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PyroMark Q24 Advanced User Manual

1. Plaque signal tique a l arri re de l appareil et de tous les autres produits EN Fabricant l gal gt E E Bi A l int rieur de l appareil EN Avertissement se reporter au manuel d utilisation D 8 PyroMark Q24 Advanced User Manual 06 2013 Appendix D Symbole Emplacement Langue Description ly Cartouche EN Tenir l abri de la Tem Ex TF lumi re et des rayons du soleil oN Cartouche EN Num ro de la m thode INSTRUM PARAM Cartouche EN Param tres de l appareil PyroMark Q24 Advanced User Manual 06 2013 D 9 Appendix D This page intentionally left blank D 10 PyroMark Q24 Advanced User Manual 06 2013 Appendix E Appendix E 1 Sicherheitshinweise Der PyroMark Q24 Advanced besteht aus dem PyroMark Q24 Instrument und der PyroMark Q24 Advanced Software Vor der Inbetriebnahme des PyroMark Q24 Advanced 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 um einen sicheren Betrieb des PyroMark Q24 Advanced 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 A Der Begriff WARNUNG WARNING weist Sie auf Situationen hin in den
2. 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 PyroMark Q24 Advanced User Manual 06 2013 E 5 Appendix E 1 5 Gefahren durch mechanische Teile Der Deckel des PyroMark Q24 Instrument muss w hrend des Betriebs geschlossen sein Ein akustisches Warnsignal weist Sie ggf darauf hin dass der Deckel noch nicht geschlossen ist und ein sicherer Betrieb nicht gew hrleistet ist WARNUNG A Sich bewegende Ger teteile W7 Um einen Kontakt mit sich bewegenden Teilen beim Betrieb des PyroMark Q24 Instrument 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 Q24 Instrument auftreten sollte WARNUNG A Scharfe Nadeln wg Ber hren Sie nicht die scharfen Nadeln am Boden der Reagenzienkartusche Gehen Sie vorsichtig mit den Nadeln um Kleine Partikel oder Fasern k nnen die Nadeln verstopfen ACHTUNG A Wartung der Lichtleiter C1 Verwenden Sie ein fusselfreies Tuch um die Fl che zwischen dem Heizblock und dem
3. menu option is used for service applications Only run a service application when instructed by QIAGEN Technical Services Setting up a run Before setting up a run it is recommended that you familiarize yourself with the safety information by referring to Section 1 Detailed instructions on setting up a run can be found in the PyroMark Q24 Advanced Software User Guide Starting PyroMark Q24 Advanced Software In the Windows Start menu select All Programs PyroMark PyroMark Q24 Advanced The PyroMark Q24 Advanced Software User Guide can be accessed at any time by pressing the F1 key when in the software Setting up an assay Zr I NewAQAssay Ctri A ZX NewSNP Assay Ctrl N im New CpG Assay Ctrl G Wee NewSEQAssay Ctri Q 1 Click in the toolbar and select the required assay A new assay file is created For analyzing methylation at CpN sites create a New CpG Assay and then enable the CpN mode by checking the box next to CpN mode enabled Alternatively you can create a new assay file in the shortcut browser by right clicking the folder you wish to place it in and selecting New Assay followed by the desired assay type from the context menu PyroMark Q24 Advanced User Manual 06 2013 Operating Procedures Note To add a shortcut to a folder in the shortcut browser click Add Folder Shortcut 2 Enter the sequence to analyze Note If creating a CpG assay w
4. ACHTUNG Der PyroMark Q24 Instrument ist sehr schwer und sollte nicht von einer Person angehoben werden Heben Sie das A Ger t nicht allein an um eine Verletzung und oder Besch digung des Ger ts zu vermeiden WARNUNG Verletzungsgefahr und Besch digung des Ger ts ms ACHTUNG Bewegen Sie den PyroMark Q24 Instrument auf keinen A Fall w hrend des Betriebs E 2 PyroMark Q24 Advanced User Manual 06 2013 Appendix E 1 2 Schutz vor Stromschlag Ziehen Sie die Netzanschlusskabel aus den Steckdosen bevor Sie Wartungsarbeiten am Ger t vornehmen WARNUNG Gefahr durch Stromschlag w4 A 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 Offnen der Abdeckungen oder das Entfernen von Geh useteilen k nnen spannungsf hrende Komponenten freigelegt werden Um einen zufriedenstellenden und sicheren Betrieb des PyroMark Q24 Instrument zu gew hrleisten befolgen Sie bitte die nachstehenden Hinweise M Die Ger te Netzkabel m ssen an Wechselstrom Steckdosen mit Schutzleiter Erdungs Masseleiter angeschlossen werden M So
5. 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 1 5 WARNING A Mechanical hazards The lid of the PyroMark Q24 Instrument must remain closed during operation of the instrument An audible warning signal will alert you if the lid is opened when it is not safe Moving parts W7 To avoid contact with moving parts during operation of the PyroMark Q24 Instrument 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 Q24 Instrument contact QIAGEN Technical Services immediately 1 4 PyroMark Q24 Advanced User Manual 06 2013 Safety Information WARNING Sharp needles wg 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 CAUTION Light guide maintenance
6. maintain the system in a safe condition About this user manual This user manual provides information about the PyroMark Q24 Advanced in the following sections 1 Safety Information Introduction General Description Installation Procedures Operating Procedures Maintenance Procedures Troubleshooting GR a 1 ee Glossary Appendices The appendices include the following Technical data Environmental conditions WEEE recycling requirements Assay design and validation Warranty terms PyroMark Q24 Advanced User Manual 06 2013 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 Service 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 Q24 Advanced or QIAGEN products in general please 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 well 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 Technica
7. 1 6 2 2 6 3 6 3 1 6 3 2 6 3 3 6 3 4 6 3 5 7 7 1 7 2 Annealing of sequencing primer to samples 5 20 Processing a run on the PyroMark Q24 Advanced 5 20 Instrument software 5 21 Starting the instrument 5 22 Starting the run 5 22 Monitoring the run 5 23 After the run 5 25 Analyzing the run 5 27 Viewing the analysis results 5 29 Analysis reports 5 32 Finishing work and shutting down 5 32 Shutting down the instrument 5 32 Emptying the waste container and troughs 5 33 Checking the instrument 5 34 Backup of PyroMark Q24 Advanced files 5 34 Maintenance 6 1 Checking the performance of the PyroMark Q24 Advanced 6 1 Maintenance of the PyroMark Q24 Instrument 6 2 Cleaning the instrument 6 2 Cleaning the heating block and light guides 6 3 Maintenance of PyroMark Q24 Vacuum Workstation 6 4 Cleaning the PyroMark Q24 Vacuum Workstation 6 5 Testing and replacing the filter probes 6 5 Replacing the rubber seal 6 6 Replacing the tubing 6 7 Replacing the waste filter 6 8 Troubleshooting 7 1 Analysis related errors 7 2 Analysis software related errors 7 4 PyroMark Q24 Advanced User Manual 06 2013 Contents 3 Contents 7 3 Instrument related errors 7 5 7 A Vacuum workstation related errors 7 6 7 5 Verification of correct installation and operation 7 7 8 Glossary 8 1 Appendix A A 1 Technical data A 1 Environmental conditions A 1 Waste Electrical and Electronic Equipment WEEE A 5 FCC declaration A 6 Declaration of conformity A 7 Ap
8. 35 Quality assessments The plate overview in the Overview tab gives a quick overview of the quality assessments cm Shows the quality assessment of all variable positions in the well or of all the bases in the base called sequence PyroMark Q24 Advanced User Manual 06 2013 5 29 Operating Procedures Quality colors LT Blue Passed C Yellow Check BI Red Failed L White Not analyzed Either analysis is not supported by the software e g analysis of SNP when in the CpG mode or the variable position has been deselected by the user AQ SNP and CpG assays only AQ analysis results The allele frequencies are displayed in Pyrogram for example amp and a InDel The quality assessment is displayed by the background color of the result A3 GAYGGGYGGGTTYGTTTTTTTGAGAGYGAYGYGAGTTAATGGGAAGGTTTTGGGGTGATATTATGGGTTATTTTTAGGG on ses 1008 558 ss 788 5 10 15 20 25 30 35 Example Pyrogram for a CpG assay Variable positions in AQ and CpG assays are highlighted with a blue gray background color and bisulfite treatment controls in CpG assays with a light orange background color CpG analysis results The methylation percentage 28 or average methylation percentage Average 25 are displayed in the Pyrogram The quality assessment is displayed by the background color of the result 5 30 PyroMark Q24 Advanced User Manual 06 2013 Operating Procedures A methylation bar i
9. Installation Procedures 4 3 2 6 When the software has been successfully installed click Exit Setup in the CD menu 7 Please use Windows Update www update microsoft com to check for any critical updates to the NET Framework 4 0 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 To view reports generated by PyroMark Q24 Advanced Software in PDF format a PDF reader must be installed on the computer Adobe Reader can be downloaded at www adobe com Uninstalling PyroMark Q24 Advanced Software 1 Select Control Panel in the Windows Start menu 2 In the Control Panel click Programs then click Programs and Features 3 In the list of programs select PyroMark Q24 Advanced 4 Click Uninstall 5 Repeat steps 3 and 4 for PyroMark Launcher PyroMark Q24 Advanced User Manual 06 2013 5 1 5 1 1 5 1 2 Operating Procedures Operating Procedures This section describes how to operate PyroMark Q24 Advanced Before proceeding it is recommended that you familiarize yourself with the features of PyroMark Q24 Instrument by referring to Section 3 4 Instrument administration Setting date and time Setting the date and time correctly ensures an accurate date and time stamp in the instrument and run logs and the analysis repo
10. Mg concentration is required use of the 25 mM MgCl provided in the kit is recommended The recommended annealing temperature is 60 C and 56 C for genomic DNA and bisulfite treated DNA respectively when using PyroMark ADSW 2 0 PyroMark Q24 Advanced User Manual 06 2013 B 3 Appendix B Addition of Q Solution provided with the PyroMark PCR Kit can 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 quantification 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 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 Q24 Advanced 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
11. 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 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 Testing and replacing the filter probes Function test for filter probes The function test for the filter probes is described in Section Boek 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 PyroMark Q24 Advanced User Manual 06 2013 6 5 Maintenance 6 3 3 6 6 The following items are required M Powder free gloves E 2 mm Allen key supplied with the system E High purity water Milli Q 18 2 MQ x cm or equivalent E New filter probes QIAGEN 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 Remove the tool from the tubing 4 Loosen the four screws using the 2 mm Allen key supplied with the system 5 Pul
12. 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 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 2 Discard any solutions left in the troughs 3 Rinse the troughs with high purity water or replace them if necessary 4 Empty the waste container Note The cap can be removed without disconnecting the tubing 5 If the vacuum workstation must be cleaned e g from dust or spillage follow the instructions in Section 6 3 PyroMark Q24 Advanced User Manual 06 2013 5 33 Operating Procedures 5 5 3 5 6 5 34 Checking the instrument Check the instrument for dust and spillage If the instrument needs to be cleaned follow the instructions in Section 6 2 1 At the rear of the instrument press the light button see image below and check that the coolant level is visible in the window If it is not contact QIAGEN Technical Services Model PyroMary ong Backup of PyroM
13. block and placing the PyroMark Q24 plate in the PyroMark Q24 Instrument and starting the run should not exceed 30 seconds Processing a run on the PyroMark Q24 Advanced The lid of the PyroMark Q24 Instrument must remain closed during operation of the instrument An audible warning signal will alert you if the lid is opened when it is not safe PyroMark Q24 Advanced User Manual 06 2013 Operating Procedures WARNING A Moving parts W7 To avoid contact with moving parts during operation of the PyroMark Q24 Instrument 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 Q24 Instrument contact QIAGEN Technical Services immediately A WARNING Sharp needles w8 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 5 4 1 Instrument software y The instrument is controlled via the six buttons underneath the screen Runs are started and monitored through the instrument software During processing of a run the software displays the following information PyroMark Q24 Advanced User Manual 06 2013 5 21 Operating Procedures 1 Run name Validation run 080214 Selected well Current time Instrument status Pyrogram Warning messages o
14. by double clicking the run file Ed in the shortcut browser If several assay types are included select analysis mode in the dialog box that opens The analysis modes displayed in the dialog box are limited to the assay types on the plate Note To update the contents of a folder in the shortcut browser right click it and select Refresh from the context menu or press the F5 key Note It is also possible to open the run file by double clicking it in Windows Explorer 3 In the Overview tab analyze selected wells with a valid analysis setup for the current analysis mode PyroMark Q24 Advanced User Manual 06 2013 5 27 Operating Procedures Analysis modes PyroMark Q24 Advanced Software has four analysis modes AQ SNP CpG and SEQ To toggle between the modes select AQ SNP CpG or SEQ in the toolbar Note How the analysis is performed can be modified using the Analysis Setup tab 5 28 PyroMark Q24 Advanced User Manual 06 2013 5 4 7 Operating Procedures Viewing the analysis results By selecting an analyzed well in the Overview tab the corresponding Pyrogram is displayed in the Pyrogram area and the well information including analysis warnings is listed in the Well Information area TGGCCGGGTCACGATGCCCTATTTATAGCT GAGGGGTGGGGIGJATGGAG L Fe BERLIN ole Lait eyes REZ EEE ET s ss Lio tri
