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1. i PyroMark instrument Clean the heating block and the light started without a plate guides lens array according to instructions in inserted the user manual for the instrument PyroMark Control Oligo Follow the instruction in the protocols for not correctly prepared preparing the PyroMark Control Oligo Make sure to dilute the PyroMark Control Oligo in the dilution buffer as described in the protocols Make sure that the 10x dilution buffer provided is first diluted to 1x using high purity water k Contaminated sample Change buffers Only use buffers that are leads to unusually high supplied by QIAGEN or QIAGEN authorized consumption of substrate distributors mixture noted as a high E ed Use the zoom in function to check if any peaks presequencing signal have been generated select a section of Pyrogram with the left mouse button Warning regarding signal to noise Various See points a to k in Small or missing peaks above Quantification results Comments and suggestions Poor or incorrect sequence a PyroMark Control Oligo Follow the instruction in the protocols for not correctly prepared preparing the PyroMark Control Oligo Make sure to dilute the PyroMark Control Oligo in the dilution buffer as described in the protocols Make sure that the 10x dilution buffer provided is first diluted to 1x using high purity water b Incorrect sequence to Check that the correct sequence was typed in analyze or dis
2. Histogram from PyroMark Q96 ID Software Nucleotide additions 1 2 3 5 and 11 are blank dispensations and serve as negative controls The seventh and the eighth dispensations analyze the variable position wobbled degenerated base 5 10 Figure 13 Histogram from PyroMark CpG Software Nucleotide additions 1 2 3 5 and 11 are blank dispensations and serve as negative controls The seventh and the eighth dispensations analyze the variable position wobbled degenerated base 4 Save the entry PyroMark Q96ID Software assay PyroMark CpG Software 5 Open a new run and allocate the created entry assay to appropriate wells Choose instrument parameters supplied by QIAGEN according to the reagents and cartridge that will be used for the run and save the run We recommend using 16 wells 8 wells for samples prepared using the PyroMark Q96 Vacuum Workstation and 8 samples added directly to PyroMark Q96 Plate Low Note A well is color coded according to the assay type loaded to the well For more information on how to create a Run Setup file in PyroMark Q96 ID Software see the PyroMark Q96 ID Software Online Help For more information on how to create a Run Setup file in PyroMark CpG Software see the PyroMark CpG Software Online Help PyroMark Control Oligo Handbook 07 2009 29 6 Print a list of required volumes of enzyme mix substrate mix and nucleotides and the plate setup Note To generate a list of required volumes
3. 425 Ireland Orders 1800 555 049 Fax 1800 555 048 Technical 1800 555 061 ltaly 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 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 CILLA USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 QIAGEN BS Sample amp Assay Technologies
4. Do not leave the PCR plate used in the Streptavidin Sepharose immobilization step for longer than 1 min after High Performance mixing is finished If necessary mix for an extra beads in some wells or minute before capturing the beads tubes when capturing the beads containing immobilized template to the filter probes d Leakage of the Ensure that the tubing is connected properly and PyroMark Q24 or Q96 that there is no leakage The waste filter might be Vacuum Workstation wet and need to be replaced 52 PyroMark Control Oligo Handbook 07 2009 Appendix A Preparation of the PyroMark Q24 Vacuum Workstation This protocol is a description of how to prepare the PyroMark Q24 Vacuum Workstation before using it for preparation of single stranded DNA Procedure 1 Fill 5 separate troughs supplied with the PyroMark Q24 Vacuum Workstation as follows Approximately 50 ml ethanol 70 1 Approximately 40 ml PyroMark Denaturation Solution 2 Approximately 50 ml PyroMark Wash Buffer 3 Approximately 50 ml high purity water 4 Approximately 70 ml high purity water 5 A suggested setup is shown in Figure 22 Refill the troughs to these levels whenever necessary Figure 22 Positions on the PyroMark Q24 Vacuum Workstation 2 Switch on the vacuum pump o Apply vacuum to the tool by opening the vacuum switch 4 Wash the filter probes by lowering the probes into high purity water trough 5
5. a master mix for DNA immobilization according to Table 4 Prepare a volume 1096 greater than that required for the total number of reactions to be performed 22 PyroMark Control Oligo Handbook 07 2009 Table 4 Master mix for DNA immobilization Number of samples 1 9 Streptavidin Sepharose High Performance di I5 PyroMark Binding Buffer 40 ul 360 ul High purity water 13 ul 117 ul Total volume 55 pl 495 pl Provides a sufficient amount for the 8 samples required 11 Dilute the PyroMark Control Oligo to 0 04 uM as shown in Table 5 Table 5 Dilution of the PyroMark Control Oligo Component Volume Concentration PyroMark Control Oligo 10 ul 20 uM 1x Dilution buffer 90 ul First serial dilution 100 pl 2 uM First serial dilution from above 30 ul 2 uM 1x Dilution buffer 1470 ul Final dilution 1500 yl 0 04 uM Make sure that the 10x dilution buffer supplied with the PyroMark Control Oligo is diluted with high purity water before use See Things to do before starting page 21 12 Shake the tube containing the master mix and add 55 ul of the master mix and 25 ul of the diluted 0 04 uM PyroMark Control Oligo to 8 wells of a 24 well PCR plate or strips 13 Seal the PCR plate or strips Using strip caps 14 Agitate the PCR plate at room temperature 15 25 C for 5 10 min at 1400 rpm Sepharose beads sediment quickly Capturing of beads must take place immediately following agitat
6. discarded and the PyroMark Q96 Vacuum Workstation should be checked for dust and spillage see Appendix C page 55 30 Heat the PyroMark Q96 HS Plate with the samples at 80 C for 2 min using a heating block and the prewarmed PyroMark HS Q96 Sample Prep Thermo Plate Kit Note Use one Sample Prep Thermo Plate as lid on the plate to prevent evaporation of the samples 31 Remove the PyroMark Q96 HS Plate from the thermo plate and let the samples cool to room temperature 15 25 C for at least 5 min 32 Load the nucleotide and reagent tips in the PyroMark Q96 Dispensing Tip Holder with the appropriate volumes of PyroMark Gold Q96 Reagents Note To generate a list of required volumes in the PyroMark Q96 MD Software see View and Run To generate a list of required volumes in the PyroMark CpG Software open the run file and select Volume Information from the Tools menv 33 Switch on the instrument 34 Open the process chamber lid using either software 35 Place the PyroMark Q96 HS Plate on the heating block Close the process chamber lid 36 Open the dispensing unit cover by releasing the latch With the two RDTs furthest away from you insert the filled dispensing tip holder into position 37 Close the dispensing unit cover Make sure that the latch snaps into its locked position 38 Close the instrument lid 39 Perform a run see PyroMark Q96 MD User Manual or PyroMark CpG Software Online Help 40 After ru
