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Glossary

9-4

PyroMark Q96 ID User Manual 01/2016

Term

Description

Streptavidin

A protein that can bind very strongly to biotin.

Substrate

A molecule acted upon by an enzyme. Pyrosequencing

technology uses a mixture of the substrates Adenosine 5'
phosphosulfate (APS) and Luciferin in the sequencing

reaction.

Theoretical
outcome

The possible genotypes for a specific polymorphic position
are calculated on the basis of the sequence to analyze and

the dispensation order.

Summary of Contents for PyroMark Q96 ID

Page 1: ...Version 5 January 2016 Sample Assay Technologies PyroMark Q96 ID User Manual...

Page 2: ......

Page 3: ...mation 2 2 2 2 1 Technical assistance 2 2 2 2 2 Policy statement 2 2 2 2 3 Version management 2 2 2 3 Intended use of the PyroMark Q96 ID 2 3 2 3 1 Requirements for PyroMark Q96 ID users 2 4 3 General...

Page 4: ...instrument and computer 4 5 5 Operating Procedures 5 1 5 1 Required items 5 1 5 2 Starting the system 5 1 5 2 1 Starting the PyroMark Q96 ID 5 1 5 2 2 Starting the PyroMark Q96 Software 5 2 5 3 Prepa...

Page 5: ...g the instrument 5 24 5 7 5 Emptying the waste container and troughs 5 24 6 PyroMark Q96 Software 6 1 6 1 System requirements 6 1 6 1 1 Operator s and office computers 6 1 6 1 2 Printer 6 1 6 1 3 Data...

Page 6: ...tation 7 8 7 8 Testing and replacing the filter probes 7 9 7 8 1 Function test for filter probes 7 9 7 8 2 Replacing filter probes 7 10 7 9 Replacing the tubing 7 10 8 Troubleshooting 8 1 8 1 Unexpect...

Page 7: ...Contents PyroMark Q96 ID User Manual 01 2016 Contents 5 Appendix C C 1 Liability clause C 1 Appendix D D 1 Informations de s curit D 1 Appendix E E 1 Sicherheitshinweise E 1 Index Index 1...

Page 8: ...Contents Contents 6 PyroMark Q96 ID User Manual 01 2016 This page intentionally left blank...

Page 9: ...other 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 instrument or other...

Page 10: ...isconnection of the protective conductor terminal is likely to make the instrument dangerous Intentional interruption is prohibited Lethal voltages inside the instrument When the instrument is connect...

Page 11: ...disposal of wastes must be in accordance with all national state and local health and safety regulations and laws OSHA Occupational Safety and Health Administration United States of America ACGIH Ame...

Page 12: ...them when they are hot CAUTION Risk of overheating C1 To ensure proper ventilation maintain a minimum clearance of 10 cm 4 in at the sides and rear of the PyroMark Q96 ID Slits and openings that ensu...

Page 13: ...al and Electronic Equipment WEEE Type plate on the back of the instrument EN FCC mark of the United States Federal Communications Commission Type plate on the back of the instrument EN RCM mark for Au...

Page 14: ...Safety Information 1 6 PyroMark Q96 ID User Manual 01 2016 This page intentionally left blank...

Page 15: ...nd to maintain the instrument in a safe condition A French and German translation of the safety information can be found in Appendices D and E respectively 2 1 About this user manual This user manual...

Page 16: ...ul 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 tec...

Page 17: ...QIAGEN kits indicated for use with the PyroMark Q96 ID Instrument for the applications described in the kit handbooks If the PyroMark Q96 ID Instrument and PyroMark Q96 Vacuum Workstation are used wit...

Page 18: ...pecial requirements No special requirements Installation QIAGEN Field Service Specialists only Routine use running protocols Laboratory technicians or equivalent Appropriately trained and experienced...

Page 19: ...nstrument includes an embedded computer module with hard disk and network interface 3 1 PyroMark Q96 ID definitions PyroMark Q96 ID Instrument Instrument only PyroMark Q96 Software Software only PyroM...

Page 20: ...n 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 incor...

Page 21: ...es is placed in the dispensing unit 2 The run is then started when the user clicks the Run button in the Run Setup dialog box The operator s computer sends all information required for the analysis to...

Page 22: ...ting process chamber is operational throughout the sequencing run 9 The dispensation pressure is increased 10 The nucleotides are predispensed into the rectangular sink well at the right hand edge of...

