site unpack the instrument and remove the transport protection block from under the instrument lid 10 Follow the reinstallation procedures in Section 4 4 for stand alone installation or Section 4 5 for network installation Reinstalling the PyroMark Q96 ID Stand alone When delivered the PyroMark Q96 ID is configured as a peer to peer network The operator s computer acts as the host and PyroMark Q96 ID as the guest Install a stand alone system as follows 1 Identify the cable marked Cross connected TP provided with the PyroMark Q96 ID Connect one end of this cable to the network connector on PyroMark Q96 ID net Note The keyboard mouse and monitor connectors are for the use of QIAGEN Field Service Specialists only 2 Connect the other end of the Ethernet cable to the network card connector on the operator s computer 3 Connect
88. tective conductor earth ground E Keep mains plugs easily accessible in case the equipment needs to be disconnected quickly from mains power M Use only power supplies and cords supplied with the instrument M When replacing the mains fuse replace only with the type and current rating specified on the rating label 1 2 PyroMark Q96 ID User Manual 12 2012 Safety Information 1 3 Chemical hazards WARNING Hazardous chemicals WA The Denaturation Solution used with the Vacuum A Workstation contains sodium hydroxide which is irritating to eyes and skin Always wear safety glasses gloves and a lab coat The responsible body e g laboratory manager must take the necessary precautions to ensure that the surrounding workplace is safe and that the instrument operators are not exposed to hazardous levels of toxic substances chemical or biological as defined in the applicable Safety Data Sheets SDSs or OSHA ACGIHt or COSHH documents For more information visit www giagen com safety Venting for fumes and disposal of wastes must be in accordance with all national state and local health and safety regulations and laws 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 1 4 Mechanical hazards The lid of the PyroMark
89. th the PyroMark CpG Software do not need to be converted These runs are in a format that is compatible with the new PyroMark Q96 Software Runs containing multiplex assay data cannot be converted and are skipped during the conversion procedure 1 Open the PyroMark Data Converter and open the Database Settings window by clicking Connect or selecting Connect from the File menu PyroMark Q96 ID User Manual 12 2012 6 3 PyroMark Q96 Software Step Establish Database Correction Fe mm Step 2 Select Dats Select ai data Deselect at date Step 2 Specty Directoy Era EE 2 Enter the requested information the database name TNS user name and password into the dialog box and click the OK button to connect to the specified database ET _21x Insert Database Settings TNS orcl User Pyo 23 2 Check Connection Ok Cancel Note Click Check Connection to determine if a connection is possible with the information entered Note The database connection can be disconnected by clicking Disconnect 3 Select Pyrosequencing data for conversion by checking the box next to individual runs or folders or using the Select all data or Deselect all data buttons 6 4 PyroMark Q96 ID User Manual 12 2012 PyroMark Q96 Software 6 5 6 6 CORRE U E fe Hab 33308 QUGEN Step Establish Database Connection Connected t
90. the plate holding frame Place the PyroMark Q96 Plate Low on the heating block Close the plate holding frame and the process chamber PyroMark Q96 ID User Manual 12 2012 5 15 Operating Procedures 5 5 2 Loading the reagent cartridge 1 Open the dispensing unit cover Release the latch and then open the cover 2 Insert the filled reagent cartridge into position It will only fit in one orientation the label should be facing the user 3 Close the dispensing unit cover Ensure the latch snaps into its locked position 4 Close the instrument lid 5 16 PyroMark Q96 ID User Manual 12 2012 Operating Procedures 5 5 3 5 5 4 Starting the run Start the run by clicking The Run view becomes active The run can be monitored paused or stopped in this view Note The Start icon is enabled only if an instrument connection has been configured instrument parameters have been defined for the run file and at least one well has an assay Monitoring the run The Run view displays the following information regarding the progress of a run M Last nucleotide or reagent dispensation to each well M Well specific information M Instrument specific information in the Instrument Status Monitor A diagram of a 96 well plate displays all wells active in the run During the course of the run each well is colored to reflect the last reagent Enzyme Mix or Substrate Mix or nucleotide A C C or T