7. how to create an Assay Setup file see the PyroMark Q24 Software User Guide PyroMark Control Oligo Handbook 07 2009 21 Figure 8 Histogram for AQ mode Nucleotide additions 1 2 3 5 and 11 are blank and serve as negative controls The seventh and the eight dispensations analyze the variable position 5 Click amp in the toolbar to save the assay 6 Create a Run Setup by importing the assay parameters to the appropriate wells We recommend using 16 wells 8 wells for samples prepared using the PyroMark Q24 Vacuum Workstation and 8 samples added directly to PyroMark Q24 Plate To add an assay to a well you can either Wm Right click the well and select Load Assay from the context menu m Select the assay in the shortcut browser and click and drag the assay to the well m Recommended Fill in Sample ID Plate ID Barcode Reagent ID and Run Note A well is color coded according to the assay type loaded to the well For more information on how to create a Run Setup file see the PyroMark Q24 Software User Guide 7 Save the Run Setup to a USB memory stick supplied with the PyroMark Q24 system 8 Print a list of required volumes of enzyme mix substrate mix and nucleotides and the plate setup Select Pre Run Information from the Tools menu and when the report appears click 3 9 Gently shake the bottle containing Streptavidin Sepharose High Performance until it is a homogeneous solution 10 Prepare
8. in the PyroMark Q96 ID Software click View and Run in the browser area To generate a list of required volumes in the PyroMark CpG Software open the run file and select Volume Information from the Tools menu 7 Gently shake the bottle containing Streptavidin Sepharose High Performance until it is a homogeneous solution 8 Prepare a master mix for DNA immobilization according to Table 6 Prepare a volume 10 greater than that required for the total number of reactions to be performed Note Gently shake the bottle containing Streptavidin Sepharose High Performance until it is a homogeneous solution Table 6 Master mix for DNA immobilization Component Volume sample Streptavidin Sepharose High Performance 3 ul PyroMark Binding Buffer 37 ul Total volume 40 pl 9 Dilute the PyroMark Control Oligo to 0 05 uM as shown in Table 7 Table 7 Dilution of the PyroMark Control Oligo Component Volume Concentration PyroMark Control Oligo 10 ul 20 uM 1x Dilution buffer 90 ul First serial dilution 100 pl 2 uM First serial dilution from above 20 ul 2 UM 1x Dilution buffer 780 ul Final dilution 800 ul 0 05 uM Make sure that the 10x dilution buffer supplied with the PyroMark Control Oligo is diluted with high purity water before use See Things to do before starting page 28 30 PyroMark Control Oligo Handbook 07 2009 10 Add 40 ul of the master mix and 40 ul of the diluted 0 05 uM Pyr
9. menu Save the data in a suitable format csv or tsv Open this file in Microsoft Excel Delimited and calculate the mean single peak height and background for each well see below Perform a quality assessment All wells should give Passed quality If the quality assessment is Check or Failed look in Well info PyroMark Q96 MD Software Well Information PyroMark CpG Software for explanations Evaluate the quantification results PyroMark Q96 MD Software Quantification results with standard deviation are shown in AQ Statistics under the Statistics Tab Select all wells by holding down the Ctrl or Shift key and click in the wells one by one to mark 42 PyroMark Control Oligo Handbook 07 2009 them Selected wells appear with a square around them Click the Statistics button under the Statistics Tab PyroMark CpG Software Quantification results with standard deviation are shown in the CpG Statistics Report that can be selected from the Reports menu The C should be in the range 40 to 60 The standard deviation should not exceed 2 units Evaluate single peak heights The mean single peak height should be at least 350 RLU Sum single peaks dispensation 4 6 12 13 Mean single peak height i Evaluate the background Background from blank dispensations should not exceed 3 Sum blanks dispensation 1 2 3 5 Background 96 z x 100 Sum single peaks dispensation 4 6 12
10. run in the PyroMark Q24 Software and analyze all wells The peak pattern for Run 1 should look like the one in Figure 3 To obtain peak height values select Export Peak Heights from the Tools menu Save the data in a suitable format csv or tsv Open this file in Microsoft Excel Delimited and calculate the mean single peak height for each well as described on the next page m Perform a quality assessment All wells should give Passed quality shown as a blue bar in the bottom field of the well when looking at the overview tab and with C indicated in a blue rectangle in the Pyrogram If the quality assessment is Check or Failed look in Well Information for explanations m Evaluate peak heights The single peak height dispensations 2 4 and 9 should approximately be 50 RLU If the values are within the set limits the system is properly installed If the results are not as stated above see Troubleshooting Guide page 44 for possible reasons for the failure and rerun Run 1 If the repeat of Run 1 fails 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 giagen com C 5296 T 4896 250 200 150 100 50 E S C T G A Cc T G T G Figure 3 Pyrogram of Run 1 PyroMark Control Oligo Handbook 07 2009 13 Protocol Verifying the Function of the PyroMark Q
11. 009 31 16 Wash the filter probes by lowering the probes into high purity water parking position Let approximately 180 ml of water flush through the filter probes i e empty the trough 17 Refill the parking position with 180 ml high purity water 18 Carefully lower the filter probes into the PCR plate or strips to capture the beads containing immobilized template Hold the probes in place for 15 s Take care when picking up the tool Note Sepharose 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 19 Transfer the tool to the trough containing 70 ethanol trough 1 Flush the filter probes for 5 s 20 Transfer the tool to the trough containing PyroMark Denaturation Solution trough 2 Flush the filter probes for 5 s 21 Transfer the tool to the trough containing PyroMark Wash Buffer trough 3 Flush the filter probes for 10 s 22 Raise the tool up and back beyond 90 vertical for 5 s to drain liquid from the filter probes Figure 15 Illustration of the vacuum tool raised to beyond 90 vertical 23 While the tool is held over the PyroMark Q96 Plate Low close the vacuum switch on the vacuum workstation Off 24 Release the beads in the plate containing 40 pl PyroMark Annealing Buffer by shaking the tool from side to side Allow the filter probes to rest on the bottom of the wells 25 Transfer the tool to the tro