Page 23: ...anual 01 2016 3 5 3 4 External features of the PyroMark Q96 ID The PyroMark Q96 ID Instrument lid Standby switch Indicator LED Note The PyroMark Q96 ID is delivered with a laptop to serve as the opera...

Page 24: ...mperature of the PyroMark Q96 Plate Low and its contents An integral mixer enables rapid mixing of sample and reagents in the PyroMark Q96 Plate Low 3 7 Dispensing unit Reagents are manually added to...

Page 25: ...ter During the start up sequence All the lights illuminate The instrument software loads takes 1 3 minutes and the Busy light goes off and the Information light starts flashing This indicates that the...

Page 26: ...or s computer The computer used for data analysis should have the following as minimum specifications Microsoft Windows XP 32 bit English version or Microsoft Windows 7 32 bit English version Operatin...

Page 27: ...ing CD ROM 4 2 Requirements Site The PyroMark Q96 ID and PyroMark Q96 Vacuum Workstation must be located out of direct sunlight away from heat sources and away from sources of vibration and electrical...

Page 28: ...Q96 ID must not be covered Power requirements The PyroMark Q96 ID operates at 100 240 V AC 50 60 Hz 1 1 0 45 A Depending on the unit purchased the PyroMark Q96 Vacuum Workstation operates at 100 V AC...

Page 29: ...new location within the same laboratory go to step 4 To move the instrument to another site go to step 7 4 Place the instrument onto a trolley and move to the new location Note The instrument weighs 5...

Page 30: ...k Q96 ID Connect one end of this cable to the network connector on PyroMark Q96 ID net Note The keyboard mouse and monitor connectors are for the use of QIAGEN Field Service Specialists only 2 Connect...

Page 31: ...a unique IP address to the second network adapter for communication with the LAN 4 6 Connecting mains power 1 Ensure the standby switch on the front of the PyroMark Q96 ID is in position press the lo...

Page 32: ...Installation Procedures 4 6 PyroMark Q96 ID User Manual 01 2016 This page intentionally left blank...

Page 33: ...Plate Low High purity water Milli Q 18 2 M x cm or equivalent Powder free gloves Lint free tissues 5 2 Starting the system The PyroMark Q96 ID requires a warm up time of 90 minutes for the CCD camera...

Page 34: ...ou want to place the assay in and select New Assay followed by the desired assay type AQ CpG SNP or SQA from the context menu Note To add a shortcut to a folder or drive click Add Folder Shortcut 2 Cl...

Page 35: ...he analysis parameters 5 3 2 Setting up a run 1 In the shortcut browser right click the folder you want to place the run file in and select New Run from the context menu Note To add a shortcut to a fo...

Page 36: ...k 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 5 3 3 Managing instrument parameters In...

Page 37: ...ent parameters for the PyroMark Q96 ID found under the User Support tab In the provided table find the dispensing pressures and pulse time settings that correspond to the Code number printed on the Py...

Page 38: ...nsation of nucleotides Nucleotide pulse time Dispensation time milliseconds for nucleotides Note Note about the instrument method optional 5 4 Sample preparation Equipment and reagents to be supplied...

Page 39: ...angled blue connector on the cap of the liquid waste container Attach the other end to the connector on the back of the PyroMark Q96 Vacuum Workstation 4 With a third piece of tubing attach the Vacuum...

Page 40: ...r of the Streptavidin Sepharose HP For lot number 10057037 or higher use 1 5 l For lot numbers lower than 10057037 use 3 l The volume of water depends on the amount of PCR product used Allow all reage...

Page 41: ...e next section 5 4 4 Strand separation WARNING Hazardous chemicals W4 The Denaturation Solution used with the vacuum workstation contains sodium hydroxide which is irritating to eyes and skin Always w...

Page 42: ...ould be replaced after preparation of approximately 100 plates or when needed Note Check and empty the waste container if necessary see Section 5 7 5 2 Fill five separate troughs supplied with the Pyr...

Page 43: ...r is being transferred to the waste container If it is not ensure the tubing is connected correctly and is not broken Broken tubing should be replaced see Section 7 9 Ensure that the tubing is connect...

Page 44: ...mmobilized template Hold the probes in place for 15 s Take care when picking up the tool 12 Ensure that all liquid is aspirated from the wells and that all beads have been captured onto the filter pro...