91. tion appropriee AVERTISSEMENT ATTENTION A Risque d accident corporel et de d t rioration du mat riel L utilisation inappropri e du PyroMark Q96 ID peut entrainer des accidents corporels ou une deterioration de l appareil L utilisation du PyroMark Q96 ID est r serv e exclusivement au personnel qualifi ayant t convenablement form L entretien du PyroMark Q96 ID doit tre effectu uniquement par des sp cialistes de l entretien sur site QIAGEN Proc der la maintenance comme d crit la Section 7 QIAGEN facture les r parations rendues n cessaires suite une maintenance inappropri e AVERTISSEMENT ATTENTION A Risque d accident corporel et de deterioration du materiel L appareil PyroMark Q96 ID est trop lourd pour tre soulev par une seule personne Afin d viter tout accident corporel et toute d t rioration du mat riel ne pas soulever l appareil seul PyroMark Q96 ID User Manual 12 2012 Appendix D 1 2 Securite electrique Avant l entretien d brancher les cordons d alimentation des prises de courant AVERTISSEMENT Danger lectrique Toute interruption du conducteur de protection A conducteur de terre de masse l int rieur ou l ext rieur de l appareil ou toute d connexion de la borne du conducteur de protection est susceptible de rendre l appareil dangereux Toute interruption intentionnelle est interdite Tensions mortell
92. to eyes and skin Always wear safety glasses gloves and a lab coat The responsible body e g laboratory manager must take the necessary precautions to ensure that the surrounding workplace is safe and that the instrument operators are not exposed to hazardous levels of toxic substances chemical or biological as defined in the applicable Safety Data Sheets SDSs or OSHA ACGIH or COSHH documents For more information visit www giagen com safety Venting for fumes and disposal of wastes must be in accordance with all national state and local health and safety regulations and laws OSHA Occupational Safety and Health Administration United States of America t ACGIH American Conference of Government Industrial Hygienists United States of America COSHH Control of Substances Hazardous to Health United Kingdom 5 24 PyroMark Q96 ID User Manual 12 2012 Operating Procedures Be sure to observe all federal state and local environmental regulations for the disposal of laboratory waste The following item is required E High purity water Milli Q 18 2 MQ x cm or equivalent 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 Discard any solutions left in the troughs Rinse the troughs with high purity water or replace them if necessary Empty the waste container Note The cap can be
93. tool for 10 s 20 Wash the filter probes by lowering the probes into high purity water Parking Position and applying vacuum Flush the probes with 180 ml high purity water 21 Raise the tool to beyond 90 vertical for 5 s to drain liquid from the filter probes 22 Close the vacuum switch on the worktable Off and place the tool in the Parking Position 23 If more than one plate is prepared at once refill the troughs step 2 and repeat the procedure from step 8 24 Turn off the vacuum pump Note The filter probes should be left to air dry when not in use i e empty the trough and ensure there is no water in the Parking Position when the tool is left there PyroMark Q96 ID User Manual 12 2012 5 13 Operating Procedures 5 4 5 Annealing of sequencing primer to samples WARNING Hot surface wg The thermoplate and the heating block for annealing can A reach temperatures of up to 80 C 176 F Avoid touching them when they are hot 1 Place the PyroMark Q96 Plate Low in a prewarmed PyroMark Q96 Sample Prep Thermoplate Low and heat the samples on a heating block at 80 C for 2 min 2 Remove the plate from the thermoplate and allow the samples to cool to room temperature 15 25 C for at least 5 min The plate can now be processed in the PyroMark Q96 ID 5 4 6 Reagent preparation 1 Prepare reagents according to instructions included with the PyroMark Gold Q96 Reagents 2 Open a reagent cartri
94. ulation As the substrate is dispensed into a well indicated by an contaminants such as ATP or PPi will be converted to light Too high a substrate peak indicates that high levels of a contaminant might be present in for example buffers Pyrogram The graph resulting from a sequencing reaction performed using Pyrosequencing technology Each incorporated nucleotide is shown as a peak in Pyrogram 9 2 PyroMark Q96 ID User Manual 12 2012 Glossary Term Reference peak RLU Sepharose beads Sequence to analyze Sequencing primer Shift Signal to noise ratio Single nucleotide polymorphism SNP Description Nonvariable peaks i e peaks that are not a part of an SNP site are referred to as reference peaks Reference peaks are used in the analysis both as references when calculating the single peak height and as internal controls when assessing the quality Relative Light Unit entity used in Pyrosequencing to define peak heights in Pyrogram Streptavidin coated beads can be used for preparation of biotinylated PCR products A short part of a DNA sequence in your sample starting directly after the sequencing primer which contains one or several variable positions to be analyzed using Pyrosequencing instrument platforms The sequencing primer is annealed to the template during the sample preparation The 3 end of the sequencing primer serves as the starting point for the extension b