15. 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 Le terme AVERTISSEMENT signale des situations A 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 Le terme ATTENTION signale des situations risquant A 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 Q24 Advanced User Manual 06 2013 D 1 Appendix D 1 1 Utilisation appropri e AVERTISSEMENT ATTENTION A Risque d accident corporel et de d t rioration du mat riel w1 L utilisation inappropri e du PyroMark Q24 Instrument peut entra ner des accidents corporels ou une d t rioration de l appareil L utilisation du PyroMark Q24 Instrument est r serv e exclusivement au personnel qualifi ayant t convenablement form L entretien du PyroMark Q24 Instrument doit tre effectu
16. immediately after use and ensure that it can be used for analysis It is recommended that the reagent cartridge is used a maximum of 30 times WARNING Sharp needles wg 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 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 PyroMark Q24 Advanced User Manual 06 2013 5 25 Operating Procedures High purity water Milli Q 18 2 MQ x cm or equivalent Beaker not always required Lint free tissues To clean and check that the reagent cartridge can be used for analysis 1 2 5a 5b Discard any solutions remaining in cartridge Rinse the cartridge compartments 4 times with high purity water Spray the outside of the needles using high purity water Check that the cartridge needles are not blocked or damaged Completely fill the compartments 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 each needle If a needle is blocked follow step 5a If the jet of water comes out of a needle at an angle not parallel to the direction of the needle follow step 5b If all needle
17. lid open Allow at least 10 cm 4 in of free space behind the instrument for cabling Remove the foam dispensing module transport lock Keep the transport lock for future transportation of the instrument The PyroMark Q24 Instrument must be placed within approximately 1 5 m 59 in of two properly grounded earthed AC power outlets The power lines to the PyroMark Q24 Instrument should be voltage regulated and surge protected Note It is recommended to plug the instrument directly into its own power outlets and not to share the power outlets with other lab equipment Do not place PyroMark Q24 Instrument on a vibrating surface or near vibrating objects Risk of overheating C2 To ensure proper ventilation maintain a minimum clearance of 10 cm at the sides and rear of the PyroMark Q24 Instrument Slits and openings that ensure the ventilation of the PyroMark Q24 Instrument must not be covered 4 2 Power requirements The PyroMark Q24 Instrument operates at M Input 100 240 V AC 50 60 Hz 160 VA M Instrument rating 24 V DC 40 W M Cooler rating 12 VDC 60 W The PyroMark Q24 Vacuum Workstation operates at M 100 VAC 50 60 Hz 25 VA M 115 VAC 60 Hz 25 VA M 230 V AC 50 Hz 25 VA Make sure that the voltage rating of the PyroMark Q24 Advanced is compatible with the AC voltage available at the PyroMark Q24 Advanced User Manual 06 2013 Installation Procedures 4 3 4 3 1 installation site Mains supp
18. of adenosine 5 phosphosulfate 6 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 y light APS PPi ATP luciferin oxyluciferin Luciferase ATP Light time nucleotide incorporation generates light seen as a peak in Pyrogram 7 Apyrase a nucleotide degrading enzyme continuously degrades unincorporated nucleotides and ATP When degradation is complete another nucleotide is added dntp AP GNDP dNMP phosphate ATP Apyrase D ADP AMP phosphate 8 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 9 As the process continues the complementary sequence is built up and the nucleotide sequence is determined from the peak in the Pyrogram 3 2 PyroMark Q24 Advanced User Manual 06 2013 General Description nucleotide sequence nucleotide added 3 3 PyroMark Q24 principle The PyroMark Q24 Advanced performs DNA sequencing using Pyrosequencing technology 1 The PyroMark Q24 Plate containing the samples is placed on the heating block inside the ins
19. und Gesetze zu Gesundheitsschutz und Sicherheit am Arbeitsplatz auf bernationaler 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 Q24 Advanced User Manual 06 2013 Appendix E 1 4 Chemikalien WARNUNG Gef hrliche Chemikalien We Die Denaturierungsl sung die zusammen mit der A 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 nationaler und regionaler Ebene eingehalten werden
20. 4 Advanced User Manual 06 2013 Operating Procedures We recommend that the reagent cartridge is used a maximum of 30 times If the reagent cartridge has not been used for an extended period of time e g it has been stored check that it can be used for analysis by performing the function test steps 4 6 in Section 5 4 5 PyroMark Q24 Cartridge compartments from above 5 3 5 Separation of DNA strands and release of samples into the PyroMark Q24 Plate WARNING Hazardous chemicals w A The Denaturation Solution used with the vacuum workstation contains sodium hydroxide which is irritating to eyes and skin Always wear safety glasses gloves and a lab coat The responsible body e g laboratory manager must take the necessary precautions to ensure that the surrounding workplace is safe and that the 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 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 t ACGIH American Conference of Government Industrial Hygienists United States of America COSHH Control of Substances Hazardous to Health United Kingd
21. European Union the European Directive 2002 96 EC on WEEE requires proper disposal of electrical and electronic equipment when it reaches its end of life The separate collection and recycling of waste 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 follow up information for scheduling collection of the electronic waste or to provide you with an individual quote PyroMark Q24 Advanced User Manual 06 2013 A 5 Appendix A A 6 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 of the 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
22. FRG Setting up a run 1 Create a new Run Setup by one of the following methods m Click in the toolbar m Select New Run from the File menu m Press the R key while holding down the Ctrl key m Right click a folder in the shortcut browser and select New Run from the context menu Enter a run name and press Enter To add a shortcut to a folder in the shortcut browser click Add Folder Shortcut m 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 be copied If the new run has not yet been saved click tal to enter a run name and save the file in the desired folder In the Instrument Methods drop down list select the instrument methods file that corresponds to the PyroMark Q24 Cartridge to be used See Section 5 2 4 for detailed instructions Set up the plate in the plate layout of the run file by adding assays to wells and if desired entering the sample ID and note for each used well Add an assay to each well used by m Dragging an assay from the shortcut browser to a well or a selection of wells m Right clicking one well and selecting Load Assay from the context menu PyroMark Q24 Advanced User Manual 06 2013 Operating Procedures A well is colored according to the assay loaded into the well Wells with AQ
23. June 2013 PyroMark Q24 Advanced User Manual Sample amp Assay Technologies Trademarks QIAGEN DNeasy EpiTect PyroMark Pyrosequencing Pyrogram QlAamp QlAprep Q Solution RNeasy QIAGEN Group Adobe Reader Adobe Systems Inc ABgene Thermo Fisher Scientific Eppendorf Eppendorf AG Microsoft Windows Microsoft Corporation Millipore Milli Q Merck KGaA Pentium Intel Corporation Sepharose GE Healthcare Variomag Thermo Electron LED GmbH Registered names trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law 2012 2013 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 Biological safety 1 3 1 4 Chemicals 1 4 1 5 Mechanical hazards 1 4 1 6 Heat hazard 1 5 1 7 Consumables 1 5 1 8 Symbols used on PyroMark Q24 products 1 6 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 PyroMark Q24 Advanced 2 3 2 3 1 Requirements for PyroMark Q24 Advanced users 2 3 3 General Description 3 1 3 1 PyroMark Q24 definitions 3 1 3 2 Pyrosequencing principle 3 1 3 3 PyroMark Q24 principle 3 3 3 4 PyroMark Q24 Instrument 3 4 3 4 1 Process chamber 3 5 3 4 2 Dispensing unit 3 5 3 5 Sample preparat
24. Lichtleiter Block im Ger teinneren zu reinigen Verwenden Sie keine Papiert cher E 6 PyroMark Q24 Advanced User Manual 06 2013 Appendix E 1 6 berhitzungsgefahr WARNUNG A HeiBe Oberflachen w9 Der Platten Halter und der Heizblock f r das Annealing k nnen sehr hei e Temperaturen von bis zu 80 C erreichen Ber hren Sie diese Teile nicht wenn sie aufgeheizt sind ACHTUNG A 1 7 berhitzungsgefahr C2 Vergewissern Sie sich dass ein Mindestabstand von 10 cm zwischen Seitenw nden und R ckseite des PyroMark Q24 Instrument und der Wand eingehalten wird damit eine ausreichende Bel ftung des Ger ts gew hrleistet ist Die L ftungsschlitze und Offnungen die f r die Be und Entl ftung des PyroMark Q24 Instrument bestimmt sind d rfen nicht verdeckt werden Verbrauchsartikel ACHTUNG A Nicht unterst tzte Verbrauchsartikel C3 Verwenden Sie keine anderen Verbrauchsartikel oder Zubeh rteile und schlie en Sie keine anderen externen Ger te an als die die in diesem Handbuch angegeben sind PyroMark Q24 Advanced User Manual 06 2013 E 7 Appendix E 1 8 Symbole auf den PyroMark Q24 Produkten Symbol Ort Sprache Beschreibung Typenschild auf EN CE Kennzeichnung der Ger ter ckseite Typenschild auf EN Entspricht US SP der amerikanischen und Ger ter ckseite kanadischen C US Sicherheitsstandards Typenschild
25. USB stick Do not remove the USB stick until the instrument confirms that the run file has been saved Finished The run has been completed and the run data have been transferred to the USB stick Pyrogram and warnings The run name and selected well are displayed in the top left corner To select another well use the and screen buttons Any instrument warnings are displayed below the Pyrogram area the three latest warnings are displayed For suggested actions see Section 7 3 Cancel the run To cancel the run press Stop PyroMark Q24 Advanced User Manual 06 2013 Operating Procedures 5 4 5 After the run 1 When the instrument confirms that the run file has been saved to the USB stick press Close 2 Remove the USB stick Open the instrument lid 4 Open the cartridge gate and remove the reagent cartridge by lifting it up and pulling it out 5 Close the gate 6 Open the plate holding frame and remove the plate from the heating block 7 Close the plate holding frame and the instrument lid Discard the plate 9 If the reagent cartridge is to be reused clean it according to the instructions below 10 If this was the last run for the day follow the instructions in Section 5 5 Note Be sure to observe all federal state and local environmental regulations for the disposal of laboratory waste Cleaning and testing the reagent cartridge If the reagent cartridge is to be reused clean it
26. ains 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 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 A The Denaturation Solution used with the vacuum 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 6 4 PyroMark Q24 Advanced User Manual 06 2013 Maintenance 6 3 1 6 3 2 Cleaning the PyroMark Q24 Vacuum Workstation If the vacuum workstation needs to be cleaned to remove dust and spillage follow the instructions below The following items are required M Powder free gloves M High purity water Milli Q 18 2 MQ x cm or equivalent M A mild detergent if necessary M Clean lint free cloths Procedure 1 Ensure that no vacuum is applied to the vacuum prep tool i e the vacuum switch is closed
27. anual 06 2013 Appendix A M Consult the dealer or an experienced radio T V technician for help QIAGEN GmbH Germany is not responsible for any radio television interference caused by unauthorized modifications of this equipment or the substitution or attachment of connection cables and equipment other than those specified by QIAGEN GmbH Germany The correction of interference caused by such unauthorized modification substitution or attachment will be the responsibility of the user Declaration of conformity Name and address of the company QIAGEN GmbH QIAGEN Strasse 1 40724 Hilden Germany We herewith declare that the product PyroMark Q24 meets all applicable requirements of the following European Directives Low Voltage Directive LVD 2006 95 EC Electromagnetic Compatibility 2004 108 EC Directive EMC Hombrechtikon July 27 2009 N L Pit Muggli Director Business Excellence QIAGEN Instruments AG PyroMark Q24 Advanced User Manual 06 2013 A 7 Appendix A This page intentionally left blank A 8 PyroMark Q24 Advanced User Manual 06 2013 Appendix B Appendix B Assay design and validation Assay design For design of Pyrosequencing assays use 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 Pyroseq
28. ark Q24 Advanced files PyroMark Q24 Advanced Software supports the following file types M AQ assay paq E SNP assay psnp E CpG assay pcpg M SEQ assay pseq M Plate setup pset M Processed run prun The data generated by PyroMark Q24 Advanced Software are stored on the computer as files with these suffixes Note Files created in PyroMark Q24 2 0 Software or in PyroMark Q96 2 5 Software are not supported PyroMark Q24 Advanced User Manual 06 2013 Operating Procedures Data backup should be performed frequently This can be done by copying PyroMark Q24 Advanced files to another location The alternative location should be another physical drive or permanent medium For more information about backup contact your system administrator PyroMark Q24 Advanced User Manual 06 2013 5 35 Operating Procedures This page intentionally left blank 5 36 PyroMark Q24 Advanced User Manual 06 2013 Maintenance 6 1 Maintenance The following maintenance procedures must be carried out to ensure reliable operation of the PyroMark Q24 Advanced M Regular performance checks M Cleaning of the instrument Following these procedures ensures that the PyroMark Q24 Instrument is free of dust and liquid spills Before undertaking maintenance procedures it is recommended that you familiarize yourself with the safety information by referring to Section 1 Important Disconnect the instrument from mains power before cleanin