12. 00V PyroMark Gold Q24 Reagents 5 x 24 cat no 970802 For use with the PyroMark Q96 ID System PyroMark Q96 ID Instrument cat no 9001525 PyroMark Q96 ID Software cat no 9019083 PyroMark Q96 Plate Low 100 cat no 979002 PyroMark Q96 Cartridge 3 cat no 979004 PyroMark Gold Q96 Reagents 5x96 cat no 972804 PyroMark Q96 Vacuum Workstation cat no 9001529 220 V 9001528 110V 9001740 UK or use with the PyroMark Q96 MD System PyroMark Q96 MD Instrument cat no 9001526 PyroMark Q96 MD Software cat no 9019085 PyroMark Q96 HS Plate 100 cat no 979101 PyroMark Q96 HS Dispensing Tip Holder cat no 9019075 PyroMark Q96 HS Reagent Tips 4 cat no 979102 PyroMark Q96 HS Nucleotide Tips 8 cat no 979103 PyroMark Gold Q96 Reagents 5 x 96 cat no 972804 PyroMark Q96 Vacuum Workstation cat no 9001529 220 V 9001528 110V 9001740 UK o EHEHE ERN E Additional equipment needed for all systems M PyroMark Binding Buffer cat no 979006 E PPyroMark Denaturation Solution cat no 979007 8 PyroMark Control Oligo Handbook 07 2009 E PyroMark Wash Buffer concentrate cat no 979008 E PyroMark Annealing Buffer cat no 979009 W Plate mixer for immobilization to beads E Heating block capable of attaining 80 C BE 24 or 96 well PCR plate or strips EB Strip caps E 1 5 ml or 2 ml microcentrifuge tubes for dilution of the PyroMark Control Oligo E Streptavidin
13. 07 2009 them Selected wells appear with a square around them Click the Statistics button under the Statistics Tab PyroMark CpG Software Quantification results with standard deviation are shown in the CpG Statistics Report that can be selected from the Reports menu The C should be in the range 40 to 60 The standard deviation should not exceed 2 units Evaluate single peak heights The mean single peak height should ideally be 35 10 RLU Sum single peaks dispensation 4 6 12 13 Mean single peak height i Evaluate the background Background from blank dispensations should not exceed 5 Sum blanks dispensation 1 2 3 5 Background 96 i i x 100 Sum single peaks dispensation 4 6 12 13 Evaluate the difference in peak heights with and without sample preparation The reduction in peak height between samples prepared using the PyroMark Q96 Vacuum Workstation compared with PyroMark Control Oligo added directly to the PyroMark Q96 Plate Low should not be more than 2096 If the values are within the set limits the system is properly installed If the results are not as stated above see Troubleshooting guide page 44 for possible causes and actions to be taken If the troubleshooting guide does not explain the problem please see our Technical Support Center at www qiagen com Support or call one of the QIAGEN Technical Service Departments or local distributors see bac
14. 1 min has elapsed since the plate or strips was agitated agitate again for 1 min before capturing the beads 19 Transfer the tool to the trough containing 7096 ethanol trough 1 Flush the filter probes for 5 s 20 Transfer the tool to the trough containing PyroMark Denaturation Solution trough 2 Flush the filter probes for 5 s 21 Transfer the tool to the trough containing PyroMark Wash Buffer trough 3 Flush the filter probes for 10 s 22 Raise the tool up and back beyond 90 vertical for 5 s to drain liquid from the filter probes see Figure 10 24 PyroMark Control Oligo Handbook 07 2009 23 24 25 26 27 28 29 30 31 32 Figure 10 Illustration of the vacuum tool raised to beyond 90 vertical While the tool is held over the PyroMark Q24 Plate close the vacuum switch on the tool Off Release the beads in the plate containing 25 pl PyroMark Annealing Buffer by shaking the tool from side to side Allow the filter probes to rest on the bottom of the wells Transfer the tool to the first trough containing high purity water trough 4 and agitate the tool for 10 s Wash the filter probes by lowering the probes into the second trough with high purity water trough 5 and applying vacuum Flush the probes with 70 ml high purity water Raise the tool up and back beyond 90 vertical for 5 s to drain liquid from the filter probes see Figure 10 Close the vacuum switch
15. 13 Evaluate the difference in peak heights with and without sample preparation The reduction in peak height between samples prepared using the PyroMark Q96 Vacuum Workstation compared with PyroMark Control Oligo added directly to the PyroMark Q96 HS Plate should not be more than 20 If the values are within the set limits the system is properly installed If the results are not as stated above see Troubleshooting guide page 44 for possible causes and actions to be taken If the troubleshooting guide does not explain the problem 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 cover or visit www qiagen com PyroMark Control Oligo Handbook 07 2009 43 Troubleshooting Guide This troubleshooting guide may be helpful 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 giagen com Sections are included for each evaluation performed Quality assessment below Quantification results page 47 Single peak heights page 49 Background page 51 Diff
16. 24 Instrument and PyroMark Q24 Vacuum Workstation This protocol describes how to use the PyroMark Control Oligo to verify the function of the PyroMark Q24 Instrument and PyroMark Q24 Vacuum Workstation To verify the function of the PyroMark Q24 Instrument only see Protocol Verifying the Function of the PyroMark Q24 Instrument page 10 Important point before starting mB For further information on how to create an Assay Setup and a Run Setup see the PyroMark Q24 Software User Guide Things to do before starting Bm Follow the instructions in PyroMark Q24 User Manual to install the PyroMark Q24 EB The dilution buffer provided with the PyroMark Control Oligo needs to be diluted before use Prepare 1x dilution buffer by mixing 200 ul of 10x dilution buffer with 1800 ul of high purity water M Place the PyroMark Q24 Plate Holder on a heating block at 80 C for use in step 30 amp Allow all required reagents and solutions to reach room temperature 15 25 C before starting Procedure 1 Set up an assay for the PyroMark Control Oligo by using the PyroMark Q24 Software 2 Click in the toolbar and select New AQ Assay 3 Type the following sequence in Sequence to Analyze TAYGGTITGCA For more information on how to create an Assay Setup file see the PyroMark Q24 Software User Guide 4 Manually enter the following Dispensation Order ACGTTATCGTTGC For more information on how to create an Assay Setup fi
