Sample & Assay Technologies PyroMark® Gold Q24
Contents
1. 6 PyroMark Gold Q24 Reagents Handbook 10 2009 24 hour emergency information Emergency medical information in English French and German can be obtained 24 hours a day from Poison Information Center Mainz Germany Tel 49 6131 19240 PyroMark Gold Q24 Reagents Handbook 10 2009 7 Introduction PyroMark Gold Q24 Reagents are a set of reagents that is optimized for Pyrosequencing technology The reagents are designed to generate Pyrogram traces with sharp and distinct peaks and low background PyroMark Gold Q24 Reagents especially improve assays with longer sequencing read lengths such as with CpG methylation analysis as well as providing optimal conditions for mutation and SNP analyses PyroMark Gold Q24 Reagents contain all enzymes substrates and nucleotides that are needed in the Pyrosequencing cascade Figure 1 Proper use of the reagents results in release of pyrophosphate and generation of a detectable light signal proportional to the number of nucleotides incorporated PyroMark Gold Q24 Reagents are intended to be used together with the PyroMark Q24 Instrument and the PyroMark Q24 Cartridge Enzyme mix The enzyme mixture contains all enzymes that are needed in the Pyrosequencing cascade These include DNA polymerase for incorporation of nucleotides ATP sulfurylase for conversion of pyrophosphate to ATP luciferase for generation of the light signal and apyrase to degrade ATP and unincorpo2. Sweden Orders 020 790282 Fax 020 790582 Technical 020 798328 Switzerland Orders 055 254 22 11 Fax 055 254 22 13 Technical 055 254 22 12 UK Orders 01293 422 911 Fax 01293 422 922 Technical 01293 422 999 eoeo090 USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 QIAGEN m Sample amp Assay Technologies
3. in solving any problems that may arise For more information see also the Frequently Asked Questions page at our Technical Support Center www qgiagen com FAQ FAQList aspx The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocols in this handbook or sample and assay technologies for contact information see back cover or visit www qiagen com Refer to the PyroMark Q24 User Manual for general troubleshooting of the instrument and the PyroMark PCR Handbook for PCR troubleshooting Comments and suggestions Small or missing peaks in the Pyrogram a Reagents incorrectly diluted or stored b One or more of the nucleotide compartments in the PyroMark Q24 Cartridge not loaded correctly c Blocked or damaged nucleotide needles in the PyroMark Q24 Cartridge d No enzyme or substrate added to the well noted as a missing presequencing signal and missing peaks in the Pyrogram Obstructed or damaged reagent cartridge needles D Be sure to follow the instructions in Storage page 4 and Protocol Loading PyroMark Gold Q24 Reagents into the PyroMark Q24 Cartridge page 11 Be sure to add enough reagents select Pre Run Information from the Tools menu of the PyroMark Q24 software Follow the kit handbook supplied with the PyroMark Kit used Clean the PyroMark Q24 Cartridge and check that it is working corr
4. our product literature The purchaser must determine the suitability of the product for its particular use Should any product fail to perform satisfactorily due to any reason other than misuse QIAGEN will replace it free of charge or refund the purchase price We reserve the right to change alter or modify any product to enhance its performance and design If a QIAGEN product does not meet your expectations simply call your local Technical Service Department or distributor We will credit your account or exchange the product as you wish Separate conditions apply to QIAGEN scientific instruments service products and to products shipped on dry ice Please inquire for more information A copy of QIAGEN terms and conditions can be obtained on request and is also provided on the back of our invoices If you have questions about product specifications or performance please call QIAGEN Technical Services or your local distributor see back cover or visit www qiagen com 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 PyroMark Gold Q24 Reagents or QIAGEN products in general please do not hesitate to contact us QIAGEN custome