29. assays are yellow wells with SNP assays are red wells with CpG assays are green wells with SEQ assays are purple 6 To enter a sample ID or note select the cell and enter the text A selected cell is highlighted with a blue background color 7 Click in the toolbar 8 Print the plate setup and a list of required volumes of enzyme mix substrate mix and nucleotides by selecting Pre Run Information from the Tools menu When the report opens click 3 9 Close the run file and copy it to one of the USB sticks supplied The run file can now be processed by inserting the USB stick into the USB port at the front of the PyroMark Q24 Instrument see Section 5 4 Optional If desired enter the Reagent ID i e the lot number for PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents a Plate ID of the PyroMark Q24 Plate a Barcode for the plate and a Run Note in the run file PyroMark Q24 Advanced User Manual 06 2013 5 7 Operating Procedures 5 2 4 5 8 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 Q24 Advanced Software User Guide press the F1 key when in PyroMark Q24 Advanced Software Managing instrument me
30. auf EN FCC Kennzeichen der C der Federal Ger ter ckseite Communications Commission der Vereinigten Staaten Typenschild auf EN C Tick Mark Zeichen der f r Australien Ger ter ckseite Herstellerkennung N17965 Oe Typenschild auf EN Markierung gem 257 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 Elektro und Elektronik der Altger te WEEE Ger ter ckseite Reagenzien EN Kennzeichnung der L sungen Produktions Charge Kartusche Platte E 8 PyroMark Q24 Advanced User Manual 06 2013 Appendix E Symbol Ort Sprache Beschreibung Reagenzien EN Zur Verwendung bis L sungen Kartusche Platte Reagenzien EN Zul ssiger L sungen Temperaturbereich Kartusche Platte Ger t EN Lesen Sie die Vakuum detaillierten Arbeitsstation Informationen im Reagenzien Handbuch Kartusche Typenschild auf EN Hersteller i S d der R ckseite Gesetzes ml des Ger ts und allen anderen Produkten Im EN Warnhinweis lesen Sie Ger teinneren das Handbuch N Kartusche EN Von direkter ze Sonneneinstrahlung fernhalten AY a Kartusche EN Methoden Nummer Kartusche EN Ger te Parameter PyroMark Q24 Advanced User Manual 06 2013 E 9 Appendix E This page intentionally left blank E 10 PyroMark Q24 Advanced User Manual 06 2013 Ap
31. aux dangereux d agents infectieux comme cela est d fini dans les fiches de donn es de s curit FDS ou dans les documents de l OSHA de l ACGIH t ou du COSHH applicables Pour plus d informations voir le site www qiagen com safety L vacuation 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 t ACGIH American Conference of Government Industrial Hygienists Etats 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 Q24 Advanced User Manual 06 2013 Appendix D 1 4 Produits chimiques AVERTISSEMENT Produits chimiques dangereux w La solution de d naturation utilis e avec le poste de A 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 q
32. c1 Use lint free tissues to clean the space between the heating A block and the light guide block inside the instrument Do not use paper tissues 1 6 Heat hazard WARNING Hot surface W9 The plate holder and the heating block for annealing can A reach temperatures of up to 80 C 176 F Avoid touching them when they are hot CAUTION Risk of overheating C2 A To ensure proper ventilation maintain a minimum clearance of 10 cm 3 94 in at the sides and rear of the PyroMark Q24 Instrument Slits and openings that ensure the ventilation of the PyroMark Q24 Instrument must not be covered 1 7 Consumables CAUTION A Unsupported consumables C3 Do not connect or use any consumables accessories or external equipment other than that specified PyroMark Q24 Advanced User Manual 06 2013 1 5 Safety Information 1 8 Symbols used on PyroMark Q24 products Symbol Location Language Description Type plate on EN CE mark a the back of the instrument Type plate on EN CSA listing mark for SP the back of Canada and the USA C US the instrument Type plate on EN FCC mark of the United C the back of States Federal the Communications instrument Commission Type plate on EN C Tick mark for Australia the back of supplier identification the N17965 instrument On Type plate on EN RoHS mark for China 257 the back of the restriction of the use the of c
33. ct QIAGEN Technical Services Ensure your upgrade installation files are located in a folder called Upgrade at the root of the USB stick Note For all other instrument error messages please contact QIAGEN Technical Services PyroMark Q24 Advanced User Manual 06 2013 7 5 Troubleshooting Other problems a The instrument is making unexpected noise when starting b No contact with the USB stick c USB stick cannot be inserted Comments and suggestions Check that the reagent cartridge is inserted correctly The used USB stick is damaged or not compatible with the system It is recommended that USB sticks supplied by QIAGEN are used Broken USB contact contact QIAGEN Technical Services 7 4 Vacuum workstation related errors a No vacuum is received b Vacuum lost during sample preparation c Filter probes not working properly d Liquid left in some wells in the immobilization plate 7 6 Comments and suggestions Turn off the vacuum pump and open the cap to 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 The waste filter may be wet and require replacing see Section 6 3 5 Ensure that the tubing is connected correctly and there is no leakage The waste filter may be wet and require replacing see Section 6 3 5 Ensure filter probe
34. 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 Q24 Advanced User Manual 06 2013 Appendix B Sample preparation Use 5 20 ul of a 25 ul PCR for immobilization to Streptavidin Sepharose High Performance GE Healthcare according to the instructions in Section 5 3 3 This will be equivalent to approximately 0 5 4 picomoles of PCR product depending on the PCR efficiency Pyrosequencing analysis Use default settings in the software for all assay setups if not otherwise stated Assay setup AQ SNP and CpG assays When creating an AQ SNP 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 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 ser
35. dly encompassing any of the many types of variations in DNA sequences that are found within a given population As the substrate is dispensed into a well indicated by an S in the Pyrogram 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 The graph resulting from a sequencing reaction performed using Pyrosequencing technology Incorporated nucleotides are shown as peaks in the Pyrogram PyroMark Q24 Advanced User Manual 06 2013 8 3 Glossary Term Quality control window Reference peak RLU Sepharose beads SEQ Sequence to analyze Sequencing primer Shift Description Gives an overview of the quality at the end of a defined number of bases in the base called sequence A setting in the SEQ analysis mode Nonvariable peaks i e peaks that are not a part of a variable position are referred to as reference peaks Reference peaks are used in the analysis both as references when calculating the single peak height level and as internal controls when assessing the quality Relative Light Unit entity used in Pyrosequencing to define peak heights in Pyrogram Streptavidin coated beads should be used for preparation of biotinylated PCR products Analysis mode used for base calling of unknown sequences A short part of a DNA sequence in your sample starting directly after
36. dow Help Saarai Res CpG Pyrogram Report SEQ Pyrogram Report CpG Full Report SEQ Full Report To view reports generated in PDF formot a PDF reader must be installed on the computer Adobe Reader can be downloaded at www adobe com 5 5 Finishing work and shutting down 5 5 1 Shutting down the instrument 1 When the instrument is not processing using the and vw screen buttons select Shutdown from the main menu and press OK 2 When the message It is now safe to turn off the instrument appears switch off the instrument The power switch is located at the rear of the instrument 5 32 PyroMark Q24 Advanced User Manual 06 2013 5 5 2 Operating Procedures Emptying the waste container and troughs WARNING A Hazardous chemicals we The Denaturation Solution used with the vacuum workstation contains sodium hydroxide which is irritating to eyes and skin Always wear safety glasses gloves and a lab coat The responsible body e g laboratory manager must take the necessary precautions to ensure that the surrounding workplace is safe and that the 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
37. e 2 T Technical assistance 2 2 Technical data 1 Test filter probes 5 12 Upgrade analysis software 4 3 instrument software 5 3 V Ventilation 1 5 4 2 Verify proper installation and operation 7 7 W Warnings 1 1 Waste disposal 5 Index 2 PyroMark Q24 Advanced User Manual 06 2013 www qiagen com Australia techservice au qiagen com Austria techservice at qiagen com Belgium techservice bnl qiagen com Brazil suportetecnico brasil qiagen com Canada techservice ca qiagen com China techservice cn qiagen com Denmark techservice nordic qiagen com Finland techservice nordic qiagen com France techservice fr giagen com Germany techservice de qiagen com Hong Kong techservice hk giagen com India techservice india qiagen com Ireland techservice uk qiagen com Italy techservice it qiagen com Japan techservice jp qiagen com Korea South techservice kr giagen com Luxembourg techservice bnl giagen com Mexico techservice mx qiagen com The Netherlands techservice bnl qiagen com Norway techservice nordic qiagen com Singapore techservice sg giagen com Sweden techservice nordic qiagen com Switzerland techservice ch qiagen com UK techservice uk qiagen com USA techservice us qiagen com sn iia Sample amp Assay Technologies
38. e Be M Estimated remaining run time hh mm 5 4 2 Starting the instrument 1 Before switching on the instrument ensure that the mains plugs are connected to properly grounded earthed mains outlets with the correct voltage and frequency and that mains plugs are easily accessible in case the instrument needs to be disconnected quickly from mains power 2 Switch on the instrument The power switch is located at the rear of the instrument see image in Section 3 4 5 4 3 Starting the run Load the reagent cartridge and the plate 1 When the instrument is not processing open the instrument lid An audible warning signal will alert you if the lid is opened when it is not safe 2 Open the cartridge gate and insert the filled reagent cartridge with the label facing out Push the cartridge in fully and then push it down see image below 3 Ensure the cartridge is properly inserted with the line visible in front of the cartridge and close the gate 4 Open the plate holding frame and place the plate on the heating block inside the instrument 5 Close the plate holding frame and the instrument lid 5 22 PyroMark Q24 Advanced User Manual 06 2013 5 4 4 Operating Procedures Select the run file and start the run 1 Insert the USB stick containing the run file into the USB port at the front of the instrument 2 Using the and screen buttons select Run in the main menu and press OK 3 Select t
39. e 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 M 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 duplexes 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 any significant peak after any nucleotide addition PyroMark Q24 Advanced User Manual 06 2013 B 7 Appendix B 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 p
40. e of the substrates adenosine 5 phosphosulfate APS and luciferin in the sequencing reaction A region in the sequence that varies at one or more variable bases In PyroMark Q24 Advanced Software the variable positions are highlighted with a blue gray background color in the histogram and Pyrogram PyroMark Q24 Advanced User Manual 06 2013 8 5 Glossary This page intentionally left blank 8 6 PyroMark Q24 Advanced User Manual 06 2013 Appendix A Appendix A Technical data QIAGEN reserves the right to change specifications at any time Environmental conditions Operating conditions PyroMark Q24 Instrument Input power 100 240 VAC 50 60 Hz Mains supply voltage fluctuations are not to exceed 10 of nominal supply voltages Input power Maximum 160 VA consumption Instrument rating 24 VDC 40 W Cooler rating 12 V DC 60 W Overvoltage Il category Air temperature 15 32 C 59 90 F Relative humidity Maximum relative humidity 80 for temperatures up to 31 C decreasing linearly to 50 relative humidity at 40 C non condensing Altitude Up to 2000 m 6500 ft Place of operationFor indoor use only Draft free location not close to window Keep instrument out of direct sunlight Pollution degree 2 Environmental 3K2 IEC 60721 3 3 class PyroMark Q24 Advanced User Manual 06 2013 A 1 Appendix A Transportation conditions Air temperature 25 C to 60 C 13 F to 140 F Relative humidi
41. e recommend entering the Sequence Before Bisulfite Treatment This enables the software to automatically generate the sequence to analyze and select the most appropriate bisulfite treatment control 3 Click the Generate Dispensation Order button If creating an SEQ assay enter the Dispensation Order 4 Click lal in the toolbar Note Before running your samples validate your assay using a reference DNA sample see Appendix B Note When using QIAGEN kits use the settings stated in the kit handbooks Optional If desired enter a note about the assay in the Assay Note box Set up the variable positions in the Variable Positions tab AQ SNP and CpG assays only Lock the assay for editing by clicking the Lock Assay button at the bottom of the assay setup window If creating a CpG assay check that the software has selected a bisulfite treatment control If no bisulfite treatment control has been automatically selected add one manually if possible preferably at the beginning of the sequence A bisulfite treatment control can be added manually by adding a C before or after a T that was a C before bisulfite treatment in a forward assay or by adding a G before or after an A that was a T before bisulfite treatment in a reverse assay in the dispensation order PyroMark Q24 Advanced User Manual 06 2013 5 5 Operating Procedures 5 2 3 5 6 Histogram LIL I chan LA Be Ti 6 AS