17. 9 Single peak heights Comments and suggestions Poor or incorrect sequence a PyroMark Control Oligo Follow the instruction in the protocols for not correctly prepared preparing the PyroMark Control Oligo Make sure to dilute the PyroMark Control Oligo in the dilution buffer as described in the protocols Make sure that the 10x dilution buffer provided is first diluted to 1x using high purity water b Incorrect sequence to Check that the correct sequence was typed in analyze or dispensation the Assay Setup order c Buffers or reagents Follow the instructions supplied with the incorrectly diluted or reagents Include an empty well containing only incorrectly stored PyroMark Annealing Buffer in your run to check if background peaks are coming from the nucleotides d Dispensation error seen Clean or replace the Dispensing Unit If the for example as split problem remains contact QIAGEN Technical peaks Services for contact information see back cover or visit www giagen com e Blocked PyroMark Nucleotides are not dispensed correctly due to a Dispensing Unit blocked needle in the Dispensing Unit Clean the Dispensing Unit and check that it is working properly f Damaged PyroMark Discard the Dispensing Unit according to Dispensing Unit federal state and local environmental regulations for disposal of laboratory waste g Annealing time too long Carry out annealing for the correct time and at the temperatures described in
18. Flush the probes with 70 ml high purity water Make sure that the water is being transferred to the waste container If it is not then make sure that the tubing is connected correctly and is not broken Broken tubing should be replaced see Replacing the tubing in the PyroMark Q24 User Manual 5 Make sure that the waste filter is dry If the filter is wet it should be replaced see Replacing the waste filter in the PyroMark Q24 User Manual 6 Refill trough 5 with 70 ml high purity water 7 Close the vacuum switch on the tool Off and place the tool in the Parking P position PyroMark Control Oligo Handbook 07 2009 53 Appendix B Preparation of the PyroMark Q96 Vacuum Workstation This protocol is a description of how to prepare the PyroMark Q96 Vacuum Workstation before using it for preparation of single stranded DNA 1 Fill 5 separate troughs supplied with the PyroMark Q96 Vacuum Workstation as follows Approximately 110 ml ethanol 70 1 Approximately 90 ml PyroMark Denaturation Solution 2 Approximately 110 ml PyroMark Wash Buffer 3 Approximately 110 ml high purity water 4 Approximately 180 ml high purity water Parking position A suggested setup is shown in Figure 23 Refill the troughs to these levels whenever necessary Figure 23 Placement of PCR plate or strips and PyroMark Q96 Plate Low or PyroMark Q96 HS Plate on the PyroMark Q96 Vacuum Workstation The marked positions co
19. Instrument Pyrosequencing of 96 samples in parallel PyroMark Q96 MD Application software for PyroMark Q96 9019085 Software MD PyroMark Q96 For preparation of single stranded 9001529 Vacuum Workstation DNA from 96 samples for use with 220 V PyroMark Q96 ID or PyroMarkQ96 MD PyroMark Q96 For preparation of single stranded DNA 9001528 Vacuum Workstation 110 V template ready for sequencing by PyroMark Q96 ID or MD UK 9001518 220V 9001516 110V 9001519 100V 58 PyroMark Control Oligo Handbook 07 2009 Ordering Information Product Contents Cat no PyroMark PCR Kit For 200 reactions 2x PyroMark PCR 978703 200 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 EpiTect Bisulfite Kit 48 EpiTect Bisulfite Spin Columns 59104 48 Reaction Mix DNA Protect Buffer Carrier RNA Buffers EpiTect PCR Control Human control DNA set containing 59695 DNA Set 100 both bisulfite converted methylated and unmethylated DNA and unconverted unmethylated DNA for 100 control PCRs Other kit sizes formats available see www qiagen com 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 Te
20. July 2009 PyroMark Control Oligo Handbook For use with PyroMark systems for installation check 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 Principle and procedure Description of protocols O 0o Oo Oo Uu uu 5 b 5 BD Equipment and Reagents to Be Supplied by User Protocols eo E Verifying the Function of the PyroMark Q24 Instrument B Verifying the Function of the PyroMark Q24 Instrument and PyroMark Q24 Vacuum Workstation 14 B X Troubleshooting Procedure for the PyroMark Q24 Instrument and PyroMark Q24 Vacuum Workstation 21 B Troubleshooting Procedure of the PyroMark Q96 ID Instrument and PyroMark Q96 Vacuum Workstation 28 B X Troubleshooting Procedure of the PyroMark Q96 MD Instrumen
21. New AQ Assay 3 Type the following sequence in Sequence to Analyze TAYGGTTTGC For more information on how to create an Assay Setup file see the PyroMark Q24 Analysis Software User Guide 4 Click the Generate Dispensation Order icon to get the following nucleotide dispensation order CTGACTGTG 10 PyroMark Control Oligo Handbook 07 2009 Figure 2 Histogram for AQ mode Nucleotide additions 1 and 3 are blank dispensations and serve as negative controls The fifth and the sixth dispensations analyze the variable position wobbled degenerated base 5 Click amp in the toolbar to save the assay 6 Create a Run Setup by importing the assay parameters to all 24 wells To add an assay to a well you can either Wm Right click the well and select Load Assay from the context menu m Select the assay in the shortcut browser and click and drag the assay to the well A well is color coded according to the assay type loaded to the well For more information on how to create a Run Setup file see the PyroMark Q24 Software User Guide 7 Save the Run Setup to a USB memory stick supplied with the PyroMark Q24 system 8 Print a list of required volumes of enzyme mix substrate mix and nucleotides and the plate setup Select Pre Run Information from the Tools menu and when the report appears click 3 9 Dilute the PyroMark Control Oligo to 0 04 uM as shown in Table 1 Table 1 Dilution of the Pyr
22. Sepharose High Performance GE Healthcare cat no 17 5113 01 www gelifesciences com EB Pipets adjustable E Sterile pipet tips E High purity water Milli Q 18 2 MQ x cm or equivalent E Ethanol 70 PyroMark Control Oligo Handbook 07 2009 9 Protocol Verifying the Function of the PyroMark Q24 Instrument This protocol describes how to use the PyroMark Control Oligo to verify the function of PyroMark Q24 Instrument To verify the function of the PyroMark Q24 Instrument and the PyroMark Q24 Vacuum Workstation see Protocol Verifying the function of the PyroMark Q24 Instrument and the PyroMark Q24 Vacuum Workstation page 14 Important point before starting mB For further information on how to create an Assay Setup and a Run Setup see the PyroMark Q24 Software User Guide Things to do before starting mB Follow the instructions in PyroMark Q24 User Manual to install the PyroMark Q24 Instrument EB The dilution buffer provided with the PyroMark Control Oligo needs to be diluted before use Prepare 1x dilution buffer by mixing 200 pl of 10x dilution buffer with 1800 ul of high purity water M Place the PyroMark Q24 Plate Holder on a heating block at 80 C for use in step 11 amp Allow all required reagents and solutions to reach room temperature 15 25 C before starting Procedure 1 Set up an assay for the PyroMark Control Oligo by using the PyroMark Q24 Software 2 Click in the toolbar and select