5. October 2009 PyroMark Gold Q24 Reagents Handbook For performing Pyrosequencing reactions on the PyroMark Q24 QIAGEN Sample amp Assay Technologies QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies enabling the isolation and detection of contents of any biological sample Our advanced high quality products and services ensure success from sample to result QIAGEN sets standards in Purification of DNA RNA and proteins Nucleic acid and protein assays microRNA research and RNAi Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs For more information visit www giagen com Contents Kit Contents Storage Product Use Limitations Product Warranty and Satisfaction Guarantee Technical Assistance Quality Control Safety Information Introduction OoMmnonounuunn 525 hh Equipment and Reagents to Be Supplied by User Protocol E Loading PyroMark Gold Q24 Reagents into the PyroMark Q24 Cartridge 11 Troubleshooting Guide 14 References 16 Ordering Information 17 PyroMark Gold Q24 Reagents Handbook 10 2009 3 Kit Contents PyroMark Gold Q24 Reagents 5 x 24 Catalog no 970802 Number of preps 5x24 Enzyme Mixture 1 vial Substrate Mixture 1 vial dATPaS 1180 ul dGTP 1180 ul dCTP 1180 ul dTTP 1180 ul Handbook 1 Storage Store nucleotides lyophi
6. ULLLLALLLLLLLZZZLLLULIZIIILUIILIGHT 11111111 XI PyroMark Gold Q24 Reagents Handbook 10 2009 17 Product Contents Cat no PyroMark Q24 For performance check of system 979204 Validation Oligo PyroMark PCR Kit 200 For 200 reactions 2x PyroMark PCR 978703 Master Mix includes HotStartaq DNA Polymerase and optimized PyroMark Reaction Buffer containing 3 mM MgCl and dNTPs 10x CoralLoad Concentrate 5x Q Solution 25 mM MgCl and RNase Free Water PyroMark Assay Design Software for convenient design of PCR 9019077 SW 2 0t and sequencing primers optimized for Pyrosequencing analysis Other kit sizes formats available see www giagen com t Multiple software licenses available please inquire For up to date licensing information and product specific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www giagen com or can be requested from QIAGEN Technical Services or your local distributor SSS CANI 18 PyroMark Gold Q24 Reagents Handbook 10 2009 Trademarks QIAGEN CoralLoad PyroMark Pyrosequencing Pyrogram Q Solution QIAGEN Group Milli Q Millipore Corporation Sepharose GE Healthcare Limited License Agreement Use of this product signifies the agreement of any purchaser or user of the PyroMark Gold Q24 Reagents to the following terms 1 The PyroMark Gold Q24 Reagents may be used solely in accordance with the PyroMark Go
7. ectly Clean the PyroMark Q24 Cartridge and check that it is working correctly Clean the PyroMark Q24 Cartridge and check that it is working correctly In case of bent needles discard the PyroMark Q24 Cartridge according to federal state and local environmental regulations for disposal of laboratory waste PyroMark Gold Q24 Reagents Handbook 10 2009 Comments and suggestions f PyroMark Q24 Ensure that the cartridge is inserted correctly Cartridge incorrectly inserted High presequencing signal Contaminated sample Change buffers Only use buffers that are leads to unusually high supplied by QIAGEN or QIAGEN authorized consumption of distributors substrate mixture noted as a high presequencing signal Use the zoom in function to check if any peaks have been generated select a section of Pyrogram with the left mouse button PyroMark Gold Q24 Reagents Handbook 10 2009 15 References QIAGEN maintains a large up to date online database of scientific publications utilizing QIAGEN products Comprehensive search options allow you to find the articles you need either by a simple keyword search or by specifying the application research area title etc For a complete list of references visit the QIAGEN Reference Database online at www qiagen com RefDB search asp or contact QIAGEN Technical Services or your local distributor EA 16 PyroMark Gold Q24 Reagents Handbook 10 2009 Ordering Information P
8. ld Q24 Reagents Handbook and for use with components contained in the Reagents only QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of these Reagents with any components not included within these Reagents except as described in the PyroMark Gold Q24 Reagents Handbook and additional protocols available at www giagen com 2 Other than expressly stated licenses QIAGEN makes no warranty that these Reagents and or their use s do not infringe the rights of third parties 3 These Reagents and their components are licensed for one time use and may not be reused refurbished or resold 4 QIAGEN specifically disclaims any other licenses expressed or implied other than those expressly stated 5 The purchaser and user of the Reagents agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court and shall recover all its investigative and Court costs including attorney fees in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the Reagents and or their components For updated license terms see www giagen com 2009 QIAGEN all rights reserved www qiagen com Australia Orders 03 9840 9800 Fax 03 9840 9888 Technical 1 800 243 066 Austria Orders 0800 28 10 10 Fax 0800 28 10 19 Tech