42. en eine Verletzungsgefahr f r Sie selbst oder andere Personen besteht N here Einzelheiten ber diese Situationen werden in einem Textfeld wie diesem beschrieben A 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 Q24 Advanced User Manual 06 2013 E 1 Appendix E 1 1 Sachgem e Handhabung WARNUNG Verletzungsgefahr und Besch digung des Ger ts w1 ACHTUNG Die unsachgem e Bedienung des PyroMark Q24 A Au endiensts durchgef hrt werden Instrument kann zu einer Verletzung des Benutzers oder zur Besch digung des Ger ts f hren Die Bedienung des PyroMark Q24 Instrument darf nur durch qualifiziertes entsprechend geschultes Personal erfolgen Die Instandhaltung des PyroMark Q24 Instrument darf nur durch einen Servicespezialisten des QIAGEN F hren Sie alle Wartungsarbeiten gem den Anweisungen in Abschnitt 6 dieses Handbuchs durch QIAGEN stellt alle Reparaturen in Rechnung die nachweislich auf eine inkorrekte Wartung zur ckzuf hren sind WARNUNG Verletzungsgefahr und Besch digung des Ger ts w2
43. ersonal injury and material damage W3 CAUTION Do not attempt to move the PyroMark Q24 Instrument A during operation 1 2 Electrical safety Disconnect the line power cords from the power outlets before servicing WARNING Electrical hazard w4 Any interruption of the protective conductor earth ground A lead inside or outside the instrument or disconnection of the protective conductor terminal is likely to make the instrument dangerous Intentional interruption is prohibited Lethal voltages inside the equipment When the equipment 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 Q24 Instrument follow the advice below M The instrument s line power cords must be connected to line power outlets that have protective conductors earth ground E Keep mains plugs easily accessible in case the equipment needs to be disconnected quickly from mains power 1 2 PyroMark Q24 Advanced User Manual 06 2013 Safety Information 1 3 M Use only power supplies and cords supplied with the system Biological safety When handling biological material use safe laboratory procedures as outlined in publications such as Biosafety in Microbiological and Biomedical Laboratories HHS www cdc gov od ohs biosfty biosfty htm WARNING A Biological materials w5 Hand
44. ertain hazardous instrument substances in electrical and electronic equipment Type plate on EN Waste Electrical and the back of Electronic Equipment the WEEE instrument ll Reagents EN Identification of solutions production batch lot cartridge plate 1 6 PyroMark Q24 Advanced User Manual 06 2013 Safety Information Symbol Location Language Description Reagents EN Use by date solutions cartridge plate Reagents EN Temperature limitation solutions cartridge plate Instrument EN Read the manual VPW reagents cartridge Type plate on EN Legal manufacturer the back of ml instrument and all other products Inside the EN Warning consult user instrument manual Als Cartridge EN Keep away from TN sun light ZN D Cartridge EN Method number Cartridge EN Instrument parameters PyroMark Q24 Advanced User Manual 06 2013 1 7 Safety Information This page intentionally left blank 1 8 PyroMark Q24 Advanced User Manual 06 2013 Introduction 2 1 Introduction Thank you for choosing the PyroMark Q24 Advanced We are confident it will become an integral part of your laboratory Before using the PyroMark Q24 Advanced it is essential that you read this user manual carefully and pay particular attention to the safety information see Section 1 The instructions and safety information in the user manual must be followed to ensure safe operation of the system and to
45. ester 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 PyroMark Q24 Instrument contacter imm diatement les Services techniques de QIAGEN AVERTISSEMENT A Aiguilles pointues w8 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 ATTENTION A Maintenance des guides de lumi re c1 Nettoyer l espace entre l unit de chauffage et les guides de lumi re l int rieur de l appareil l aide de tissus non pelucheux Ne pas utiliser de mouchoirs en papier 1 6 Dangers li s la chaleur AVERTISSEMENT A Surface chaude w9 Le porte plaques et l unit de chauffage utilis e pour l hybridation peuvent atteindre une temp rature de 80 C viter de toucher ces l ments lorsqu ils sont chauds ATTENTION A Risque de surchauffe C2 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 Q24 Instrument Les fentes et les ouvertures qui assurent la ventilation de l appareil ne doivent pas tre obstru es D 6 PyroMark Q24 Advanced User Manual 06 2013 Appendix D 1 7 Consumables ATTENTION Consommables
46. fications 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 Q24 Advanced User Manual 06 2013 C 1 Appendix C This page intentionally left blank C 2 PyroMark Q24 Advanced User Manual 06 2013 Appendix D Appendix D 1 Informations de s curit Le PyroMark Q24 Advanced est la combinaison de l quipement PyroMark Q24 Instrument et du logiciel PyroMark Q24 Advanced Software Avant d utiliser le PyroMark Q24 Advanced 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 syst me en bon tat de marche il
47. forming the function test as follows 1 Add 100 ul high purity water to each well of a 24 well PCR plate 2 Fill a trough with 70 ml high purity water G9 Start the vacuum pump 4 Apply vacuum to the vacuum prep tool by opening the vacuum switch 5 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 not check the connections 6 Lower the filter probes into the PCR plate and check that the water is aspirated evenly across all wells and thot all wells are empty after a maximum of 10 s 7 Ifthe wells are not empty after 10 s repeat the procedure from step 1 If the function test fails twice replace the filter probes see Section 6 3 2 DNA amplification Amplify the DNA to be analyzed by PCR using one of the primers biotinylated To receive valid analysis data see Appendix B Immobilizing the PCR product to beads Biotinylated PCR products are immobilized on streptavidin coated Sepharose beads Streptavidin Sepharose High Performance GE Healthcare 1 Gently shake the bottle with streptavidin coated Sepharose beads from side to side until a homogenous solution is obtained 2 Mix Binding Buffer 40 ul per sample and the total amount of streptavidin coated Sepharose beads in a tube PyroMark Q24 Advanced User Manual 06 2013 Operating Procedures Note Check the lot numbe
48. g Servicing QIAGEN offers comprehensive Service Support Agreements including Warranty Extensions Full Cover Support Agreements and system application training including on site installation and annual preventative maintenance Service Support Agreements maximize productivity and ensure high performance from your system 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 Checking the performance of the PyroMark Q24 Advanced Check that PyroMark Q24 Advanced is functioning according to specifications by measuring imprecision bias and linearity for an AQ SNP or CpG assay using PyroMark Q24 Validation Oligo Perform the validation according to the handbook supplied with the product To order PyroMark Q24 Validation Oligo please contact QIAGEN PyroMark Q24 Advanced User Manual 06 2013 6 1 Maintenance 6 2 Maintenance of the PyroMark Q24 Instrument 6 2 1 Cleaning the instrument If the instrument has been contaminated by dust and spillage clean it according to the instructions below Important points before starting E Avoid harsh cleaners and chemicals and getting moisture inside the instrument M The cleaning liquid must be applied to the cloth only M Do not use any organic solvent or detergent other than ethanol w
49. gh 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 6 3 4 Ensure that the waste filter is dry If the filter is wet it should be replaced see Section 6 3 5 6 Close the vacuum switch on the tool Off and place the tool in the Parking P position PyroMark Q24 Advanced User Manual 06 2013 5 17 Operating Procedures 7 8 10 11 12 Refill trough 5 with 70 ml high purity water Dilute the sequencing primer to 0 375 uM in Annealing Buffer Add 20 ul of the solution to each well of a PyroMark Q24 Plate that is to be used Note Use one of the supplied PyroMark Q24 Plate Holders as support when preparing and moving the plate Immediately after immobilization see Section 5 3 3 place the PCR plate or the strips and PyroMark Q24 Plate on the worktable Ensure that the plate is in the same orientation as when the samples were loaded see image below for guidance Note Sepharose beads sediment quickly Capturing of beads must take place immediately following agitation If more than 1 minute has elapsed since the plate was agitated agitate again for 1 minute before capturing the beads Apply vacuum to the tool by opening the vacuum switch Carefully lower the filter probes into the PCR plate or strips to capture the beads containing immobilized tem
50. h purity water trough 5 and applying vacuum Flush the filter probes with 70 ml high purity water 21 Raise the tool to beyond 90 vertical for 5 seconds to drain liquid from the filter probes PyroMark Q24 Advanced User Manual 06 2013 5 19 Operating Procedures 5 3 6 22 Close the vacuum switch on the tool Of and place the tool in the Parking P 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 25 At the end of a working day liquid waste and any remaining solutions should be discarded and the PyroMark Q24 Vacuum Workstation should be checked for dust and spillage see Section 5 5 2 Annealing of sequencing primer to samples WARNING A Hot surface w9 The plate holder and the heating block for annealing can reach temperatures of up to 80 C 176 F Avoid touching them when they are hot 5 4 5 20 1 Heat the PyroMark Q24 Plate containing the samples at 80 C for 5 minutes using the PyroMark Q24 Plate Holder two are supplied with the vacuum workstation and a heating block 2 Remove the hot plate holder together with the PyroMark Q24 Plate from the heating block and place the plate immediately on the heating block of the PyroMark Q24 Instrument Ensure that the plate holding frame is closed Start the run immediately Note The time between removing the plate holder from the heating
51. he USB port at the front of the instrument 2 Using the and screen buttons select Administration in the main menu and press OK 3 Select Extract Damaged Runs and press OK 4 When the instrument confirms that the run files have been saved to the USB stick press Close 5 Remove the USB stick Viewing acknowledgements version and contact information View acknowledgements software and hardware versions or contact information as follows 1 Select About in the main menu using the and screen buttons and press OK 2 Select the information you want to view and press OK Upgrading the instrument software If you have received a software upgrade from QIAGEN upgrade the software as follows 1 Save the upgrade files on one of the USB sticks supplied The files should be saved in a folder named Upgrade in the root directory of the USB stick 2 When the instrument is not processing insert the USB stick into the USB port at the front of the instrument Do not remove it until the upgrade is completed 3 Using the and screen buttons select Administration in the main menu and click OK 4 Select Upgrade Software and click OK 5 Follow the instructions on the screen PyroMark Q24 Advanced User Manual 06 2013 5 3 Operating Procedures 5 1 8 5 2 5 2 1 5 2 2 Run an external application The Run External Application
52. he run file using the and screen buttons To view the contents of a folder select the folder and press Select To go back to the previous view press Back 4 When the run file is selected press Select to start the run PyroMark Q24 10 33 Select file to run 10 34 y Administration E upgrade About Instr_SW_test Shutdown ok Monitoring the run The instrument will start dispensing reagents when the pressure in the dispensing unit the speed of the mixer and the temperatures of the heating block process chamber lid and the coolant liquid have reached their preset levels PyroMark Q24 Advanced User Manual 06 2013 5 23 Operating Procedures 5 24 Instrument status The instrument status is displayed in the top right hand corner of the Pyrogram area Environment Waiting for the pressure in the dispensing unit the speed of the mixer and the temperatures of the heating block process chamber lid and the coolant liquid to reach their preset levels may take several minutes Priming Priming the needles of the reagent cartridge to ensure that the dispensation capillaries are flushed and filled with solution Running The enzyme mix and substrate mix are dispensed to all wells used Then the nucleotides are dispensed to the wells according to their dispensation order which is defined in the assay file Stopped The run has been canceled Saving The run data is transferred to the
53. hen cleaning the screen The following items are required M Ethanol 70 E High purity water Milli Q 18 2 MQ x cm or equivalent M Clean non abrasive lint free cloths Procedure 1 When the instrument is not processing using the and screen buttons select Shutdown in the main menu and press OK 2 When the message It is now safe to turn off the instrument appears switch off the instrument The power switch is located at the rear of the instrument 3 Disconnect the instrument from the mains power There are two mains plugs 4 Open the instrument lid 5 Clean the area around the dispensing unit the process chamber and the heating block using a clean lint free cloth lightly moistened with 70 ethanol 6 Clean the screen by wiping with a clean non abrasive lint free cloth lightly moistened with water If this does not clean the screen properly apply a small amount of 70 ethanol to the cloth Do not allow ethanol to soak into the gaps around the screen protection 6 2 PyroMark Q24 Advanced User Manual 06 2013 Maintenance 6 2 2 7 If necessary clean the exterior of the instrument using a clean lint free cloth lightly moistened with water 8 After cleaning wipe the surfaces dry with a clean dry non abrasive lint free cloth 9 Reconnect the instrument to the mains power Cleaning the heating block and light guides In case of spillage on the heating block inside the instru
