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Qiagen PyroMark Q24 MDx, User Manual

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Glossary 

8-2

    

                  

PyroMark Q24 MDx User Manual    01/2016

 

Term 

Description 

Drop off 

A continual  decrease in peak height normally seen in the 

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, luciferase, and apyrase is used in 

the sequencing  reaction. 

Histogram 

The theoretical representation  of the expected 

Pyrosequencing peak pattern. 

Homopolymer 

A stretch of identical  bases in DNA. In Pyrosequencing 

technology, a stretch of more than two identical bases is 
regarded as a homopolymer. 

InDels 

Insertion and/or deletions. 

Instrument 

methods 

A method that describes physical settings for the 

instrument, such as mixer speed, block  temperature, and 
pulse time settings. 

Summary of Contents for PyroMark Q24 MDx

Page 1: ...Version 1 January 2016 Sample Assay Technologies PyroMark Q24 MDx User Manual IVD MAT 1063078EN QIAGEN GmbH QIAGEN Strasse 1 40724 Hilden GERMANY R4 ...

Page 2: ...ems Incorporated Intel Pentium Intel Corporation Microsoft Windows Microsoft Corporation Millex Millipore Milli Q Merck KGaA Sepharose GE Healthcare Registered names trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law HB 0332 004 2011 2016 QIAGEN all rights reserved ...

Page 3: ...rk 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 MDx 2 3 2 3 1 Requirements for PyroMark Q24 MDx users 2 3 3 General Description 3 1 3 1 PyroMark Q24 MDx definitions 3 1 3 2 Pyrosequencing principle 3 1 3 3 PyroMark Q24 MDx princip...

Page 4: ...tration 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 MDx Software 5 4 5 2 2 ...

Page 5: ... 5 5 8 Analysis reports 5 27 5 6 Finishing work and shutting down 5 28 5 6 1 Shutting down the instrument 5 28 5 6 2 Emptying the waste container and troughs 5 29 5 6 3 Checking the instrument 5 30 5 7 Backup of PyroMark Q24 files 5 30 6 Maintenance 6 1 6 1 Checking the performance of the PyroMark Q24 MDx 6 1 6 2 Maintenance of the PyroMark Q24 MDx Instrument 6 2 6 2 1 Cleaning the instrument 6 2 ...

Page 6: ...7 5 7 4 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 EC declaration of conformity A 7 Appendix B B 8 Assay design and validation B 8 Appendix C C 1 Liability clause C 1 Index Index 1 ...

Page 7: ...iven 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 inju...

Page 8: ...nductor earth ground 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 parts is likely to expose live parts To ensure satisfactory and safe ...

Page 9: ...ry 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 Material Safety Data Sheets MSDSs or OSHA ACGIH or COSHH documents For more information visit www qiagen com support msds aspx Venting for fumes and disposal of wastes must b...

Page 10: ...isposal of wastes must be in accordance with all national state and local health and safety regulations and laws OSHA Occupational Safety and Health Administration United States of America ACGIH American Conference of Government Industrial Hygienists United States of America COSHH Control of Substances Hazardous to Health United Kingdom 1 5 Mechanical hazards The lid of the PyroMark Q24 MDx Instru...

Page 11: ... paper tissues 1 6 Heat hazard WARNING Hot surface 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 CAUTION Risk of overheating To ensure proper ventilation maintain a minimum clearance of 10 cm 3 94 in at the sides and rear of the PyroMark Q24 MDx Instrument Slits and openings that ensure the ventilation of the P...

Page 12: ...ck 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 Australia supplier identification N17965 Type plate on the back of the instrument EN RoHS mark for China the restriction of the use of certain hazardous substances in electrical and electronic equipment Type plate on the back of the instrument EN Waste El...

Page 13: ...ns EN Contains reagents for N tests Reagents solutions cartridge plate EN Catalog number Reagents solutions cartridge plate EN Material number Reagents solutions cartridge plate EN Component Reagents solutions cartridge plate EN Contains Reagents solutions cartridge plate EN Number Reagents solutions cartridge plate EN Use by date Reagents solutions cartridge plate EN Temperature limitation ...

Page 14: ...ion Instrument VPW reagents cartridge EN Read the manual Type plate on the back of the instrument and all other products EN Legal manufacturer Cartridge EN Keep away from sun light Cartridge EN Method number Cartridge EN Instrument parameters Inside the instrument EN Warning consult user manual ...