Page 45: ...te the tool for 10 s 20 Wash the filter probes by lowering the probes into high purity water Parking Position and applying vacuum Flush the probes with 180 ml high purity water 21 Raise the tool to be...

Page 46: ...ed with the PyroMark Gold Q96 Reagents 2 Open a reagent cartridge pack and take out a new cartridge The cartridge has to be handled with care in order to maintain accuracy 3 Remove the plastic needle...

Page 47: ...leotides PyroMark Q96 Cartridge from above with indicated compartments for enzymes substrate and nucleotides 5 5 Run 5 5 1 Loading the PyroMark Q96 Plate Low 1 Open the instrument lid Note The dispens...

Page 48: ...n the dispensing unit cover Release the latch and then open the cover 2 Insert the filled reagent cartridge into position It will only fit in one orientation the label should be facing the user 3 Clos...

Page 49: ...following information regarding the progress of a run Last nucleotide or reagent dispensation to each well Well specific information Instrument specific information in the Instrument Status Monitor A...

Page 50: ...If the connection to the instrument is lost during a run a message box will appear to inform the user Upon clicking OK the Run view will close but the instrument will continue to process the run The...

Page 51: ...current analysis mode or click the Analyze all Wells icon to analyze all wells Analyze all wells Analyze the selected wells only 5 6 2 Viewing the analysis results By selecting an analyzed light blue...

Page 52: ...sults The following well information can be viewed in the plate overview in the Overview tab AQ and SNP assays Select to show the assay name Select to show the sample ID Select to show the well note S...

Page 53: ...to display the quality assessment of all variable positions in a selected well or of all bases in the base called sequence SQA assays also include quality assessment at the end of the quality control...

Page 54: ...Setup tab Light green Below the expected range Green Within the expected range Dark green Above the expected range SQA analysis results The base called sequence is displayed in the Overview tab above...

Page 55: ...les with care Small particles and fibers may obstruct the needles Note To avoid injury replace the plastic needle cover on the reagent cartridge prior to disposal Note Be sure to observe all federal s...

Page 56: ...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 surr...

Page 57: ...nt 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...

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Page 59: ...his is required if the computer used to analyze the data is not connected to a LAN 6 2 Installing PyroMark Q96 Software Note If the computer is connected to a network network policy settings may preve...

Page 60: ...oft Windows 7 click Programs and then Programs and Features 3 In the list of programs select PyroMark Q96 4 Click Remove XP or Uninstall 7 6 4 Upgrading from PyroMark Q96 ID Software Previous versions...

Page 61: ...ool The PyroMark Data Converter tool converts Pyrosequencing runs set up and completed with the old PyroMark Q96 ID Software into a file format that is compatible with the new PyroMark Q96 Software Th...

Page 62: ...OK button to connect to the specified database Note Click Check Connection to determine if a connection is possible with the information entered Note The database connection can be disconnected by cl...

Page 63: ...he export will be displayed in the progress bar 6 5 Data backup Good data management requires that data backups are made on a regular basis Note QIAGEN is not responsible for the setup and administrat...

Page 64: ...o the PyroMark Q96 ID This takes 1 3 min The yellow Busy light on the instrument will then go out and the yellow Information light will start to flash The instrument is now ready to receive instructio...

Page 65: ...96 Plate Low on the heating block 13 Close the process chamber lid 14 Open the dispensing unit cover and insert an empty reagent cartridge 15 Close the dispensing unit cover and press the latch down i...

Page 66: ...The run progress bar displays Run and dispensation of the nucleotides begins 21 Function test Open the instrument lid There will be an audible warning signal and the yellow Information light will illu...

Page 67: ...t lasts for 1 year beginning on the date of shipment The warranty includes all repairs due to mechanical breakdown Application development software upgrades accessories and disposable items are not in...

Page 68: ...layed 5 Turn off the standby switch on the front of PyroMark Q96 ID and check that the Power indicator lamp is turned off 7 2 Routine maintenance of the PyroMark Q96 ID The PyroMark Q96 ID should be c...

Page 69: ...5 Carefully clean the lens array with a cloth moistened with 70 ethanol 6 Let the lens array dry before remounting the heating block 7 Place the heating block on top of the lens array Ensure that the...

Page 70: ...the standby switch at the front of the instrument and disconnect the mains power cable 2 If necessary carefully turn the instrument so that the rear panel can be easily accessed 3 Use a small screwdr...