23. aise the tool up and back beyond 90 vertical for 5 s to drain liquid from the filter probes see Figure 6 Figure 6 Illustration of the vacuum tool raised to beyond 90 vertical While the tool is held over the PyroMark Q24 Plate close the vacuum switch on the tool Off PyroMark Control Oligo Handbook 07 2009 17 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 Release the beads in the plate containing 25 pl PyroMark Annealing Buffer by shaking the tool from side to side Allow the filter probes to rest on the bottom of the wells Transfer the tool to the first trough containing high purity water trough 4 and agitate the tool for 10 s Wash the filter probes by lowering the probes into the second trough with high purity water trough 5 and applying vacuum Flush the probes with 70 ml high purity water Raise the tool up and back beyond 90 vertical for 5 s to drain liquid from the filter probes see Figure 6 Close the vacuum switch on the tool Off and place the tool in the Parking P position Turn off the vacuum pump At the end of a working day liquid waste and remaining solutions should be discarded and the PyroMark Q24 Vacuum Workstation should be checked for dust and spillage see Appendix C page 55 Heat the PyroMark Q24 Plate with the samples at 80 C for 2 min using a heating block and the prewarmed PyroMark Q24 Plate Holder Remov
24. ark Control Oligo Handbook 07 2009 5 Introduction The PyroMark Control Oligo provides a means to verify proper installation of the PyroMark systems In addition the PyroMark Control Oligo can be used in troubleshooting to determine if an unexpected result is related to the instrument to the PyroMark Vacuum Workstations or to the assay Principle and procedure The PyroMark Control Oligo is a biotinylated oligonucleotide which allows the user to verify that all the PyroMark instruments and PyroMark Vacuum Workstations are functioning properly Under defined conditions the oligonucleotide can form an internal stem loop structure This structure enables self priming of the oligonucleotide for extension by the DNA polymerase and eliminates the need for a sequencing primer in the Pyrosequencing reaction The sequenced region includes single bases of all nucleotides homopolymers of 2 and 3 bases and a wobbled degenerated base This variable position is automatically analyzed by the software and results are presented as C and T Figure 1 shows the structure of the oligonucleotide A 3 ATRCCAAACG 9 5 Biotin B Sequenced region 3 TAYGGTTTGC du ATRCCAAACG 99 Biotin Figure 1 Structure of the PyroMark Control Oligo IN The open structure of the oligonucleotide The self primed structure of the oligonucleotide with the analyzed sequence indicated Description of protocols It is recommended that 2 runs be performed
25. ation results single peak heights and background To obtain peak height values select Export Peak Heights from the Tools menu Save the data in a suitable format csv or tsv Open this file in Microsoft Excel Delimited and calculate the mean single peak height and background for each well as described below m Perform a quality assessment All wells should give Passed quality shown as a blue bar in the bottom field of the well when looking at the overview tab and with C indicated in a blue rectangle in the Pyrogram If the quality assessment is Check or Failed look in Well Information for explanations m Evaluate the quantification results Select the AQ Analysis Statistics Report from the Reports menu Quantification results are given in the report with standard deviation The C should be in the range 40 60 The standard deviation should not exceed 2 percentage units m Evaluate single peak heights The mean single peak height should ideally be 50 15 RLU Sum single peaks dispensation 4 6 12 13 Mean single peak height 4 m Evaluate the background Background from blank dispensations should not exceed 3 Sum blanks dispensation 1 2 3 5 Background x 100 Sum single peaks dispensation 4 6 12 13 51 Evaluate the difference in peak heights with and without sample preparation The reduction in peak height between samples prepared usin
26. chnical Services or your local distributor F GR as AAA RSARA X X XE BR SES RR t PER SSS PR PyroMark Control Oligo Handbook 07 2009 59 Notes 60 PyroMark Control Oligo Handbook 07 2009 Notes PyroMark Control Oligo Handbook 07 2009 61 Notes 62 PyroMark Control Oligo Handbook 07 2009 Trademarks QIAGEN CoralLoad EpiTect PyroMark Pyrosequencing Pyrogram Q Solution QIAGEN Group Microsoft Microsoft Corporation 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 Control Oligo to the following terms 1 The PyroMark Control Oligo may be used solely in accordance with the PyroMark Control Oligo Handbook and for use with components contained in the Product only QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this Product with any components not included within this Product except as described in the PyroMark Control Oligo Handbook and additional protocols available at www giagen com Other than expressly stated licenses QIAGEN makes no warranty that this Product and or their use s do not infringe the rights of third parties This product and its components are licensed for one time use and may not be reused refurbished or resold QIAGEN specifically disclaims any other licenses expressed or implied other tha
27. e Streptavidin Sepharose High Performance 2 ul PyroMark Binding Buffer 40 ul High purity water 26 ul Total volume 68 pl 9 Dilute the PyroMark Control Oligo to 0 06 uM as shown in Table 9 Table 9 Dilution of the PyroMark Control Oligo Component Volume Concentration PyroMark Control Oligo 10ul 20 uM 1x Dilution buffer 90 ul First serial dilution 100 ul 2 uM First serial dilution from above 104l 2 uM 1x Dilution buffer 340 ul Final dilution 350 pl 0 06 uM Make sure that the 10x dilution buffer supplied with the PyroMark Control Oligo is diluted with high purity water before use See Things to do before starting page 36 38 PyroMark Control Oligo Handbook 07 2009 10 Add 68 pl of the master mix and 12 pl of the diluted 0 06 uM PyroMark Control Oligo to 8 wells of a 96 well PCR plate or strips 11 Seal the PCR plate or strips using strip caps 12 Agitate the PCR plate at room temperature 15 25 C for 5 10 min at 1400 rpm Note Sepharose beads sediment quickly Capturing of beads must take place immediately after agitation During this step prepare the PyroMark Q96 Vacuum Workstation for sample preparation see Appendix B page 54 13 Add 12 pl of PyroMark Annealing Buffer to the wells of the PyroMark Q96 HS Plate that will be used with the immobilized PyroMark Control Oligo to be processed with the PyroMark Q96 Vacuum Workstation Add 12 pl of the diluted 0 06 uM P