Page 15: ...information in the user manual must be followed to ensure safe operation of the system and to maintain the system in a safe condition 2 1 About this user manual This user manual provides information about the PyroMark Q24 MDx in the following sections 1 Safety Information 2 Introduction 3 General Description 4 Installation Procedures 5 Operating Procedures 6 Maintenance Procedures 7 Troubleshootin...

Page 16: ... 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 Technical Support Center at www qiagen com Support or call one of the QIAGEN Technical Service Departments or local distributors see back co...

Page 17: ... in combination with QIAGEN kits indicated for use with the PyroMark Q24 MDx Instrument for the applications described in the kit handbooks The PyroMark Q24 MDx System is intended for use by professional users such as technicians and physicians trained in molecular biological techniques and the operation of the PyroMark Q24 MDx System 2 3 1 Requirements for PyroMark Q24 MDx users The table below c...

Page 18: ...trained and experienced personnel familiar with use of computers and automation in general Assay design and validation Scientist or equivalent Appropriately trained and experienced personnel familiar with molecular biological techniques Preventive maintenance Laboratory technicians or equivalent Appropriately trained and experienced personnel familiar with use of computers and automation in genera...

Page 19: ...ftware only PyroMark Q24 MDx Vacuum Workstation Vacuum Workstation only PyroMark Q24 MDx System All of the above plus any PyroMark Kits 3 2 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 substr...

Page 20: ...ight signal is proportional to the number of nucleotides incorporated 7 Apyrase a nucleotide degrading enzyme continuously degrades unincorporated nucleotides and ATP When degradation is complete another nucleotide is added 8 Nucleotides are added one at a time Note Deoxyadenosine alfa thio triphosphate dATPαS is used instead of natural deoxyadenosine triphosphate dATP since it is used efficiently...

Page 21: ...is then started by the user 3 The dispensing unit pressure mixer speed and temperatures of the heating block process chamber lid and coolant liquid are adjusted to preset levels 4 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 5 Enzyme mixture and then substrate mixture ...

Page 22: ...If the USB stick has been removed during a run data can be retrieved manually from the instrument 3 4 PyroMark Q24 MDx Instrument Instrument lid Screen Menu buttons USB port Power switch LED is lit when the cooling device is receiving power USB port inactive Light button for the coolant level window Window showing the coolant level Instrument power connector 24V Cooler power connector 12V PyroMark...

Page 23: ...lected 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 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 cartrid...

Page 24: ...then immobilized on streptavidin coated Sepharose beads Samples to be analyzed using the PyroMark Q24 MDx Instrument should be prepared according to the instructions in Section 5 3 using the PyroMark Q24 MDx Vacuum Workstation 3 6 Analysis software The PyroMark Q24 MDx is shipped with PyroMark Q24 MDx Software The computer used for setup of runs and data analysis should have the following as minim...

Page 25: ...PyroMark Q24 MDx Software User Guide PyroMarkQ24 Plate Holder Reagents and other accessories can be ordered separately visit www qiagen com products PyromarkQ24MDx aspx 4 2 Requirements Site The PyroMark Q24 MDx Instrument and PyroMark Q24 MDx Vacuum Workstation must be located out of direct sunlight away from heat sources and away from sources of vibration and electrical interference Refer to App...

Page 26: ...ulated and surge protected Note We recommended plugging the instrument directly into its own power outlets and not to share the power outlets with other lab equipment Do not place PyroMark Q24 MDx on a vibrating surface or near vibrating objects CAUTION Risk of overheating To ensure proper ventilation maintain a minimum clearance of 10 cm at the sides and rear of the PyroMark Q24 MDx Instrument Sl...

Page 27: ...ed to a network network policy settings may prevent you from completing this procedure For more information contact your system administrator 1 Ensure that the computer meets the minimum requirements see Section 3 6 2 Close any programs running on the computer 3 Insert the PyroMark Q24 MDx Software CD into the CD drive 4 In the CD menu click Install PyroMark Q24 MDx Software If the CD menu does no...

Page 28: ...cal updates to the NET Framework 3 5 To view reports generated by PyroMark Q24 MDx Software in PDF format a PDF reader must be installed on the computer Adobe Reader can be downloaded at www adobe com 4 3 2 Uninstalling PyroMark Q24 MDx Software 1 Select Control Panel in the Windows Start menu 2 In the Control Panel click Uninstall a program under the Programs category 3 In the list of programs se...