54. imer 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 give insufficient yield and cause background problems in Pyrosequencing reactions due to excess 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 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 Q24 Advanced User Manual 06 2013 Appendix B These include the QlAprep system for rapid plasmid p
55. in a forward assay or by adding a G before or after an A that was a T before bisulfite treatment in a reverse assay in the dispensation order Refer to PyroMark Q24 Advanced Software User Guide SEQ assays Experience with sequencing large numbers of templates indicates that the dispensation order n CTGA gives on average the best sequencing quality Individual templates may however give better results with other dispensation orders Where possible SEQ assays that involve resolution of different sequences should be designed such that resolution does not depend 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 of 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 mixed sequence that cannot be analyzed PyroMark Q24 Advanced User Manual 06 2013 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 Q24 Advanced Softwar
56. incompatibles C3 Ne pas relier ni utiliser de consommables d accessoires ni A d quipement externe autres que ceux sp cifi s 1 8 Symboles sur les produits PyroMark Q24 Symbole Emplacement Langue Description Plaque EN Marquage CE signal tique l arri re de l appareil Plaque EN Conforme aux normes SP signal tique a de s curit des l arri re de Etats Unis et du a ua l appareil Canada Plaque EN Label FCC de la C signal tique Federal l arri re de Communications l appareil Commission des Etats Unis Plaque EN Label C Tick pour signal tique l Australie identifiant du l arri re de fournisseur N17965 l appareil Oy Plaque EN Label RoHS pour la 257 signal tique Chine restriction de l arri re de l utilisation de certaines l appareil substances dangereuses dans l quipement lectrique et lectronique PyroMark Q24 Advanced User Manual 06 2013 Appendix D Symbole Emplacement Langue Description Plaque signal tique l arri re de l appareil EN D chets d quipements lectriques et lectroniques DEEE R actifs solutions cartouche plaque EN Identification du lot de production R actifs solutions cartouche plaque EN A utiliser avant R actifs solutions cartouche plaque EN Limitations de temp rature Appareil poste de travail sous vide r actifs cartouche EN Lire le manuel
57. interference that may cause undesired operation This Class B digital apparatus complies with Canadian ICES 0003 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 of the FCC Rules and meets all requirements of the Canadian Interference Causing Equipment Standard ICES 003 for digital apparatus 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 the interference will not occur in a particular installation If this equipment does 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 PyroMark Q24 Advanced User M
58. ion The PyroMark Q24 Advanced uses proven real time sequence based Pyrosequencing technology for sequence based detection and quantification in genetic analysis and epigenetic methylation studies The system can analyze up to 24 samples simultaneously An easy to use protocol is used to prepare samples PyroMark Q24 definitions M PyroMark Q24 Instrument Instrument only M PyroMark Q24 Advanced Software Software only M PyroMark Q24 Vacuum Workstation Vacuum Workstation only M PyroMark Q24 Advanced Instrument software and installation M PyroMark Q24 Advanced 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 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 4 Each incorporation event is accompanied by release of pyrophosphate PPi in an equimolar quantity to the amount of nucleotide incorporated Polymerase DNA dNTP DNA PPi n 1 PyroMark Q24 Advanced User Manual 06 2013 3 1 General Description 5 ATP sulfurylase quantitatively converts PPi to ATP in the presence
59. ion with the PyroMark Q24 Vacuum Workstation 3 6 PyroMark Q24 Advanced User Manual 06 2013 Contents 1 Contents 3 6 Analysis software 3 6 4 Installation Procedures 4 1 4 1 System delivery and installation 4 1 4 2 Requirements 4 1 4 3 Installation of the analysis software 4 3 4 3 1 Installing or upgrading PyroMark Q24 Advanced Software 4 3 4 3 2 Uninstalling PyroMark Q24 Advanced Software 4 4 5 Operating Procedures 5 1 5 1 Instrument administration 5 1 5 1 1 Setting date and time 5 1 5 1 2 Copying unsaved runs 5 1 5 1 3 Copying recently saved runs 5 2 5 1 4 Copying log files 5 2 5 1 5 Extracting damaged runs 5 3 5 1 6 Viewing acknowledgements version and contact information 5 3 5 1 7 Upgrading the instrument software 5 3 5 1 8 Run an external application 5 4 5 2 Setting up a run 5 4 5 2 1 Starting PyroMark Q24 Advanced Software 5 4 5 2 2 Setting up an assay 5 4 5 2 3 Setting up a run 5 6 5 2 4 Managing instrument methods 5 8 5 3 Sample and reagent preparation 5 9 5 3 1 PyroMark Q24 Vacuum Workstation function test 5 12 5 3 2 DNA amplification 5 12 5 3 3 Immobilizing the PCR product to beads 5 12 5 3 4 Loading reagents into the PyroMark Q24 Cartridge5 14 5 3 5 Separation of DNA strands and release of samples into the PyroMark Q24 Plate 5 15 Contents 2 PyroMark Q24 Advanced User Manual 06 2013 Contents 5 3 6 5 4 5 4 1 5 4 2 5 4 3 5 4 4 5 4 5 5 4 6 5 4 7 5 4 8 5 5 5 5 1 5 5 2 5 5 3 5 6 6 1 6 2 6 2
60. ips 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 Note During immobilization prepare the vacuum workstation for the sample preparation steps 1 7 in Section 5 3 1 5 3 4 Loading reagents into the PyroMark Q24 Cartridge WARNING Sharp needles wg A 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 1 Allow the reagents to reach ambient temperature 20 25 C 2 Fillthe PyroMark Q24 Cartridge according to the handbook supplied with the reagents and place the cartridge into the instrument Note Information on volume is given in the Pre Run information report found in the Tools menu at run setup see Section 5 2 3 Note If pipetting small volumes of reagents into the cartridge e g below 50 ul per well make sure that all liquid is collected at the bottom of the cartridge For example this can be accomplished by gently tapping the cartridge several times on a smooth work bench Important Avoid tapping the cartridge too hard or on uneven surfaces as this can damage the needles Alternatively the volume of the liquid used can be increased Note Ensure any reused reagent cartridges are cleaned thoroughly according to the instructions in Section 5 4 5 PyroMark Q2
61. ir temperature Relative humidity 10 C to 40 C 50 F to 104 F Max 75 noncondensing Mechanical data and hardware features Dimensions worktable Clearance space Mass Capacity Chemical resistance Width 295 mm 11 61 in Height 68 mm 2 68 in Depth 353 mm 13 90 in Width 350 mm 13 78 in or 700 mm 27 56 in Height 400 mm 15 74 in Depth 700 mm 27 56 in or 350 mm 13 78 11 kg 24 26 lb includes filled troughs and a full waste container Up to 24 samples per plate pH 4 to pH 9 common detergents 0 5 M sodium hydroxide 70 ethanol PyroMark Q24 Advanced Software Operating system Microsoft Windows 7 32 bit English version Processor RAM Free hard disk space Graphics card Monitor Pointer device Interfaces A 4 Intel Pentium IV 3 GHz or higher 1 GB 100 MB Supporting the resolution of the monitor 1280 x 800 pixels True Color 32 bit Mouse or similar USB port and CD ROM PyroMark Q24 Advanced User Manual 06 2013 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 local laws and regulations In the
62. l Support Center at www qiagen com Support or call one of the QIAGEN Technical Service Departments or local distributors see back cover or visit www giagen 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 Q24 Advanced User Manual version 2 0 2 2 PyroMark Q24 Advanced User Manual 06 2013 Introduction 2 3 2 3 1 Intended use of PyroMark Q24 Advanced The PyroMark Q24 Advanced is the PyroMark Q24 Instrument running the PyroMark Q24 Advanced Software The PyroMark Q24 Advanced System is the PyroMark Q24 Advanced plus any PyroMark kits The PyroMark Q24 Advanced System is designed to detect changes in specified variable positions in or base calling of DNA prepared from biological samples in molecular biology applications The PyroMark Q24 Instrument and PyroMark Q24 Vacuum Workstation are intended to be used only in combination with QIAGEN kits indicated for use with the PyroMark Q24 Instrument for the applications described in the kit handbooks If the PyroMark Q24 Instrument and PyroMark Q24 Vacuum Workstation are used with other than QIAGEN kits it is the user
63. l out the old filter probes 6 Gently insert new filter probes without pressing on the filter tips 7 Fasten the four screws and reconnect the vacuum pump to the mains power Replacing the rubber seal If the filter probes are loose and or fall out there are two possible causes MB The four screws are not tight enough M The rubber seal needs to be replaced If the rubber seal needs to be replaced the following items are required E Powder free gloves PyroMark Q24 Advanced User Manual 06 2013 Maintenance 6 3 4 MB 2 mm Allen key supplied with the system M New rubber seal QIAGEN Procedure 1 9 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 tool from the tubing Remove the four screws using the 2 mm Allen key supplied with the system Gently remove the filter probes Avoid contaminating the filter probes Remove the metal plate and replace the rubber seal Reassemble the tool and reconnect the vacuum pump to the mains power Check thot the filter probes are functioning properly by performing the function test as described in Section 5 3 1 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 fo
64. le biological material with the greatest of care and in accordance with the required safety regulations Always wear safety glasses 2 pairs of 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 operators are suitably trained and not exposed to hazardous levels of infectious agents as defined in the applicable Safety Data Sheets SDSs or OSHA ACGIH 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 t ACGIH American Conference of Government Industrial Hygienists United States of America COSHH Control of Substances Hazardous to Health United Kingdom PyroMark Q24 Advanced User Manual 06 2013 1 3 Safety Information 1 4 Chemicals WARNING A Hazardous chemicals we The Denaturation Solution used with the vacuum workstation contains sodium hydroxide which is irritating to eyes and skin Always wear safety glasses gloves and a lab coat The responsible body e g laboratory manager must take the necessary precautions to ensure that the surrounding workplace is safe and that the operators are not exposed to hazardous levels of toxic substances
65. llowing items are required E New tubing QIAGEN M Beaker PyroMark Q24 Advanced User Manual 06 2013 6 7 Maintenance 6 3 5 6 8 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 Remove the broken tubing at one end and empty any liquid waste into an empty beaker 4 Disconnect the other end of the tubing and discard the tubing and any liquid waste 5 Cut the new vacuum tubing into three pieces and assemble it Ensure that the tubing is connected to the pump s Vacuum fitting 6 Reconnect the vacuum pump to the mains power Replacing the waste filter If the waste filter is wet e g if the waste container is full no vacuum is attained and the filter must be replaced Be sure to observe all federal state and local environmental regulations for the disposal of laboratory waste The following items are required M New waste filter M Beaker Note Two waste filters are supplied with the vacuum workstation Filters can be ordered at www millipore com Millipore Millex FG50 Filter Unit catalogue number SLFGO5010 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 Remove the tubing from the fil
66. ly voltage fluctuations are not to exceed 10 of nominal supply voltages Grounding requirements To protect operating personnel the PyroMark Q24 Instrument must be correctly grounded earthed The PyroMark Q24 Instrument is equipped with two 3 conductor AC power cords To preserve this protection feature do not operate the PyroMark Q24 Instrument from AC power outlets that have no ground earth connection Installation of the analysis software Installing or upgrading PyroMark Q24 Advanced Software To install or upgrade PyroMark Q24 Advanced Software 1 Ensure that the computer meets the minimum requirements see Section 3 6 2 Close any programs running on the computer Insert the PyroMark Q24 Advanced Software CD into the CD drive 4 Inthe CD menu click Install PyroMark Q24 Advanced 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 and select Explore Double click the file autorun exe 5 Follow the instructions that appear in the Setup Wizard Note If NET Framework 4 0 has to be installed installation is prompted by the Setup Wizard the installation has to be restarted when the NET Framework installation has been completed i e open the CD menu see step 4 and click Install PyroMark Q24 Advanced Software PyroMark Q24 Advanced User Manual 06 2013 4 3