28. e the PyroMark Q24 Plate from the plate holder and let the samples cool to room temperature 15 25 C for at least 5 min Load the PyroMark Q24 Cartridge with the appropriate volumes of PyroMark Gold Q24 Reagents as given in the Pre Run Information report from step 8 The Pre Run Information report found in the Tools menu at run setup see the PyroMark Q24 Software User Guide provides information about the volume of nucleotides enzyme mixture and substrate mixture needed for the assay Open the cartridge gate and insert the filled PyroMark Q24 Cartridge with the label facing out Push the cartridge in fully and then push it down Ensure the cartridge is properly inserted and close the gate Refer to the PyroMark Q24 User Manual for more information Open the plate holding frame and place the plate on the heating block Close the plate holding frame and the instrument lid Insert the USB memory stick containing the run file into the USB port at the front of the instrument Do not remove the USB memory stick before the run is finished Select Run in the main menu using the and screen buttons g and press OK PyroMark Control Oligo Handbook 07 2009 39 40 41 42 43 44 45 46 47 48 49 50 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 p
29. erence in peak height with and without sample preparation page 52 Refer to the PyroMark Q24 User Manual for general troubleshooting of PyroMark Q24 Refer to the PyroMark Q96 ID User Manual for general troubleshooting of the PyroMark Q96 ID Refer to the PyroMark Q96 MD User Manual for general troubleshooting of the PyroMark Q96 MD In the text below the term Dispensing Unit includes Cartridges NDTs and CDTs used to dispense reagents in the various systems Quality assessment Comments and suggestions Warning from software about broad peaks Concentration of Follow the relevant protocol Make sure to PyroMark Control Oligo dilute the PyroMark Control Oligo in dilution too high buffer as described in the protocols 44 PyroMark Control Oligo Handbook 07 2009 Comments and suggestions High substrate peak Contaminated sample Change buffers Only use buffers that are leads to unusually high supplied by QIAGEN or QIAGEN authorized consumption of substrate distributors mixture noted as a high ier Use the zoom in function to check if any peaks presequencing signal have been generated select a section of Pyrogram with the left mouse button Poor or incorrect sequence a PyroMark Control Oligo Follow the instruction in the protocols for not correctly prepared preparing the PyroMark Control Oligo Make sure to dilute the PyroMark Control Oligo in the dilution buffer as described in the protocols Make sure that t
30. esearch 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 EE E gt gt gt UMMM 56 PyroMark Control Oligo Handbook 07 2009 Ordering Information Product PyroMark Control Oligo Accessories PyroMark Gold Q24 Reagents 5 x 24 PyroMark Annealing Buffer 250 ml PyroMark Binding Buffer 200 ml PyroMark Denaturation Solution 500 ml PyroMark Wash Buffer concentrate 200 ml PyroMark Q24 Plate 100 PyroMark Q24 Cartridge 3 PyroMark Gold Q96 Reagents 5 x 96 PyroMark Q96 Plate Low 100 PyroMark Q96 HS Plate 100 PyroMark Q96 Cartridge Contents For installation check of PyroMark systems For 5 x 24 samples for use on the PyroMark Q24 Enzyme Mixture Substrate Mixture and Nucleotides For annealing sequencing primer to single stranded PCR product and for Pyrosequencing reaction For binding of biotinylated PCR product to Sepharose beads For denaturation of double stranded PCR product into single stranded template DNA For washing of single stranded DNA 24 well sequencing reaction plate Cartridges for dispensing nucleotides and reagents For performing Pyrosequencing reactions on the PyroMark Q96 ID 5 x 96 and PyroMark Q96 MD 15 x 96 96 well sequencing reaction plate for use with PyroMark Q96 ID 100
31. f the PyroMark Q96 ID Troubleshooting Run Confirm the proper installation of the system and use of the reagents by evaluating the quality assessment quantification results background and difference in peak height with and without sample preparation PyroMark Q96 ID Software Select all wells by holding down the Ctrl or Shift key and click in the wells to mark them Selected wells appear with a square around them Click the arrow next to the Peak Heights Button on the Peak Heights Tab Select Export from the drop down list and save the data Open this file in Microsoft Excel Delimited and calculate the mean single peak height and background for each well see below PyroMark CpG Software Select Peak Heights from the Reports menu Save the data in a suitable format csv or tsv Open this file in Microsoft Excel Delimited and calculate the mean single peak height and background for each well see below Perform a quality assessment All wells should give Passed quality If the quality assessment is Check or Failed look in Well info PyroMark Q96 ID Software Well Information PyroMark CpG Software for explanations Evaluate the quantification results PyroMark Q96 ID Software Quantification results with standard deviation are shown in AQ Statistics under the Statistics Tab Select all wells by holding down the Ctrl or Shift key and click in the wells one by one to mark PyroMark Control Oligo Handbook
32. 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 Be sure to observe federal state and local environmental regulations for the disposal of laboratory waste The following item is required Bm High purity water Milli Q 18 2 MO x cm www millipore com or equivalent Procedure 1 Ensure that no vacuum is applied to the vacuum tool 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 The cap can be removed without disconnecting the tubing 5 If the PyroMark Q24 Vacuum Workstation must be cleaned for dust or spillage follow the instructions in Cleaning the PyroMark Q24 Vacuum Workstation in the PyroMark Q24 User Manual PyroMark Control Oligo Handbook 07 2009 55 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 r
33. g out Push the cartridge in fully and then push it down Ensure that the cartridge is properly inserted and close the gate Refer to the PyroMark Q24 User Manual for more information Open the plate holding frame and place the plate on the heating block Close the plate holding frame and the instrument lid Insert the USB memory stick containing the run file into the USB port at the front of the instrument Do not remove the USB memory stick before the run is finished Select Run in the main menu using the and screen buttons and press OK 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 When the run file is selected press Select to start the run When the run is finished and the instrument confirms that the run file has been saved to the USB memory stick press Close Remove the USB memory stick Open the instrument lid 25 Open the cartridge gate and remove the PyroMark Q24 Cartridge by lifting it up and pulling it out Close the gate Open the plate holding frame and remove the PyroMark Q24 Plate from the heating block PyroMark Control Oligo Handbook 07 2009 28 Close the plate holding frame and the instrument lid 29 Discard the PyroMark Q24 Plate and clean the PyroMark Q24 Cartridge see the PyroMark Gold Q24 Reagents Handbook 30 Open the