Page 29: ... 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 and 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 5 1 2 Copying unsaved runs If the USB stick is removed before the run is finished retrieve the run dat...

Page 30: ...t 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 5 1 4 Copying log files If you need to sen...

Page 31: ...nformation 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 5 1 7 Upgrading the instrument software If you have received a software upgrade from QIAGEN upgrade the software as follows 1 Save the upgrade files on one of the USB sticks...

Page 32: ...e PyroMark Q24 MDx Software User Guide can be accessed at any time by pressing the F1 key when in the software 5 2 2 Setting up an assay 1 In the shortcut browser right click the folder you want to place the assay file in and select New Assay followed by the desired assay type AQ CpG or SQA from the context menu Note To add a shortcut to a folder in the shortcut browser click Add Folder Shortcut 2...

Page 33: ...le as controls or not To add a control left click a bold orange T or A in the histogram preferably at the beginning of the sequence 5 2 3 Setting up a run 1 In the shortcut browser right click the folder you want to place the run file in and select New Run from the context menu Note To add a shortcut to a folder in the shortcut browser click Add Folder Shortcut 2 Enter the file name and press Ente...

Page 34: ...n now be processed by inserting the USB stick into the USB port at the front of the PyroMark Q24 MDx Instrument see Section 5 5 Optional If desired enter the Reagent ID i e the lot number for PyroMark Gold Q24 Reagents a Plate ID a Barcode for the plate and a Run Note in the run file Further information There are several ways to set up a plate For example it is possible to import and paste a sampl...

Page 35: ...en com Products PyroMarkQ24MDx aspx Note It is recommended that only methods supplied by QIAGEN are used To import a new method 1 From the Web site above download the method file corresponding to the method number printed on the cartridge label Save it to the computer running the PyroMark Q24 MDx Software 2 In the Instrument Methods dialog box click Import The Find Instrument Method dialog box ope...

Page 36: ...lse time Dispensation time milliseconds for nucleotides Note Note about the instrument method optional 5 3 Sample preparation Samples to be analyzed using the PyroMark Q24 MDx Instrument should be prepared according to the instructions below The following equipment and reagents are required for sample preparation All reagents and solutions should be at room temperature 15 25 C before starting All ...

Page 37: ...ly by performing the function test as follows 1 Add 100 µl high purity water to each well of a 24 well PCR plate 2 Fill a trough with 70 ml high purity water 3 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...

Page 38: ...ng the PCR product to be added in step 4 The amount of water depends on the amount of PCR product used For example If using 15 µl of PCR product 2 µl of beads and 40 µl of Binding Buffer 23 µl of high purity water must be added 3 Add the solution prepared in step 2 to a 24 well PCR plate or strips 4 Add 5 20 µl of a well optimized biotinylated PCR product to each well of the PCR plate or strips ac...

Page 39: ...safe and that the operators are not exposed to hazardous levels of toxic substances chemical or biological as defined in the applicable Material Safety Data Sheets MSDSs or OSHA ACGIH or COSHH documents For more information visit www qiagen com support msds aspx Venting for fumes and disposal of wastes must be in accordance with all national state and local health and safety regulations and laws O...

Page 40: ... be replaced after preparation of approximately 100 plates Note Empty the waste container if necessary see Section 5 6 2 2 Fill five separate troughs supplied with the PyroMark Q24 MDx Vacuum Workstation as follows Approximately 50 ml ethanol 70 1 Approximately 40 ml Denaturation Solution 2 Approximately 50 ml 1x Wash Buffer 3 Approximately 50 ml high purity water 4 Approximately 70 ml high purity...

Page 41: ... 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 position 7 Refill trough 5 with 70 ml high purity water 8 Dilute the sequencing primer to 0 3 µM in Annealing Buffer Add 25 µl of the sol...

Page 42: ...e beads sediment quickly If more than 1 min has elapsed since the plate or strips was agitated agitate again for 1 min before capturing the beads 12 Ensure that all liquid is aspirated from the wells and that all beads have been captured onto the filter probe tips Note If the wells still contain liquid or white beads remain the filter probes may need replacing see Section 6 3 2 13 Transfer the too...