67. m 5 ml GE Healthcare Sequencing primer High purity water Milli Q 18 2 MQ x cm or equivalent Ethanol 70 PyroMark Binding Buffer PyroMark Denaturation Solution PyroMark Wash Buffer concentrate PyroMark Annealing Buffer Recommended plate mixers Orbital plate mixers from these manufacturers are recommended for use with the PyroMark Q24 Advanced System PyroMark Q24 Advanced User Manual 06 2013 Eppendorf Operating Procedures Thermomixer comfort cat no 5355 000 011 basic device Thermoblock for MTP cat no 5363 000 012 Adapter plate for 96 x cat no 5363 007 009 0 2 ml PCR tubes to insert in blocks for microtiter plates H P Labortechnik GmbH cat no 51410 115 V 51410 U cat no 51110 115 V 51110 U Variomag Teleshake Variomag Monoshake Recommended 24 well plates The 24 well plates from these manufacturers are recommended for use with the PyroMark Q24 Advanced System ABgene Thermo Fast Thermo PCR plate Scientific 24 well PCR Agen Microplate FrameStar Break a way ania 96 wells clear tubes Quali PCR Kisker Plates without frame PyroMark Q24 Advanced User Manual 06 2013 cat no AB 0624 cat no PCR 24 C cat no Ati 1000 cat no G030 Operating Procedures 5 3 1 5 3 2 5 3 3 PyroMark Q24 Vacuum Workstation function test Before using the PyroMark Q24 Vacuum Workstation check that the filter probes are working properly by per
68. ment clean the heating block and the light guides underneath the block The following items are required M Cotton swabs M Ethanol 70 M clean non abrasive lint free cloth e g a camera lens cloth Procedure 1 When the instrument is not processing using the and screen buttons select Shutdown in the main menu and press OK 2 When the message It is now safe to turn off the instrument appears switch off the instrument The power switch is located at the rear of the instrument 3 Disconnect the instrument from the mains power There are two mains plugs 4 Open the instrument lid 5 Open the plate holding frame 6 Clean each well hole light guide carefully using cotton swabs lightly moistened with 70 ethanol see image next page 7 Clean the space between the heating block and the light guide block by carefully inserting a clean non abrasive lint free cloth lightly moistened with 70 ethanol see image next page 8 Close the plate holding frame and the instrument lid and reconnect the instrument to the mains power PyroMark Q24 Advanced User Manual 06 2013 6 3 Maintenance CAUTION Light guide maintenance c1 Use lint free tissues to clean the space between the heating A block and the light guide block inside the instrument Do not use paper tissues 6 3 Maintenance of PyroMark Q24 Vacuum Workstation WARNING Hazardous chemicals we workstation cont
69. n saved to a USB stick see Section 5 1 2 will not be deleted Copy recently saved runs as follows 1 When the instrument is not processing insert one of the USB sticks supplied into the USB port at the front of the instrument 2 Using the and screen buttons select Administration in the main menu and press OK 3 Select Copy Recently Saved Runs and press OK 4 Using the and screen buttons select the run file for retrieval and press Select 5 When the instrument confirms that the run file has been saved to the USB stick press Close 6 Remove the USB stick Copying log files If you need to send log files to QIAGEN Technical Services copy files as follows 1 When the instrument is not processing insert one of the USB sticks supplied into the USB port at the front of the instrument 2 Using the and screen buttons select Administration in the main menu and press OK 3 Select Copy Log Files and press OK 4 When the instrument confirms that the log files have been saved to the USB stick press Close 5 Remove the USB stick PyroMark Q24 Advanced User Manual 06 2013 5 1 5 5 1 6 5 1 7 Operating Procedures Extracting damaged runs If runs are damaged e g if the instrument was switched off during a run extract run files as follows 1 When the instrument is not processing insert one of the USB sticks supplied into t
70. n the plate overview shows the methylation level for each CpG site in the well El Light green Below the expected range El Green Within the expected range EI Dark green Above the expected range SEQ analysis results The base called sequence is displayed in the Overview tab The bases in the base called sequence and the peaks in the compensated Pyrogram are colored according to their quality assessments eII CCCTTAACTGGATTGGTATCCCCAGCTGTAAGCTGTTGTATTGGCTGCTGAGGCTCACAGCTGCA a 7 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 1 aE un Im GCE 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 161 Example of a base called sequence and Pyrogram for a SEQ assay PyroMark Q24 Advanced User Manual 06 2013 5 31 Operating Procedures 5 4 8 Analysis reports To generate a report select the desired report from the Reports menu For more information about the reports see the View Print and Save Analysis Reports section of the PyroMark Q24 Advanced Software User Guide press the F1 key when in PyroMark Q24 Advanced Software Reports Window Help AQ Analysis Statistics Reports Window Help il Sans AQ Pyrogram Report SNP Pyrogram Report AQ Full Report SNP Full Report Reports Window Help il CpG Analysis Statistics Reports Win
71. nucleotide used in Pyrosequencing reactions binds less stringently to dUTP r Plus shift Change the dispensation order s Minus shift Ensure that homopolymers are followed by an extra dispensation 7 2 Analysis software related errors For errors related to the analysis software see the Troubleshooting Guide section of the PyroMark Q24 Advanced Software User Guide 7 4 PyroMark Q24 Advanced User Manual 06 2013 Troubleshooting 7 3 Instrument related errors Error messages a Too many unsaved runs in the instrument Please go to folder Unsaved Runs and save them to USB memory b The required value was not reached The run will be stopped c Run name is invalid d Could not copy file to USB memory e Failed to connect to the hardware the connection to the hardware is lost please restart the instrument f No valid upgrade folders files found on USB memory Comments and suggestions Transfer unsaved runs to a USB stick see Section 5 1 2 Restart the run If the room temperature is high and a temperature problem remains M Ensure the cooling device is receiving power a light indicator at the rear is lit If not check your connections M Check the coolant level Ensure that the run file is created in PyroMark Q24 Advanced Software Try another USB stick It is recommended that USB sticks supplied by QIAGEN are used Restart the instrument If the problem remains conta
72. om PyroMark Q24 Advanced User Manual 06 2013 5 15 Operating Procedures Things to do before starting Prewarm one of the supplied PyroMark Q24 Plate Holders to be used in Section 5 3 6 by placing it without a plate on a heating block at 80 C Procedure 1 Ensure that the PyroMark Q24 Vacuum Workstation has been assembled correctly and securely amp ue 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 5 3 1 AI filter probes should be replaced after preparation of approximately 100 plates Note Empty the waste container if necessary see Section 5 5 2 2 Fill five separate troughs supplied with the PyroMark Q24 Vacuum Workstation as follows PyroMark Q24 Advanced User Manual 06 2013 Operating Procedures Approximately 40 ml ethanol 70 1 Approximately 40 ml Denaturation Solution 2 Approximately 50 ml 1x Wash Buffer 3 Approximately 50 ml high purity water 4 m Approximately 70 ml high purity water 5 A suggested setup is shown below Refill the troughs to these levels whenever necessary 3 Switch on the vacuum pump 4 Apply vacuum to the tool by opening the vacuum switch 5 Wash the filter probes by lowering the probes into high purity water trough 5 Flush the probes with 70 ml hi
73. ositions in an AQ SNP or CpG assay or the sequence in the quality control window of a SEQ 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 M Sufficient signal intensities Aim for a single peak height of at least 40 RLU No background in blank dispensations No background in variable positions AQ SNP and CpG Expected reference sequence pattern AQ SNP and CpG All positions AQ SNP and CpG and quality control window SEQ with quality assessment Passed The quality assessments for AQ SNP and CpG assays 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 SEQ 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 SEQ assays can improve the estimation of peak height level PyroMark Q24 Advanced User Manual 06 2013 Appendix C Appendix C Liability clause QIAGEN shall be released from all obligations under its warranty in the event repairs or modi
74. oughout this manual A 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 that could result in damage to the system 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 1 1 Proper use WARNING Risk of personal injury and material damage w1 CAUTION Improper use of the PyroMark Q24 Instrument may cause A personal injuries or damage to the system The PyroMark Q24 Instrument must only be operated by qualified personnel who have been appropriately trained Servicing of the PyroMark Q24 Instrument must only be performed by QIAGEN Field Service Specialists PyroMark Q24 Advanced User Manual 06 2013 1 1 Safety Information Perform the maintenance as described in Section 6 QIAGEN charges for repairs that are required due to incorrect maintenance WARNING Risk of personal injury and material damage w2 CAUTION The PyroMark Q24 Instrument is too heavy to be lifted by A one person To avoid personal injury or damage to the instrument do not lift the instrument alone WARNING Risk of p
75. pendix B B 1 Assay design and validation B 1 Appendix C C1 Liability clause C 1 Appendix D D 1 1 Informations de s curit D 1 1 1 Utilisation appropri e D 2 1 2 S curit lectrique D 3 1 3 S curit biologique D 3 1 4 Produits chimiques D 5 1 5 Dangers m caniques D 5 1 6 Dangers li s la chaleur D 6 1 7 Consumables D 7 1 8 Symboles sur les produits PyroMark Q24 D 7 Contents 4 PyroMark Q24 Advanced User Manual 06 2013 Contents Appendix E 1 Sicherheitshinweise 1 1 Sachgem e Handhabung 1 2 Schutz vor Stromschlag 1 3 Biologische Sicherheit 1 4 Chemikalien 1 5 Gefahren durch mechanische Teile 1 6 berhitzungsgefahr 1 7 Verbrauchsartikel 1 8 Symbole auf den PyroMark Q24 Produkten Index PyroMark Q24 Advanced User Manual 06 2013 E 1 E 1 E 2 E 3 E 4 E 5 E 6 E 7 E 7 E 8 Contents 5 Contents This page intentionally left blank Contents 6 PyroMark Q24 Advanced User Manual 06 2013 Safety Information Safety Information The PyroMark Q24 Advanced is the PyroMark Q24 Instrument running the PyroMark Q24 Advanced Software Before using the PyroMark Q24 Advanced it is essential that you read this user manual carefully and pay particular attention to the safety information The instructions and safety information in the user manual must be followed to ensure safe operation of the system and to maintain the system in a safe condition The following types of safety information appear thr
76. pendix E Index A Abort a run 5 24 Administration backup PyroMark Q24 files 5 34 instrument 5 1 Analysis modes 5 28 reports 5 32 software 3 6 Analyze a run 5 27 Assay design and validation 1 setup 5 B Backup of PyroMark Q24 files 5 34 C Cautions 1 1 Check coolant level 5 34 system performance 6 1 Clean heating block and light guides 6 3 instrument 6 2 reagent cartridge 5 25 vacuum workstation 6 5 Colors methylation levels 5 30 quality assessments 5 29 Copy log files 5 2 recently saved runs 5 2 unsaved runs 5 1 Installation analysis software 4 3 grounding requirements 4 3 power requirements 4 2 site requirements 4 1 system 4 1 M Maintenance procedures 6 1 O Operating procedures 5 1 P PCR 1 optimization 3 setup 2 PyroMark Q24 3 4 PyroMark Q24 principle 3 3 Pyrosequencing principle 3 1 Q Quality assessments 8 R Replace filter probes 6 5 rubber seal 6 6 tubing 6 7 waste filter 6 8 Run abort 5 24 analysis 5 27 finishing and shutting down 5 32 G monitoring 5 23 processing 5 20 Glossary 8 1 setup 5 4 I Informations de s curit 1 PyroMark Q24 Advanced User Manual 06 2013 E 1 Index S Safety information 1 1 reagent cartridge 5 25 Troubleshooting 7 1 Sample and reagent preparation 5 9 U Sample preparation 3 6 Servicing 6 1 Set up assay 5 PCR 2 run 5 4 Sicherheitshinweise 1 Starting templat
77. peratures of the heating block process chamber lid and the coolant liquid reach their preset levels this may take several minutes During the run the reagent cartridge is positioned over each well in the PyroMark Q24 Plate and reagents are dispensed in a zigzag fashion by a pneumatic system Sample preparation with the PyroMark Q24 Vacuum Workstation The DNA to be analyzed is amplified by PCR using one of the primers biotinylated The biotinylated PCR product is then immobilized on streptavidin coated Sepharose beads Samples to be analyzed using the PyroMark Q24 Instrument should be prepared according to the instructions in Section 5 3 using the PyroMark Q24 Vacuum Workstation Analysis software The PyroMark Q24 Advanced is shipped with PyroMark Q24 Advanced Software The computer used for setup of runs and data analysis should have the following as minimum specifications M Microsoft Windows 7 32 bit English version Operating System M Pentium IV processor 3 GHz or higher M 1GBRAM M 100 MB free hard drive capacity M Display resolution of 1280 x 800 pixels True Color 32 bit M USB port M CD ROM M Pointer device mouse or similar 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 PyroMark Q24 Advanced User Manual 06 2013 Installation Procedures 4 2 Installation Procedures System delivery and ins
78. plate Hold the filter probes in place for 15 seconds Take care when picking up the tool Note Sepharose beads sediment quickly If more than 1 minute has elapsed since the plate or strips was agitated agitate again for 1 minute before capturing the beads Ensure that all liquid is aspirated from the wells and that all beads have been captured onto the filter probe tips PyroMark Q24 Advanced User Manual 06 2013 Operating Procedures Note If the wells still contain liquid or white beads remain the filter probes may need replacing see Section 6 3 2 13 Transfer the tool to the trough containing 70 ethanol trough 1 Flush the filter probes for 5 seconds 14 Transfer the tool to the trough containing Denaturation Solution trough 2 Flush the filter probes for 5 seconds 15 Transfer the tool to the trough containing Wash Buffer trough 3 Flush the filter probes for 10 seconds 16 Raise the tool to beyond 90 vertical for 5 seconds to drain liquid from the filter probes see image below 17 While holding the tool over the PyroMark Q24 Plate close the vacuum switch on the tool Off 18 Release the beads in the plate containing sequencing primer by shaking the tool gently from side to side 19 With the vacuum switch closed Off transfer the tool to the trough containing high purity water trough 4 and agitate the tool for 10 seconds 20 Wash the filter probes by lowering the probes into hig