Page 71: ...i Q 18 2 M x cm or equivalent Clean lint free cloths There are two possible cleaning procedures A standard cleaning procedure Section 7 6 1 and a cleaning procedure for clogged cartridges Section 7 6...

Page 72: ...ents with high purity water and immerse the cartridge in a beaker with high purity water covering the needles Leave the cartridge in the beaker for 1 hour 4 Proceed with the standard cleaning procedur...

Page 73: ...ter a cartridge has passed the function test allow it to dry completely at ambient temperature 15 25 C before using Cleaning the PyroMark Q96 Cartridge A Filling the compartments with high purity wate...

Page 74: ...ined 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...

Page 75: ...using the PyroMark Q96 Vacuum Workstation check that the filter probes are working by performing the function test as follows 1 Add 100 l high purity water to each well of a 96 well PCR plate 2 Fill...

Page 76: ...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...

Page 77: ...e 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 v...

Page 78: ...Maintenance 7 12 PyroMark Q96 ID User Manual 01 2016 This page intentionally left blank...

Page 79: ...ror Photo of error if possible Take the following action before contacting QIAGEN Technical Services 1 Check important parameters in the Run Overview in the Instrument Status tab to assess if the syst...

Page 80: ...fficient enzyme or substrate for all the wells Check the reagent cartridge was filled according to the Volume Information under the Run Info tab 8 2 Sequence errors Comments and suggestions a Some pea...

Page 81: ...sequencing primer added Wrong sequence to analyze entered Wrong dispensation order chosen 8 3 Peak errors Comments and suggestions a Wide peaks Assay specific problem due to secondary structure Conta...

Page 82: ...ning the PCR and or sequencing primer s Contaminated nucleotides Background of contaminating sequence from PCR Consider redesigning sequencing primer Contact QIAGEN Technical Services if this problem...

Page 83: ...suggestions a No contact between the operator s computer and the PyroMark Q96 ID Check mains power supply to instrument and ensure the Power light is illuminated Check the network cables are properly...

Page 84: ...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 leaka...

Page 85: ...Pyrogram Enzyme 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 lucif...

Page 86: ...C G or T Out of phase When one of the alleles is sequenced ahead of the other Polymorphism Genetic variations broadly encompassing any of the many types of variations in DNA sequences that are found...

Page 87: ...template during the sample preparation The 3 end of the sequencing primer serves as the starting point for the extension by the polymerase Shift Plus shift A small proportion of the template sequences...

Page 88: ...rate A molecule acted upon by an enzyme Pyrosequencing technology uses a mixture of the substrates Adenosine 5 phosphosulfate APS and Luciferin in the sequencing reaction Theoretical outcome The possi...

Page 89: ...n 280 VA max Overvoltage category II Air temperature 18 28 C 64 82 F Relative humidity 30 90 relative humidity noncondensing Altitude Up to 2000 m 6500 ft Place of operation For indoor use only Draft...

Page 90: ...hardware features PyroMark Q96 ID Dimensions closed Width 520 mm 20 47 in Height 490 mm 19 29 in Depth 620 mm 24 41 in Clearance space Width 540 mm 21 26 in Height 570 mm 22 44 in Depth 640 mm 25 20...

Page 91: ...Microsoft Windows 7 32 bit English version Processor Intel Core Duo processor 2 GHz or higher RAM 1 GB Free hard disk space 200 MB Graphics card Supporting the resolution of the monitor Monitor 1280 x...

Page 92: ...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...

Page 93: ...ply with the limits for a Class B digital device pursuant to part 15 of the FCC Rules These limits are designed to provide reasonable protection against harmful interference in a residential installat...

Page 94: ...s FCC Compliance Tests were conducted using QIAGEN supported peripheral devices and shielded cables such as those you receive with your system Changes or modifications not expressly approved by QIAGEN...

Page 95: ...er Manual 01 2016 A 7 Declaration of Conformity Name and address of the legal manufacturer QIAGEN GmbH QIAGEN Strasse 1 40724 Hilden Germany An up to date Declaration of Conformity can be requested fr...

Page 96: ...Appendix A A 8 PyroMark Q96 ID User Manual 01 2016 This page intentionally left blank...

Page 97: ...encing applications such as mutation detection SNP analysis methylation analysis and de novo sequencing The convenient master mix format enables specific amplification of various starting materials su...