9. lized enzyme mixture and lyophilized substrate mixture light sensitive at 2 8 C The nucleotides and lyophilized reagents are stable at 2 8 C until the expiration date The reconstituted enzyme and substrate mixtures are stable for at least 5 days at 2 8 C To minimize loss of activity it is advisable to keep both the enzyme mixture and the substrate mixture in the vials supplied Reconstituted enzyme and substrate mixtures can be frozen and stored in their vials at 20 C Frozen reagents should not be subjected to more than 3 freeze thaw cycles IMPORTANT Do not freeze the nucleotides Product Use Limitations PyroMark Gold Q24 Reagents are intended for molecular biology applications These products are neither intended for the diagnosis prevention or treatment of a disease nor have they been validated for such use either alone or in combination with other products Therefore the performance characteristics of the products for clinical use i e diagnostic prognostic therapeutic or blood banking are unknown All due care and attention should be exercised in the handling of the products We recommend all users of QIAGEN products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments or to other applicable guidelines 4 PyroMark Gold Q24 Reagents Handbook 10 2009 Product Warranty and Satisfaction Guarantee QIAGEN guarantees the performance of all products in the manner described in
10. n E IMPORTANT Do not freeze the nucleotides Procedure 1 Open the PyroMark Gold Q24 Reagents box and remove the vials containing lyophilized enzyme and substrate mixtures and the vials containing nucleotides 2 Dissolve the lyophilized enzyme and substrate mixtures in 620 pl each of high purity water Milli Q 18 2 MQ x cm or equivalent filtered through a 0 22 um filter 3 Mix by swirling the vial gently Do not vortex In order to ensure that the mixture is fully dissolved leave it at room temperature 15 25 C for 5 10 min Make sure that the solution is not turbid before filling the PyroMark Q24 Cartridge If the reagents are not to be used immediately place the reagent vials on ice or in a refrigerator 4 Allow the reagents and the PyroMark Q24 Cartridge to reach ambient temperature 20 25 C 5 Place the PyroMark Q24 Cartridge with the label facing you 6 Pipet the reagents into the PyroMark Q24 Cartridge according to Figure 2 Make sure that no air bubbles are transferred from the pipet to the cartridge PyroMark Gold Q24 Reagents Handbook 10 2009 11 a O G 4 Label Figure 2 Illustration of the PyroMark Q24 Cartridge seen from above The annotations correspond to the label on the reagent vials Add enzyme mixture E substrate mixture S and nucleotides A T C G according to the volume information given in the Pre Run information report found in the Tools menu at run setup 7 Switch o
11. n the PyroMark Q24 instrument The power switch is located at the rear of the instrument 8 Open the cartridge gate and insert the filled PyroMark Q24 Cartridge with the label facing you Push the cartridge in fully and then push it down 9 Ensure that the cartridge is properly inserted and close the gate Refer to the PyroMark Q24 User Manual for more information 10 Open the plate holding frame and place the PyroMark Q24 plate on the heating block 11 Close the plate holding frame and the instrument lid 12 Insert the USB memory stick containing the run file into the USB port at the front of the instrument Do not remove the USB port before the run is finished 13 Select Run in the main menu using the and screen buttons and press OK 14 Select the 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 15 When the run file is selected press Select to start the run 16 When the run is finished and the instrument confirms that the run file has been saved to the USB memory stick press Close 17 Remove the USB memory stick 18 Open the instrument lid 19 Open the cartridge gate and remove the PyroMark Q24 Cartridge by lifting it up and pulling it out 20 Close the gate 12 PyroMark Gold Q24 Reagents Handbook 10 2009 21 Discard solutions remaining in the PyroMark Q24 Cartridge 22 Rin