Page 43: ...the probes into the second trough of high purity water and applying vacuum Flush the filter probes with 70 ml high purity water 21 Raise the tool to beyond 90 vertical for 5 s to drain liquid from the filter probes 22 Close the vacuum switch on the tool Off 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 ...

Page 44: ...rp needles 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 Open the PyroMark Gold Q24 Reagents box and remove the vials containing freeze dried enzyme and substrate mixtures and the tubes containing nucleotides 2 Reconstitute the volumes of reagents required and fill PyroMark Q24 Cartridge acco...

Page 45: ...ert you if the lid is opened when it is not safe WARNING Moving parts To avoid contact with moving parts during operation of the PyroMark Q24 MDx 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 MDx Instrument contact QIAGEN Technical Services immediately WARNIN...

Page 46: ...ell 3 Current time 4 Instrument status 5 Pyrogram 6 Warning messages 7 Estimated remaining run time hh mm 5 5 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...

Page 47: ...operly 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 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 sel...

Page 48: ... 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 an...

Page 49: ...three latest warnings are displayed For suggested actions see Section 7 3 Abort the run To abort the run press Stop 5 5 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 3 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...

Page 50: ...observe all national state and local environmental regulations for the disposal of laboratory waste The following items are required Powder free gloves High purity water Milli Q 18 2 MΩ 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 Discard any solutions remaining in cartridge 2 Rinse the cartridge compartmen...

Page 51: ...ridge in the beaker for 1 h rinse it and repeat step 4 Proceed to step 6 5b 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 Cleaning procedure for PyroMark Q24 Cartridge Filling cartridge compartments with high purity water Checking for blocked or damaged needles Cleaning blocked needles Dr...

Page 52: ...al assay types are included select analysis mode in the dialog box that opens To add a shortcut to a file or folder in the shortcut browser click Add File Shortcut or Add Folder Shortcut 3 In the Overview tab either analyze all wells or a selection of wells with a valid analysis setup for the current analysis mode Analysis modes PyroMark Q24 MDx Software has three analysis modes AQ CpG and SQA To ...

Page 53: ...area and the well information including analysis warnings is listed in the Well Information area Quality assessments The plate overview in the Overview tab gives a quick overview of the quality assessments Shows the quality assessment of all variable positions in the well or of all the bases in the base called sequence Shows the quality assessment at the end of the quality control window SQA assay...

Page 54: ... by the background color of the result 2 1 4 1 3 E E S S G G T T C C G G A A 5 C C T T A A T T G G 10 T T C C G G A A T T 15 T T G G A A T T C C 20 A A G G T T C C G G 25 T T A A T T G G T T 30 C C G G T T A A 0 100 200 300 400 A5 YGGATAGYGATTTTTAAYGYGTAAGYGTATA Example Pyrogram for a CpG assay Variable positions in AQ and CpG assays are highlighted with a blue gray background color and bisulfite ...

Page 55: ...nce 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 Example of a base called sequence and Pyrogram for a SQA assay 5 5 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 R...

Page 56: ... downloaded at www adobe com 5 6 Finishing work and shutting down 5 6 1 Shutting down the instrument 1 When the instrument is not processing using the and 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 ...

Page 57: ...nting for fumes and disposal of wastes must be in accordance with all national state and local health and safety regulations and laws OSHA Occupational Safety and Health Administration United States of America ACGIH American Conference of Government Industrial Hygienists United States of America COSHH Control of Substances Hazardous to Health United Kingdom Be sure to observe all national state an...

Page 58: ...e below and check that the coolant level is visible in the window If it is not contact QIAGEN Technical Services 5 7 Backup of PyroMark Q24 files The data generated by PyroMark Q24 MDx Software is stored on the computer as files with the following suffixes pyrorun run files pyrosetup assay files Data backup should be performed frequently This can be done by copying PyroMark Q24 files pyrorun and p...

Page 59: ...ensions 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 distributo...

Page 60: ... 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 C...

Page 61: ...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 ...

Page 62: ... 6 4 PyroMark Q24 MDx User Manual 01 2016 CAUTION Light guide maintenance Use lint free tissues to clean the space between the heating block and the light guide block inside the instrument Do not use paper tissues ...