95. uminated during a run the instrument has lost contact with the operator s computer During the start up sequence All the lights illuminate The instrument software loads takes 1 3 minutes and the Busy light goes off and the Information light starts flashing This indicates that the instrument is ready to receive instructions from the operator s computer When an application is selected and Run is clicked in the Setup Run window the flashing Information light will turn off and the Busy light will illuminate Rear panel The following connectors are located on the rear panel Mains power input connector Should be connected to a grounded mains power outlet Keyboard Keyboard interface for service purposes This should only be used by a certified QIAGEN Field Service Specialist Mouse Mouse interface for service purposes This should only be used by a certified QIAGEN Field Service Specialist Net Ethernet network connector PyroMark Q96 ID User Manual 12 2012 3 7 General Description Monitor Monitor interface for service purposes This should only be used by a certified QIAGEN Field Service Specialist 3 9 Computer and software The PyroMark Q96 ID is shipped with a laptop that serves as the operator s computer The computer used for data analysis should have the following as minimum specifications MB Microsoft Windows XP 32 bit English version or Microsoft Windows 7 32 bit English version Operating
96. y the polymerase Plus shift A small proportion of the template sequences that incorporates more than one type of nucleotide at a time if for example there are residues left from the dispensation before and will be sequenced ahead of the rest of template sequences Minus shift A small proportion of the template sequences that fails to incorporate a nucleotide will be sequenced subsequent to the rest of template The ratio of the signal height and the noise height An indication of the clarity of the data The higher the ratio the better the data SNPs involve the change of one DNA base to another SNPs and point mutations are structurally identical differing only in their frequency Variations that occur in 1 or less of a population are considered point mutations while those occurring in more than 1 are SNPs PyroMark Q96 ID User Manual 12 2012 9 3 Glossary Term Description Streptavidin A protein that can bind very strongly to biotin Substrate A molecule acted upon by an enzyme Pyrosequencing technology uses a mixture of the substrates Adenosine 5 phosphosulfate APS and Luciferin in the sequencing reaction Theoretical The possible genotypes for a specific polymorphic position outcome are calculated on the basis of the sequence to analyze and the dispensation order 9 4 PyroMark Q96 ID User Manual 12 2012 Appendix A Appendix A Technical data QIAGEN reserves the right to change specifications
97. ylated PCR products are immobilized on streptavidin coated Sepharose beads Streptavidin Sepharose High Performance GE Healthcare For each sample the final immobilization volume is 80 ul The reaction setup is shown in the table below Volume per sample Component For lot number For lot numbers 10057037 or higher lower than 10057037 Biotinylated PCR 5 20 ul 5 20 ul product Streptavidin 1 5 ul 3 ul Sepharose HP PyroMark Binding 40 ul 40 ul Buffer High purity watert Variablet Variablet Total volume 80 ul 80 ul Check the lot number of the Streptavidin Sepharose HP For lot number 10057037 or higher use 1 5 ul For lot numbers lower than 10057037 use 3 ul The volume of water depends on the amount of PCR product used Allow all reagents and solutions to equilibrate to room temperature 15 25 C before starting All steps are performed at room temperature unless stated 1 Gently shake the bottle with streptavidin coated Sepharose beads from side to side until a homogenous solution is obtained 2 Prepare a mixture of Streptavidin Sepharose beads Binding Buffer and high purity water sufficient for all samples to be analyzed 3 Add the mixture prepared in step 2 to the wells of a 96 well PCR plate according to the plate setup see Section 5 3 1 4 Add 5 20 ul of a well optimized biotinylated PCR product to each well used Note The total volume per well should be 80 ul 5 8 PyroMark Q96 ID User

Download Pdf Manuals

Related Search

Related Contents

del Usuario myCGS  Manual de Servicio  Respectez les normes d`hygiène cutanée en un tournemain  KDC-PSW9521 KDC-M907 KDC-M9021 KDC-8021  Samsung XQB55-E86 用户手册  Allegati piano di sicurezza e coordinamento  FA-1010  WARNING    Wintools  

Copyright © All rights reserved. Failed to retrieve file