79. r des l ments sous tension Afin que le PyroMark Q24 Instrument 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 E Utiliser uniquement les fiches et cordons d alimentation fournis avec le syst me 1 3 Securite biologique Lors de la manipulation de substances biologiques employer des proc dures de laboratoire s res comme d crit dans des publications telles que Biosafety in Microbiological and Biomedical Laboratories HHS www cdc gov od ohs biosfty biosfty htm PyroMark Q24 Advanced User Manual 06 2013 D 3 Appendix D AVERTISSEMENT A Substances biologiques w5 Manipuler les substances biologiques avec la plus grande pr caution et conform ment aux r glementations de s curit en vigueur Toujours porter des lunettes de protection deux paires de 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 de l appareil sont convenablement form s et ne sont pas expos s des nive
80. r of the Streptavidin Sepharose HP For lot number 10057037 or higher use 1 ul For lot numbers lower than 10057037 use 2 ul 3 Add high purity water to a volume of 60 75 ul per well The total volume per well with the PCR product to be added in step 5 will be 80 pl See the table below The amount of water depends on the amount of PCR product used For example If using 15 ul of PCR product 2 ul of beads and 40 ul of Binding Buffer 23 ul of high purity water must be added Volume per sample For Sepharose For Sepharose lot number lot numbers 10057037 or lower than Component higher 10057037 Streptavidin Sepharose HP Le 2 ul PyroMark Binding Buffer un un High purity water Variable Variable Total volume 60 75 ul 60 75 ul 4 Add the solution prepared in step 2 to a 24 well PCR plate or strips 5 Add 5 20 ul of a well optimized biotinylated PCR product to each well of the PCR plate or strips according to the plate setup see Section 5 2 3 Note The total volume per well should be 80 ul Optional When the amount of PCR product is the same for each reaction prepare a master mix for DNA immobilization according to the table in step 3 with all components except the PCR product PyroMark Q24 Advanced User Manual 06 2013 5 13 Operating Procedures 6 Seal the PCR plate or the strips using adhesive foil or strip caps Ensure that no leakage is possible between the wells 7 Agitate the PCR plate or str
81. ratory waste Be sure to follow the instructions in the handbook supplied with PyroMark Q24 Advanced Reagents and PyroMark Q24 Advanced CpG Reagents Include an empty well Annealing Buffer only in the run setup to check whether background peaks are coming from nucleotides Clean the heating block and light guides see Section 6 2 2 Correct the sequence to analyze and if necessary rerun the samples Follow the recommendations in Appendix B the first time an assay is run Redesign the assay PyroMark Q24 Advanced User Manual 06 2013 7 3 Troubleshooting m Unusually high consumption of substrate noted as a high presequencing signal n Crosstalk light from one well appears in the neighboring well o Dispensation error p Unknown SNP in sample q dUTP used in the PCR reaction Comments and suggestions Prepare samples according to the instructions in Section 5 3 Change buffers and do not use any other buffers than those supplied by QIAGEN Check if any peaks have been generated using the zoom in function select a stretch of Pyrogram with the left mouse button Avoid placing assays with high signals close to assays with low signals Replace the reagent cartridge If the problem remains contact QIAGEN Technical Services Insert the SNP in the sequence to analyze and regenerate the dispensation order Rerun the sample with the new dispensation order Replace dUTP with dTTP since the A
82. rgen 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 PyroMark Q24 Advanced User Manual 06 2013 E 3 Appendix E 1 3 Biologische Sicherheit Wenden Sie beim Umgang mit biologischen Materialien nur sichere Laborverfahren an wie sie z B in Ver ffentlichungen wie Biosafety in Microbiological and Biomedical Laboratories HHS www cdc gov od ohs biosfty biosfty htm beschrieben werden WARNUNG Biologische Materialien W5 Gehen Sie beim Umgang mit biologischen Materialien mit A der gr tm glichen Vorsicht und gem den erforderlichen Sicherheitsbestimmungen vor Tragen Sie immer eine Schutzbrille zwei Paar 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 und die Bediener des Ger ts ausreichend geschult sind Au erdem 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
83. roMark Control Oligo see Section 7 5 Checking the run log It is advisable to check the run log to assess if the system was working properly during the run 1 Open the run file 2 Select Run Information from the Tools menu or right click the file in the shortcut browser and select Run Information from the context menu The Run Information report is opened 3 Check the run log at the end of the report for any problems during the run 4 If deviations from the preset block temperature pressure and or mixer speed values are noticed several times during a run and for longer time periods or in repeated PyroMark Q24 Advanced User Manual 06 2013 7 1 Troubleshooting 7 1 o c d e 7 2 runs please contact QIAGEN Technical Services If requested to send an Environment Dota file m Select Export Environment Data from the Tools menu m Select the destination folder for the data file from the Save in drop down list E Enter the filename in the File name text box and click Save Analysis related errors PCR failed due to low DNA quality Poorly optimized PCR Biotinylation is omitted or not added to the correct PCR primer Biotinylation is of poor quality Insufficient amount of template for immobilization to Sepharose beads Comments and suggestions Check the PCR samples using an agarose gel to confirm there is one strong specific band If no
84. rts Set the date and time as follows 1 When the instrument is not processing select Administration in the main menu 2 Select Set Date and Time using the and screen buttons and press OK 3 Select the parameter you want to edit using the 4 and gt screen buttons 4 Edit the selected parameter using the and screen buttons 5 To edit further parameters repeat steps 3 and 4 6 To save the change s press Set Copying unsaved runs If the USB stick is removed before the run is finished retrieve the run data from the instrument as follows 1 When the instrument is not processing insert one of the USB sticks supplied into the USB port at the front of the instrument 2 Using the and screen buttons select Administration in the main menu and press OK 3 Select Copy Unsaved Runs and press OK 4 Using the and screen buttons select the run file for retrieval and press Select PyroMark Q24 Advanced User Manual 06 2013 5 1 Operating Procedures 5 1 3 5 1 4 5 2 5 When the instrument confirms that the run file has been saved to the USB stick press Close 6 Remove the USB stick Copying recently saved runs Copies of run files are stored on the instrument provided there is enough free space in the internal memory Note When space becomes insufficient the run files are deleted in chronological order Files that have never bee
85. s are working properly proceed to step 6 If a needle is blocked for example if the reagent cartridge was left overnight without cleaning fill the compartments with high purity water and immerse the cartridge in a beaker with enough high purity water to cover the needles Leave the cartridge in the beaker for Th rinse it and repeat step 4 Proceed to step 6 If the jet of water comes out at an angle refill the compartment with water and repeat step 4 If the water still comes out at an angle discard the cartridge 5 26 PyroMark Q24 Advanced User Manual 06 2013 Operating Procedures Cleaning procedure for PyroMark Q24 Cartridge EI Filing cartridge compartments with high purity water Checking for blocked or damaged needles Cleaning blocked needles EI Drying the cartridge on a lint free tissue 6 When all needles have been rinsed and tested discard the water and leave the reagent cartridge to dry on a lint free tissue 7 When the reagent cartridge is dry store the reagent cartridge in a PyroMark Q96 HS Tip Holder Box to protect it from dust and sun light 5 4 6 Analyzing the run Detailed instructions for analyzing the run are available in the PyroMark Q24 Advanced Software User Guide press the F1 key when in PyroMark Q24 Advanced Software 1 Move the processed run file from the USB stick to a computer running PyroMark Q24 Advanced Software 2 Open the run file
86. s are working properly see Section 6 3 2 Replace the corresponding filter probes see Section 6 3 2 PyroMark Q24 Advanced User Manual 06 2013 Troubleshooting Comments and suggestions 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 7 5 Verification of correct installation and operation PyroMark Control Oligo is sold together with the PyroMark Q24 Advanced and is required to verify proper installation and operation of the system The PyroMark Control Oligo consists of one wobbled base measured as C single bases of all four nucleotides and homopolymers of two and three bases For information on how to use the PyroMark Control Oligo see the PyroMark Q24 Validation Oligo Handbook supplied with the product PyroMark Q24 Advanced User Manual 06 2013 7 7 Troubleshooting This page left intentionally blank 7 8 PyroMark Q24 Advanced User Manual 06 2013 Glossary Term AQ Biotin Bisulfite Bisulfite treatment control CpG CpN Cyclic dispensation order Directed dispensation order Glossary Description Analysis mode used for quantification of different alleles 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 beads HSO is referred to as bis
87. s responsibility to validate the performance of such product combination for any particular application The PyroMark Q24 Advanced System is intended for use by professional users trained in molecular biological techniques and the operation of the PyroMark Q24 Advanced System Requirements for PyroMark Q24 Advanced users The table below covers the general level of competence and training necessary for transportation installation use maintenance and servicing of the PyroMark Q24 Advanced PyroMark Q24 Advanced User Manual 06 2013 2 3 Introduction Task Personnel Training and experience Delivery Installation Routine use validation Preventive maintenance Servicing and maintenance No special requirements QIAGEN Field Service Specialists only Laboratory technicians or running protocols equivalent Assay design and Scientist or equivalent Laboratory technicians or equivalent QIAGEN Field Service annual preventive Specialists only No special requirements Appropriately trained and experienced personnel familiar with use of computers and automation in general Appropriately trained and experienced personnel familiar with molecular biological techniques Appropriately trained and experienced personnel familiar with use of computers and automation in general 2 4 PyroMark Q24 Advanced User Manual 06 2013 General Description 3 1 3 2 General Descript
88. t rerun PCR with high quality DNA The PyroMark PCR Kit is recommended for highly specific amplification of bisulfite converted DNA and gDNA from various sources Check the PCR samples using an agarose gel to confirm there is one strong specific band If not reoptimize PCR Check assay design see Appendix B Use a recommended primer supplier Ensure the biotinylated primer is HPLC purified or similar Follow the recommendations for amount of template see Appendix B PyroMark Q24 Advanced User Manual 06 2013 Troubleshooting f Too much PCR product depletes substrate leading to missing peaks at the end of the sequence g One or several of the compartments in the reagent cartridge were not correctly filled h One of the needles in the reagent cartridge is blocked or damaged noted as a missing presequencing signal and or missing peaks in Pyrogram i Reagents incorrectly dissolved or stored j Low signal due to dirty light guides k Incorrect sequence to analyze l Background peaks in Pyrogram Comments and suggestions Use less PCR product Be sure to add sufficient reagents open the run setup and select Pre Run Information from the Tools menu Follow the instructions provided in the handbooks supplied with the products In case of bent needles discard the reagent cartridge Be sure to observe all federal state and local environmental regulations for disposal of labo
89. tallation The unpacking and installation of the PyroMark Q24 Advanced is carried out by a certified QIAGEN Field Service Specialist A person who is familiar with your laboratory and computer equipment should be present during the installation The following items are delivered PyroMark Q24 Instrument including two USB sticks PyroMark Q24 Vacuum Workstation purchased separately PyroMark Q24 Advanced User Manual PyroMark Q24 Advanced Software User Guide PyroMark Q24 Plate Holder Reagents and other accessories can be ordered separately visit www giagen com Requirements Site The PyroMark Q24 Instrument and PyroMark Q24 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 dimensions of the PyroMark Q24 Instrument and PyroMark Q24 Vacuum Workstation PyroMark Q24 Advanced User Manual 06 2013 4 1 Installation Procedures CAUTION A Ensure that the workbench is level dry clean vibration proof and has additional space for accessories Approximately 70 cm 27 in clearance above the workbench is required to accommodate the PyroMark Q24 Instrument with the