Page 63: ... Data Sheets MSDSs or OSHA ACGIH or COSHH documents For more information visit www qiagen com support msds aspx Venting for fumes and disposal of wastes must be in accordance with all national state and local health and safety regulations and laws OSHA Occupational Safety and Health Administration United States of America ACGIH American Conference of Government Industrial Hygienists United States ...

Page 64: ...er probes dry using a clean lint free cloth 5 Reconnect the vacuum pump to the mains power 6 3 2 Testing and replacing the filter probes Function test for filter probes The function test for the filter probes is described in Section 5 3 1 Replacing filter probes Each filter probe can be replaced individually To ensure proper flow rate trough the filter probes all probes should be replaced after pr...

Page 65: ...ied with the system 5 Pull 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 6 3 3 Replacing the rubber seal If the filter probes are loose and or fall out there are two possible causes The four screws are not tight enough The rubber seal needs to be replaced If the rubber seal n...

Page 66: ...6 Remove the metal plate and replace the rubber seal 7 Reassemble the tool and reconnect the vacuum pump to the mains power 8 Check that the filter probes are functioning properly by performing the function test as described in Section 5 3 1 6 3 4 Replacing the tubing If the tubing is broken or distorted replace it Be sure to observe all national state and local environmental regulations for the d...

Page 67: ...erve all national state and local environmental regulations for the disposal of laboratory waste The following items are required New waste filter Beaker Note Two waste filters are supplied with the vacuum workstation Filters can be ordered at www millipore com Millipore Millex FG50 Filter Unit cat no SLFG05010 Procedure 1 Ensure that no vacuum is applied to the vacuum prep tool i e the vacuum swi...

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Page 69: ...lowing 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 PyroMark Control Oligo Checking the run log It is advisable to check the run log to assess if the system was working proper...

Page 70: ...ne strong specific band If not rerun PCR with high quality DNA The PyroMark PCR Kit is recommended for highly specific amplification of bisulfite converted DNA and genomic DNA from various sources b Poorly optimized PCR Check the PCR samples using an agarose gel to confirm there is one strong specific band If not reoptimize PCR c Biotinylation is omitted or not added to the correct PCR primer Chec...

Page 71: ...al and or missing peaks in Pyrogram In case of bent needles discard the reagent cartridge Be sure to observe all national state and local environmental regulations for disposal of laboratory waste i Reagents incorrectly dissolved or stored Be sure to follow the instructions in the handbook supplied with PyroMark Gold Q24 Reagents Include an empty well Annealing Buffer only in the run setup to chec...

Page 72: ...als close to assays with low signals o Dispensation error Replace the reagent cartridge If the problem remains contact QIAGEN Technical Services p Unknown SNP in sample Insert the SNP in the sequence to analyze and regenerate the dispensation order Rerun the sample with the new dispensation order q dUTP used in the PCR reaction Replace dUTP with dTTP since the A nucleotide used in Pyrosequencing r...

Page 73: ...he rear is lit If not check your connections Check the coolant level c Run name is invalid Ensure that the run file is created in PyroMark Q24 MDx Software d Could not copy file to USB memory Try another USB stick We recommend using the USB sticks supplied by QIAGEN e Failed to connect to the hardware the connection to the hardware is lost please restart the instrument Restart the instrument If th...

Page 74: ...a No vacuum is received 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 b Vacuum lost during sample preparation Ensure that the tubing is conne...

Page 75: ...5 Verification of correct installation and operation PyroMark Control Oligo is sold together with the PyroMark Q24 MDx and 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 th...

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Page 77: ... 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 CpG Analysis mode used for analysis of CpG methylation Cyclic dispensation order A repetitive dispensation order for nucleotide dispe...

Page 78: ... Klenow polymerase sulfurylase luciferase and apyrase is used in the sequencing reaction Histogram The theoretical representation of the expected Pyrosequencing peak pattern Homopolymer A stretch of identical bases in DNA In Pyrosequencing technology a stretch of more than two identical bases is regarded as a homopolymer InDels Insertion and or deletions Instrument methods A method that describes ...

Page 79: ... G 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 within a given population Presequencing signal 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...

Page 80: ...roducts SQA Analysis mode used for base calling of unknown sequences Sequence to analyze A short part of a DNA sequence in your sample starting directly after the sequencing primer which contains one or several variable positions to be analyzed using Pyrosequencing instrument platforms Sequencing primer The sequencing primer is annealed to the template during the sample preparation The 3 end of th...