34. g the PyroMark Q24 Vacuum Workstation compared with PyroMark Control Oligo added directly to the PyroMark Q24 Plate should not be more than 20 If the values are within the set limits the system is properly installed If the results are not as stated above see Troubleshooting Guide page 44 for possible causes and actions to be taken If the troubleshooting guide does not explain the problem 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 PyroMark Control Oligo Handbook 07 2009 27 Protocol Troubleshooting Procedure for the PyroMark Q96 ID Instrument and PyroMark Q96 Vacuum Workstation If an unexpected result has been obtained it is crucial to determine whether this is related to the PyroMark Q96 ID Instrument the PyroMark Q96 Vacuum Workstation or the assay This protocol describes how to use the PyroMark Control Oligo to verify the function of the PyroMark Q96 ID Instrument and to compare results of samples prepared with or without the PyroMark Q96 Vacuum Workstation Important point before starting E Assay and run files can be set up using either PyroMark Q96 ID Software or PyroMark CpG Software mB For further information on how to create an Assay Setup and a Run Setup see the PyroMark Q96 ID Software Online Help mB For further information on how to create an Assay Setup and a Ru
35. he 10x dilution buffer provided is first diluted to 1x using high purity water b Incorrect dispensation Check that the correct sequence was typed in order the Assay Setup c Buffers or reagents Follow the instructions supplied with the incorrectly diluted or reagents Include an empty well containing incorrectly stored only PyroMark Annealing Buffer in your run to check if background peaks are coming from the nucleotides d Dispensation error seen Clean or replace the PyroMark Q24 or Q96 for example as split Cartridge If the problem remains contact peaks QIAGEN Technical Services for contact information see back cover or visit www qiagen com e Blocked Dispensing Unit Nucleotides are not dispensed correctly due to a blocked needle in the Dispensing Unit Clean the Dispensing Unit and check that it is working properly f Damaged Dispensing Unit Discard the Dispensing Unit according to federal state and local environmental regulations for disposal of laboratory waste g Annealing time too long Carry out annealing for the correct time and at the temperatures described in the protocols PyroMark Control Oligo Handbook 07 2009 45 Comments and suggestions Small or missing peaks a Insufficient amount of Make sure to dilute the PyroMark Control template for Oligo correctly and use the amounts specified immobilization in the protocols b Not enough enzyme or Fill the Dispensing Unit according to the subs
36. he PyroMark Q24 Vacuum Workstation at room temperature 15 25 C and use it as support when preparing and moving the plate 16 Place the PCR plate or strips and the PyroMark Q24 Plate on the worktable of the PyroMark Q24 Vacuum Workstation see Figure 5 Ensure that the plate is in the same orientation as when samples were loaded 16 PyroMark Control Oligo Handbook 07 2009 Figure 5 Placement of PCR plate or strips and PyroMark Q24 Plate on the PyroMark Q24 Vacuum Workstation The marked positions contain 70 ethanol 1 PyroMark Denaturation Solution 2 PyroMark Wash Buffer 3 and high purity water 4 5 P Parking position 17 18 19 20 21 22 23 Apply vacuum to the vacuum tool by opening the vacuum switch Carefully lower the filter probes into the PCR plate or strips to capture the beads containing immobilized template Hold the probes in place for 15 s Take care when picking up the tool Sepharose 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 Transfer the tool to the trough containing 70 ethanol trough 1 Flush the filter probes for 5 s Transfer the tool to the trough containing PyroMark Denaturation Solution trough 2 Flush the filter probes for 5 s Transfer the tool to the trough containing PyroMark Wash Buffer trough 3 Flush the filter probes for 10 s R
37. iew tab and with C indicated in a blue rectangle in the Pyrogram If the quality assessment is Check or Failed look in Well Information for explanations Evaluate the quantification results Select the AQ Analysis Statistics Report from the Reports menu Quantification results are given in the report with standard deviation PyroMark Control Oligo Handbook 07 2009 19 The C should be in the range 40 60 The standard deviation should not exceed 2 units m Evaluate single peak heights The mean single peak height should ideally be 50 15 RLU Sum single peaks dispensation 4 6 12 13 Mean single peak height i m Evaluate the background Background from blank dispensations should not exceed 3 Sum blanks dispensation 1 2 3 5 Background 96 x 100 Sum single peaks dispensation 4 6 12 13 If the values are within the set limits the system is properly installed If the results are not as stated above see Troubleshooting Guide page 44 for possible causes and actions to be taken If the troubleshooting guide does not explain the problem 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 cover or visit www qiagen com 20 PyroMark Control Oligo Handbook 07 2009 Protocol Troubleshooting Procedure for the PyroMark Q24 Instrument and PyroMark Q24 Vacuum Wor
38. ining 70 ethanol trough 1 Flush the filter probes for 5 s 20 Transfer the tool to the trough containing PyroMark Denaturation Solution trough 2 Flush the filter probes for 5 s 21 Transfer the tool to the trough containing PyroMark Wash Buffer trough 3 Flush the filter probes for 10 s 22 Raise the tool up and back beyond 90 vertical for 5 s to drain liquid from the filter probes Figure 20 Illustration of the vacuum tool raised to beyond 90 vertical 23 While the tool is held over the PyroMark Q96 HS Plate close the vacuum switch on the vacuum workstation Off 24 Release the beads in the plate containing 12 ul PyroMark Annealing Buffer by shaking the tool from side to side Allow the filter probes to rest on the bottom of the wells 25 Transfer the tool to the trough containing high purity water trough 4 and agitate the tool for 10 s 40 PyroMark Control Oligo Handbook 07 2009 26 Wash the filter probes by lowering the probes into the second trough with high purity water parking position and applying vacuum Flush the probes with 180 ml high purity water 27 Raise the tool up and back beyond 90 vertical for 5 s to drain liquid from the filter probes see Figure 20 28 Turn off the vacuum switch on the vacuum workstation Off and place the tool in the Parking P position 29 Turn off the vacuum pump Note At the end of a working day liquid waste and remaining solutions should be