90. ter 3 Change the method settings in the dialog box to match those posted on www giagen com support pyromarkparameters aspx 4 Click Save Method parameters In the Instrument Methods dialog box the following parameters are available Reagent pressure Enzyme pulse time Substrate pulse time Nucleotide pressure Nucleotide pulse time Note 5 3 Pressure millibar for dispensation of the enzyme mix and substrate mix Dispensation time milliseconds for the enzyme mix Dispensation time milliseconds for the substrate mix Pressure millibar from the dispensation of nucleotides Dispensation time milliseconds for nucleotides Note about the instrument method optional Sample and reagent preparation Samples to be analyzed using the PyroMark Q24 Advanced should be prepared according to the instructions below PyroMark Q24 Advanced User Manual 06 2013 5 9 Operating Procedures The following equipment and reagents are required for sample preparation All steps are performed at room temperature unless otherwise stated Equipment and reagents to be supplied by the user PyroMark Q24 Vacuum Workstation Plate mixer for immobilization to beads see table below Heating block capable of attaining 80 C PyroMark Q24 Plate 24 well PCR plate or strips see table below Adhesive foil or strip caps PyroMark Q24 Cartridge Streptavidin Sepharose High Performance 34 u
91. ter fittings and empty any liquid waste into a beaker 4 Discard the filter PyroMark Q24 Advanced User Manual 06 2013 Maintenance 5 Push the tubing onto the fittings of the new filter 6 If necessary empty the waste container Note The cap can be removed without disconnecting the tubing 7 Reconnect the vacuum pump to the mains power PyroMark Q24 Advanced User Manual 06 2013 6 9 Maintenance This page intentionally left blank 6 10 PyroMark Q24 Advanced User Manual 06 2013 Troubleshooting 7 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 Services 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 M Photo of error if possible Take the following action before contacting QIAGEN Technical Services 1 Check the run log in the Run Information report to assess if the system was working properly during the run 2 Consult the Troubleshooting sections below 3 Verify proper installation and operation of your system using Py
92. 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 by the DNA 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 PyroMark Q24 Advanced User Manual 06 2013 Glossary Term Signal to noise ratio Single nucleotide polymorphism SNP Streptavidin Substrate Variable position Description 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 A protein that can bind very strongly to biotin A molecule acted upon by an enzyme Pyrosequencing technology uses a mixtur
93. thods Tools Reports Window ft Instrumen Methods The instrument method should be selected according to the reagent cartridge that will be used for the run The code number printed on the PyroMark Q24 Cartridge corresponds to specific parameter settings provided at http www giagen com support pyromarkparameters aspx Important To access instrument methods settings online you must register your instrument with a valid serial number see back of instrument at www giagen com To import a new method 1 Go to the Web site above to download instrument method files for the PyroMark Q24 Instrument 2 Locate the instrument method file that corresponds to the method number printed on the label of the cartridge to be used 3 Save the instrument method file to the computer running the PyroMark Q24 Advanced Software 4 Open the Instrument Methods dialog box from the Tools menu 5 Click the Import button and locate the downloaded instrument method file 6 The downloaded instrument method file will now appear in the Instrument Methods dialog box under Methods PyroMark Q24 Advanced User Manual 06 2013 Operating Procedures provided by user and can be selected in the drop down list when creating a new Run Setup To create a new method 1 In the Instrument Methods dialog box select an existing method and click Save As 2 Enter a name for the new method and press En
94. tion Defines the nucleotides and the order in which they should be dispensed in Pyrosequencing runs A continual decrease in peak 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 Insertion and or deletions A method that describes physical settings for the instrument such as mixer speed block temperature and pulse time settings PyroMark Q24 Advanced User Manual 06 2013 Glossary Term IUPAC Out of phase Polymorphism Presequencing signal Pyrogram Description International Union of Pure and Applied Chemistry An organization providing recommendations on organic and biochemical nomenclature symbols terminology etc IUPAC codes Adenine W TorA C Cytosine S CorG G Guanine B C T or G not A T Thymine D A T or G not C R Purine A or G H A T or C not G Y Pyrimidine C or T V A C or G not T M CorA N Any base A C G or T K TorG When one of the alleles is sequenced ahead of the other Genetic variations broa
95. trument and the PyroMark Q24 Cartridge is filled with PyroMark Q24 Advanced Reagents and placed in the dispensing unit The USB stick containing the run file created using PyroMark Q24 Advanced Software is inserted into the USB port at the front of the instrument The run is then started by the user The dispensing unit pressure mixer speed and temperatures of the heating block process chamber lid and coolant liquid are adjusted to preset levels Enzyme and substrate mixtures are dispensed into the plate s priming well the rectangular well to ensure that the dispensation capillaries are flushed and filled with solution Enzyme mixture and then substrate mixture are dispensed into all wells used The pressure in the dispensing unit is increased Nucleotides are dispensed into the priming well before being dispensed into the wells Nucleotides are added in a predefined order and 65 seconds elapses between the additions of each nucleotide to ensure all enzymatic reactions are completed PyroMark Q24 Advanced User Manual 06 2013 3 3 General Description 8 The instrument collects data simultaneously from all wells using 24 CCDs located under the heating block Data are stored on the instrument 9 After the run data are automatically transferred to the USB stick If the USB stick has been removed during a run data can be retrieved manually from the instrument 3 4 PyroMark Q24 Instrument PyroMark Q24 Instr
96. ty Max 75 noncondensing Storage conditions Air temperature 10 C to 40 C 50 F to 104 F Relative Max 75 noncondensing humidity Mechanical data and hardware features Dimensions Width 390 mm 15 35 in closed Height 420 mm 16 54 in Depth 525 mm 20 67 in Clearance space Width 700 mm 27 56 in Height 700 mm 27 56 in Depth 600 mm 23 62 in Mass 28 kg 61 74 lb Capacity Up to 24 samples per run Chemical pH 4 to pH 9 common detergents 0 5 M sodium resistance hydroxide 70 ethanol A 2 PyroMark Q24 Advanced User Manual 06 2013 Appendix A Operating conditions PyroMark Q24 Vacuum Workstation Power 100 V AC 50 60 Hz or 115 V AC 60 Hz or 230 V AC 50 Hz Power consumption Maximum 25 VA Mains supply voltage fluctuations are not to exceed 10 of nominal supply voltages Overvoltage Il category Air temperature 15 32 C 59 90 F Relative humidity Maximum relative humidity 80 for temperatures up to 31 C decreasing linearly to 50 relative humidity at 40 C non condensing Altitude Up to 2000 m 6500 ft Place of operationFor indoor use only Normal laboratory conditions use adequate ventilation Pollution degree 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 75 noncondensing PyroMark Q24 Advanced User Manual 06 2013 A 3 Appendix A Storage conditions A
97. ue les op rateurs 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 t ACGIH American Conference of Government Industrial Hygienists Etats 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 1 5 Dangers m caniques Quand l appareil PyroMark Q24 Instrument est en marche son couvercle doit rester ferm En cas d ouverture du couvercle un moment inopportun un signal sonore retentit pour avertit du danger PyroMark Q24 Advanced User Manual 06 2013 D 5 Appendix D AVERTISSEMENT A Pi ces mobiles w7 Afin d viter le contact avec les pi ces en mouvement lorsque l appareil PyroMark Q24 Instrument est en marche son couvercle doit r
98. uencing analysis Ensure you use the correct assay type in PyroMark ADSW PCR For PCR amplification we recommend using PyroMark PCR Kit QIAGEN 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 base calling The convenient master mix format enables specific amplification of various starting materials such as genomic DNA from a variety of species as well as bisulfite converted DNA using only one protocol PCR primers One of the primers must be biotin labeled to enable immobilization to streptavidin coated beads Streptavidin Sepharose High Performance GE Healthcare during the preparation of a single stranded DNA template The orientation of the assay can either be forward or reverse The primer that needs to be biotinylated is indicated by the PyroMark ADSW 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 Q24 Advanced User Manual 06 2013 B 1 Appendix B 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 pr
99. ulfite or hydrogen sulfite In the bisulfite reaction DNA is treated with sodium bisulfite to convert cytosine residues to uracil under conditions whereby methylated cytosines remain non reactive Pyrosequencing assays can contain an internal control to assess successful bisulfite treatment C bases that are not followed by G in the sequence are normally not methylated and should therefore be fully converted to T after bisulfite treatment and PCR As a result of successful bisulfite treatment all templates should show only Ts and no Cs in these positions For reverse assays all templates should show only As and no Gs in these positions Analysis mode used for analysis of CoG methylation Analysis capability within CpG mode for analysis of methylation at CpN sites A repetitive dispensation order for nucleotide dispensation Normally used in Pyrosequencing technology for sequencing unknown DNA sequences For example CTGA or TCGA can be used and repeated for the desired number of times Non cyclic order of dispensation that follows the known sequence It can be used in Pyrosequencing technology when you know the sequence to be analyzed For example the sequence TCCAGAA can be analyzed with the dispensation order TCAGA PyroMark Q24 Advanced User Manual 06 2013 8 1 Glossary Term Dispensation order Drop off Enzyme Histogram Homopolymer InDels Instrument methods 8 2 Descrip
100. ument E Instrument lid EI Screen EI Menu buttons El USB port El Power switch EI LED is lit when the cooling device is receiving power USB port inactive EI Light button for the coolant level window E Window showing the coolant level E Instrument power connector 24V El Cooler power connector 12V 3 4 PyroMark Q24 Advanced User Manual 06 2013 General Description 3 4 1 Process chamber Heating block The process chamber contains a heating block that maintains the correct temperature of the plate and its contents If the room temperature is too high the heating block is cooled by the PyroMark Q24 Instrument Cooling Device preinstalled Data are collected from all the wells simultaneously by 24 CCDs underneath the heating block In wells where there is a positive reaction with the added nucleotide light is emitted giving rise to a peak in the Pyrogram To enable rapid mixing of samples and reagents in the plate the heating block inside the process chamber is constantly vibrated during the run 3 4 2 Dispensing unit Dispensing unit A reagent cartridge the PyroMark Q24 Cartridge filled with the required volumes of PyroMark Q24 Advanced Reagents is inserted into the dispensing unit The instrument starts PyroMark Q24 Advanced User Manual 06 2013 3 5 General Description 3 5 3 6 3 6 dispensing reagents when the pressure in the dispensing unit the speed of the mixer and the tem
101. uniquement par des sp cialistes de l entretien sur site QIAGEN Proc der la maintenance comme d crit la Section 6 QIAGEN facture les r parations rendues n cessaires suite une maintenance inappropri e AVERTISSEMENT ATTENTION A Risque d accident corporel et de d t rioration du mat riel W2 L appareil PyroMark Q24 Instrument 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 AVERTISSEMENT ATTENTION Risque d accident corporel et de d t rioration du mat riel W3 Ne pas essayer de d placer le PyroMark Q24 Instrument pendant qu il est en marche PyroMark Q24 Advanced User Manual 06 2013 Appendix D 1 2 S curit lectrique Avant l entretien d brancher les cordons d alimentation des prises de courant AVERTISSEMENT Danger lectrique w4 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 mortelles 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 expose
102. urification 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 qiagen 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 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 contact one of our Technical Service Departments see back cover or visit www giagen 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
103. ve as built in quality controls for the assay Never exclude the blank dispensations as they function as excellent indicators of unspecific nucleotide incorporation When manually generating a dispensation order include an appropriate number of blank dispensations If possible start the dispensation order with one blank dispensation and have at least the same number of blank dispensations as the number of variable positions Pay attention to tips and warnings indicated by the red icon and make suitable modifications when the icon appears PyroMark Q24 Advanced User Manual 06 2013 B 5 Appendix B B 6 Bisulfite treatment controls in CpG assays CpG assays should contain an internal control to assess successful bisulfite treatment preferably at the beginning of the sequence Cytosines not followed by a Guanine are usually not methylated and should be fully converted to Thymine after bisulfite treatment and PCR As a result of successful bisulfite treatment all templates should show only Ts and no Cs in these positions For reverse assays all templates should show As and no Gs in these positions We recommend directly entering the sequence before bisulfite treatment because this enables the software to generate the sequence to analyze and automatically select one appropriate bisulfite treatment control A bisulfite treatment control can be added manually by adding a C before or after a T that was a C before bisulfite treatment

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