12. nical 0800 28 10 11 Belgium Orders 0800 79612 Fax 0800 79611 Technical 0800 79556 Brazil Orders 0800 557779 Fax 55 11 5079 4001 Technical 0800 557779 Canada Orders 800 572 9613 Fax 800 713 5951 Technical 800 DNA PREP 800 362 7737 China Orders 021 3865 3865 Fax 021 3865 3965 Technical 800 988 0325 Denmark Orders 80 885945 Fax 80 885944 Technical 80 885942 Finland Orders 0800 914416 Fax 0800 914415 Technical 0800 914413 France Orders 01 60 920 926 Fax 01 60 920 925 Technical 01 60 920 930 Offers 01 60 920 928 Germany Orders 02103 29 12000 Fax 02103 29 22000 Technical 02103 29 12400 Hong Kong Orders 800 933 965 Fax 800 930 439 Technical 800 930 425 Ireland Orders 1800 555 049 Fax 1800 555 048 Technical 1800 555 061 Italy Orders 02 33430 420 Fax 02 33430 426 Technical 800 787980 Japan Telephone 03 6890 7300 Fax 03 5547 0818 Technical 03 6890 7300 Korea South Orders 1544 7145 Fax 1544 7146 Technical 1544 7145 Luxembourg Orders 8002 2076 Fax 8002 2073 Technical 8002 2067 Mexico Orders 01 800 7742 639 Fax 01 800 1122 330 Technical 01 800 7742 639 The Netherlands Orders 0800 0229592 Fax 0800 0229593 Technical 0800 0229602 Norway Orders 800 18859 Fax 800 18817 Technical 800 18712 Singapore Orders 65 67775366 Fax 65 67785177 Technical 65 67775366 Spain Orders 91 630 7050 Fax 91 630 5145 Technical 91 630 7050
13. rated nucleotides which switches off the light signal and regenerates the reaction solution In addition single stranded binding protein SSB has been added to prevent secondary structures in the template and primers Substrate mixture The substrate mixture consists of adenosine 5 phosphosulfate APS needed for generation of ATP and luciferin which serves as a substrate for luciferase This ATP drives the luciferase mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP Nucleotides Nucleotides included in the PyroMark Gold Q24 Reagents are dissolved in a well balanced buffer to prevent degradation of the nucleotides It should be noted that deoxyadenosine alpha thio triphosphate dATPaS is used as a substitute for the natural deoxyadensine triphosphate dATP since it is efficiently used by the DNA polymerase but not recognized by luciferase eS 8 PyroMark Gold Q24 Reagents Handbook 10 2009 Polymerase 3 CGTCCGGAGG CCAAGTTCCA TITITTWEEDDDDTITIIMTTT LLL S 5 GCAGGCCT 3 T 4 T PPi Sulfurylase s T Apyrase T ATP 7 Luciferase 9 time Figure 1 Schematic illustration of the Pyrosequencing cascade As nucleotides are incorporated into the analyzed DNA strand pyrophosphate is released and converted to ATP Generated ATP drives the light reaction detected as a peak on the Pyrogram Apyrase degrades any unincorporated nucleo
14. rdous levels of toxic substances chemical or biological as defined in the applicable Material Safety Data Sheets MSDSs or OSHA ACGIH or COSHH documents 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 The following risk phrases apply to components of PyroMark Gold Q24 Reagents Substrate mixture Contains dithiothreitol Harmful if swallowed irritating to eyes respiratory system and skin Risk phrases R22 36 37 38 Contains luciferin Toxic by inhalation in contact with skin and if swallowed causes eye skin and respiratory tract irritation Risk phrases R23 24 25 36 37 38 Enzyme mixture Contains tris hydroxymethyl aminomethane Irritating to eyes respiratory system and skin danger of cumulative effects Risk phrases R33 36 37 38 Contains dithiothreitol Harmful if swallowed irritating to eyes respiratory system and skin Risk phrases R22 36 37 38 R22 Harmful if swallowed R23 24 25 Toxic by inhalation in contact with skin and if swallowed R33 Danger of cumulative effects R36 37 38 Irritating to eyes respiratory system and skin SESE
15. roduct Contents Cat no PyroMark Gold Q24 For 5 x 24 samples for use on the 970802 Reagents 5 x 24 PyroMark Q24 Enzyme Mixture Substrate Mixture and Nucleotides Accessories PyroMark Q24 Cartridges for dispensing nucleotides 979202 Cartridge 3 and reagents on the PyroMark Q24 Related products PyroMark Q24 Sequence based detection platform for 9001514 Pyrosequencing of 24 samples in parallel PyroMark Q24 Vacuum Workstation for preparing single Varies Workstation stranded DNA from 24 samples PyroMark Q24 Application software 9019062 Software PyroMark Annealing For annealing sequencing primer to 979009 Buffer 250 ml single stranded PCR product and for Pyrosequencing reaction