Page 81: ...equency Variations that occur in 1 or less of a population are considered point mutations while those occurring in more than 1 are SNPs 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 Variable position A region ...

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Page 83: ... power 100 240 VAC 50 60 Hz Input power consumption Maximum 160 VA Instrument rating 24 V DC 40 W Cooler rating 12 V DC 60 W Overvoltage category II Air temperature 15 32ºC 59 90ºF Relative humidity 20 90 noncondensing Altitude Up to 2000 m 6500 ft Place of operation For indoor use only Draft free location not close to window Keep instrument out of direct sunlight Pollution level 2 Environmental c...

Page 84: ... 40ºC 50ºF to 104ºF Relative humidity Max 75 noncondensing Mechanical data and hardware features Dimensions closed Width 390 mm 15 35 in 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 resistance pH 4 to pH 9 common detergents 0 5 M sodium hydroxide 70 etha...

Page 85: ...erature 15 32ºC 59 90ºF Relative humidity 20 90 Altitude Up to 2000 m 6500 ft Place of operation For indoor use only Normal laboratory conditions use adequate ventilation Pollution level 2 Environmental class 3K2 IEC 60721 3 3 Transportation conditions Air temperature 25ºC to 60ºC 13ºF to 140ºF Relative humidity Max 75 noncondensing Storage conditions Air temperature 10ºC to 40ºC 50ºF to 104ºF Rel...

Page 86: ...Mass 11 kg 24 26 lb includes filled troughs and a full waste container Capacity Up to 24 samples per plate Chemical resistance pH 4 to pH 9 common detergents 0 5 M sodium hydroxide 70 ethanol PyroMark Q24 MDx Software Operating system Microsoft Windows 7 English version Processor Intel Pentium IV 3 GHz or higher RAM 1 GB Free hard disk space 100 MB Graphics card Supporting the resolution of the mo...

Page 87: ...arate 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 produ...

Page 88: ...t 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 interf...

Page 89: ...vision 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 EC declaration of conformity Name and address of the legal manufactu...

Page 90: ...uencing 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 prot...

Page 91: ...which include all necessary optimized primers PCR setup PCR reactions of 25 µl 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 pri...

Page 92: ...en 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 bisulfi...

Page 93: ...lification 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 ...

Page 94: ...if not otherwise stated Assay setup AQ and CpG assays When creating an AQ or CpG assay the sequence to analyze should contain a sufficient number of bases to generate at least five nonvariable reference peaks For InDels a few reference peaks should be included before the variable position If the sequencing primer is placed adjacent to the position to analyze include part of the sequence following ...

Page 95: ... be entered in the assay and used to see if the suggested controls are Cs converted to Ts read as Gs and As in a reverse assay and suitable as controls or not The preferable controls are those dispensations that are located at the beginning of the sequence and or represent single base incorporations SQA assays Experience with sequencing large numbers of templates indicates that the dispensation or...

Page 96: ... without template DNA This will show if the primers 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 signal in Pyrosequencing reactions Sequencing primer without any PCR product This will show if the sequencing primer can form duplexes or hairpins and giv...

Page 97: ...es Analysis results For samples and positive controls aim for 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 and CpG Expected reference sequence pattern AQ and CpG All positions AQ and CpG and quality control window SQA with quality assessment Passed The quality assessments for AQ and CpG ass...

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Page 99: ...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 re...

Page 100: ...Appendix C C 2 PyroMark Q24 MDx User Manual 01 2016 This page intentionally left blank ...

Page 101: ...vacuum workstation 6 5 Colors methylation levels 5 26 quality assessments 5 25 Copy log files 5 2 recently saved runs 5 2 unsaved runs 5 1 G Glossary 8 1 I 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 8 optimization 11 setup 9 PyroMark Q24 3 4 PyroMark Q24 principl...

Page 102: ...ssay 5 4 12 PCR 9 run 5 4 Starting template 9 T Technical assistance 2 2 Technical data 1 Test filter probes 5 9 reagent cartridge 5 22 Troubleshooting 7 1 U Upgrade analysis software 4 3 instrument software 5 3 V Ventilation 1 5 4 2 Verify proper installation and operation 7 7 View analysis results 5 25 W Warnings 1 1 Waste disposal 5 ...

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