Page 98: ...ent yield of template DNA and interfere with subsequent Pyrosequencing reactions due to the presence of unused biotinylated primer The PCR product should give one strong band with minimal excess of pr...

Page 99: ...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 on...

Page 100: ...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 Contro...

Page 101: ...CpG assays When creating an AQ or CpG assay the sequence to analyze should contain a sufficient number of bases to generate at least five nonvariable reference peaks For InDels a few reference peaks s...

Page 102: ...s converted to Ts read as Gs and As in a reverse assay and suitable as controls or not The preferable controls are those dispensations that are located at the beginning of the sequence and or represen...

Page 103: ...s interact to give a background signal in Pyrosequencing reactions PCR with template DNA but with no sequencing primer This will show if the template can loop back on itself and give a background sign...

Page 104: ...Analysis results For samples and positive controls aim for Sufficient signal intensities Aim for a single peak height of at least 20 RLU No background in blank dispensations No background in variable...

Page 105: ...ly 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 i...

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Page 107: ...e s curit suivants sont fournis tout au long du manuel AVERTISSEMENT Le terme AVERTISSEMENT signale des situations risquant d entra ner des accidents corporels dont l utilisateur ou d autres personnes...

Page 108: ...l qualifi ayant t convenablement form L entretien du PyroMark Q96 ID doit tre effectu uniquement par des sp cialistes de l entretien sur site QIAGEN Proc der la maintenance comme d crit la Section 7 Q...

Page 109: ...les bornes peuvent tre sous tension et l ouverture de capots ou le retrait de pi ces risque d exposer des l ments sous tension Afin que le PyroMark Q96 ID fonctionne de mani re satisfaisante et en to...

Page 110: ...ces 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 l ACGIH ou du COSHH applicables Pour plus d informations voir l...

Page 111: ...capot Ils ne renferment aucune pi ce r parable par l utilisateur En cas de probl me avec l appareil PyroMark Q96 ID contacter imm diatement les Services techniques de QIAGEN AVERTISSEMENT Risque de p...

Page 112: ...moins 10 cm sur les c t s et l arri re de l appareil PyroMark Q96 ID Les fentes et les ouvertures qui assurent la ventilation de l appareil ne doivent pas tre obstru es 1 6 Symboles sur le PyroMark Q9...

Page 113: ...Plaque signal tique l arri re de l appareil EN Label FCC de la Federal Communications Commission des tats Unis Plaque signal tique l arri re de l appareil EN Label RCM pour l Australie identifiant du...

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Page 115: ...folgenden beiden Kategorien von Sicherheitshinweisen verwendet WARNUNG Der Begriff WARNUNG WARNING weist Sie auf Situationen hin in denen eine Verletzungsgefahr f r Sie selbst oder andere Personen bes...

Page 116: ...e Instandhaltung des PyroMark Q96 ID darf nur durch einen Servicespezialisten des QIAGEN Au endiensts durchgef hrt werden F hren Sie alle Wartungsarbeiten gem den Anweisungen in Abschnitt 7 dieses Han...

Page 117: ...nd die Anschlussstellen spannungsf hrend Durch das ffnen der Abdeckungen oder das Entfernen von Geh useteilen k nnen spannungsf hrende Komponenten freigelegt werden Um einen zufriedenstellenden und si...

Page 118: ...zwerte 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 Informat...

Page 119: ...en 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...

Page 120: ...mit eine ausreichende Bel ftung des Ger ts gew hrleistet ist Die L ftungsschlitze und ffnungen die f r die Be und Entl ftung des PyroMark Q96 ID bestimmt sind d rfen nicht verdeckt werden 1 6 Symbole...

Page 121: ...N FCC Kennzeichen der Federal Communications Commission der Vereinigten Staaten Typenschild auf der Ger ter ckseite EN RCM Mark Zeichen f r Australien Herstellerkennung N17965 Typenschild auf der Ger...

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Page 123: ...oving the instrument 4 3 power requirements 4 2 site requirements 4 1 Intended use 2 3 L Liability clause 1 M Maintenance changing the fuses 7 4 cleaning the cartridge 7 5 cleaning the heating block 7...

Page 124: ...eise 1 Software 6 1 data backup 6 5 installation 6 1 installation testing 6 5 system requirements 6 1 upgrading software 6 2 Starting template 2 Strand separation 5 9 T Technical assistance 2 2 Techni...

Page 125: ......

Page 126: ...1100446 01 2016 Sample Assay Technologies...

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