PyroMark Binding For binding of biotinylated PCR product 979006 Buffer 200 ml t to Streptavidin Sepharose beads PyroMark Wash Buffer For washing of single stranded DNA 979008 concentrate 200 ml PyroMark Denaturation For denaturation of double stranded 979007 Solution 500 ml PCR product into single stranded template DNA PyroMark Q24 Plate 24 well sequencing reaction plate 979201 100 PyroMark Q24 Control For installation check of system 979203 Oligo 9001518 220V 9001516 110V 9001519 100V t For use with PyroMark Q24 PyroMark Q96 MD and PyroMark Q96 ID For use with PyroMark Q24 Vacuum Workstation and PyroMark Q96 Vacuum Workstation FREE Le Le LL LLULLLLLLLILUILLLLIILIILIII LE ILLI LELTLLILILLLILLLILILLLLL LILILLLLULULLLLLULLLLU
16. rs 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 Technical Support Center at www giagen com Support or call one of the QIAGEN Technical Service Departments or local distributors see back cover or visit www qiagen com Quality Control In accordance with QIAGEN s ISO certified Quality Management System each lot of PyroMark Gold Q24 Reagents is tested against predetermined specifications to ensure consistent product quality PyroMark Gold Q24 Reagents Handbook 10 2009 5 Safety Information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate material safety data sheets MSDSs These are available online in convenient and compact PDF format at www giagen com support MSDS aspx where you can find view and print the MSDS for each QIAGEN kit and kit component CAUTION Always wear safety glasses gloves and a lab coat AN The responsible body e g laboratory manager must take the necessary precautions to ensure that the surrounding workplace is safe and that the instrument operators are not exposed to haza
17. se the PyroMark Q24 Cartridge 4 times with high purity water 23 Spray the outside of the needles with high purity water 24 To rinse the needles fill the compartments completely with high purity water and hold the cartridge over a sink while pressing firmly on top of each compartment with a finger wear gloves Check that the needle is clear A jet of water should come straight out of the tip of the needle 25 If the needle is blocked follow step 25a If the jet of water comes out of the needle at an angle not parallel to the direction of the needle follow step 25b If the needle is clear proceed to step 26 25a If the needle is blocked for example if the reagent cartridge has been left overnight without cleaning fill the compartments with enough high purity water and immerse the PyroMark Q24 Cartridge in a beaker of high purity water that covers the needles Leave the reagent cartridge in the beaker for 1 h rinse it and repeat step 24 25b If the jet of water comes out at an angle refill the compartment with water and repeat If the water still comes out at an angle discard the PyroMark Q24 Cartridge 26 When all needles have been rinsed and checked discard the water and let the PyroMark Q24 Cartridge dry on a lint free tissue 27 When the PyroMark Q24 Cartridge is dry store it in a dust free place PyroMark Gold Q24 Reagents Handbook 10 2009 13 Troubleshooting Guide This troubleshooting guide may be helpful
18. tides SSS EEE PyroMark Gold Q24 Reagents Handbook 10 2009 9 Equipment and Reagents to Be Supplied by User When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier M Pipets adjustable M Sterile pipet tips with filters E PyroMark Q24 cat no 9001514 E PyroMark Q24 Vacuum Workstation cat no 9001518 220V 9001516 110V 9001519 100V PyroMark Q24 Cartridge cat no 979302 PyroMark Q24 Control Oligo cat no 979203 for installation check of PyroMark Q24 system M PyroMark Q24 Validation Oligo cat no 979204 for performance check of PyroMark Q24 system E High purity water Milli Q 18 2 MQ x cm or equivalent B Lint free tissue Ensure that instruments have been checked and calibrated according to the manufacturer s recommendations 10 PyroMark Gold Q24 Reagents Handbook 10 2009 Protocol Loading PyroMark Gold Q24 Reagents into the PyroMark Q24 Cartridge This protocol describes loading PyroMark Gold Q24 Reagents into the PyroMark Q24 Cartridge before performing Pyrosequencing analysis on the PyroMark Q24 Important points before starting M The Pre Run information report found in the Tools menu at run setup provides information about the volume of nucleotides enzyme mixture and substrate mixture needed for a specific ru
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