39. ion During this step prepare the PyroMark Q24 Vacuum Workstation for sample preparation see Appendix A page 53 PyroMark Control Oligo Handbook 07 2009 23 15 Add 25 pl of PyroMark Annealing Buffer to each well of the PyroMark Q24 Plate that will be used with the immobilized PyroMark Control Oligo to be processed with the PyroMark Q24 Vacuum Workstation Add 25 ul of the diluted 0 04 uM PyroMark Q24 Control Oligo to 8 additional wells according to the Run Setup Keep one of the PyroMark Q24 Plate Holders supplied with the PyroMark Q24 Vacuum Workstation at room temperature 15 25 C and use it as support when preparing and moving the plate 16 Place the PCR plate or strips and the PyroMark Q24 Plate on the worktable of the PyroMark Q24 Vacuum Workstation see Figure 9 Ensure that the plate is in the same orientation as when samples were loaded Figure 9 Placement of PCR plate and PyroMark Q24 Plate on the PyroMark Q24 Vacuum Workstation The marked positions contain 7096 ethanol 1 PyroMark Denaturation Solution 2 PyroMark Wash Buffer 3 and high purity water 4 5 P Parking position 17 Apply vacuum to the tool by opening the vacuum switch 18 Carefully lower the filter probes into the PCR plate or strips to capture the beads containing immobilized template Hold the probes in place for 15 s Take care when picking up the tool Sepharose beads sediment quickly If more than
40. k cover or visit www qiagen com PyroMark Control Oligo Handbook 07 2009 35 Protocol Troubleshooting Procedure for the PyroMark Q96 MD Instrument and PyroMark Q96 Vacuum Workstation If an unexpected result has been obtained it is crucial to determine whether this is related to the PyroMark Q96 MD Instrument the PyroMark Q96 Vacuum Workstation or the assay This protocol describes how to use the PyroMark Control Oligo to verify the function of the PyroMark Q96 MD Instrument comparing results of samples prepared with or without the PyroMark Q96 Vacuum Workstation Important point before starting E Assay and run files can be set up using either PyroMark Q96 MD Software or PyroMark CpG Software mB For further information on how to create an Assay Setup and a Run Setup see the PyroMark Q96 MD Software Online Help EB For further information on how to create an Assay Setup and a Run Setup with the PyroMark CpG software see the PyroMark CpG Software Online Help Things to do before starting mB Follow the instructions in PyroMark Q96 MD User Manual to install the PyroMark Q96 MD EB The dilution buffer provided with the PyroMark Control Oligo needs to be diluted before use Prepare 1x dilution buffer by mixing 200 ul of 10x dilution buffer with 1800 ul of high purity water E Place the PyroMark Q96 HS Sample Prep Thermoplate on a heating block at 80 C for use in step 30 BE Allow all required reagents and solutions to reach ro
41. kstation If an unexpected result has been obtained it is crucial to determine whether this is related to the PyroMark Q24 Instrument the PyroMark Q24 Vacuum Workstation or the assay This protocol describes how to use the PyroMark Control Oligo to verify the function of the PyroMark Q24 Instrument comparing results with or without the PyroMark Q24 Vacuum Workstation Important point before starting mB For further information on how to create an Assay Setup and a Run Setup see the PyroMark Q24 Software User Guide Things to do before starting Bm Follow the instructions in PyroMark Q24 User Manual to install the PyroMark Q24 EB The dilution buffer provided with the PyroMark Control Oligo needs to be diluted before use Prepare 1x dilution buffer by mixing 200 ul of 10x dilution buffer with 1800 ul of high purity water M Place the PyroMark Q24 Plate Holder on a heating block at 80 C for use in step 30 amp Allow all required reagents and solutions to reach room temperature 15 25 C before starting Procedure 1 Set up an assay for the PyroMark Control Oligo by using the PyroMark Q24 Software 2 Click in the toolbar and select New AQ Assay 3 Type the following sequence in Sequence to Analyze TAYGGTITGCA For more information on how to create an Assay Setup file see the PyroMark Q24 Software User Guide 4 Manually enter the following Dispensation Order ACGTTATCGTTGC For more information on
42. le see the PyroMark Q24 Software User Guide 14 PyroMark Control Oligo Handbook 07 2009 Figure 4 Histogram for AQ mode Nucleotide additions 1 2 3 5 and 11 are blank and serve as negative controls The seventh and the eight dispensations analyze the variable position 5 Click amp in the toolbar to save the assay 6 Create a Run Setup by importing the assay parameters to all 24 wells To add an assay to a well you can either m Right click the well and select Load Assay from the context menu m Select the assay in the shortcut browser and click and drag the assay to the well A well is color coded according to the assay type loaded to the well For more information on how to create a Run Setup file see the PyroMark Q24 Software User Guide 7 Save the Run Setup to a USB memory stick supplied with the PyroMark Q24 system 8 Print a list of required volumes of enzyme mix substrate mix and nucleotides and the plate setup Select Pre Run Information from the Tools menu and when the report appears click 3 9 Gently shake the bottle containing Streptavidin Sepharose High Performance until it is a homogeneous solution 10 Prepare a master mix for DNA immobilization according to Table 2 Prepare a volume 1096 greater than that required for the total number of reactions to be performed Table 2 Master mix for DNA immobilization Number of samples 1 26 Streptavidin Sepharose High Perfor
43. mance ap seu PyroMark Binding Buffer 40 ul 1040 ul High purity water 13 pl 338 ul Total volume 55 pl 1430 pl Provides a sufficient amount for the 24 samples required PyroMark Control Oligo Handbook 07 2009 15 11 Dilute the PyroMark Control Oligo to 0 04 uM as shown in Table 3 Table 3 Dilution of the PyroMark Control Oligo Component Volume Concentration PyroMark Control Oligo 10 ul 20 uM 1x Dilution buffer 90 ul First serial dilution 100 pl 2 uM First serial dilution from above 30 ul 2 uM 1x Dilution buffer 1470 ul Final dilution 1500 pl 0 04 uM Make sure that the 10x dilution buffer supplied with the PyroMark Control Oligo is diluted with high purity water before use See Things to do before starting page 14 12 Shake the tube containing the master mix and add 55 ul of the master mix and 25 ul of the diluted 0 04 uM PyroMark Control Oligo to all 24 wells of a 24 well PCR plate or strips 13 Seal the PCR plate or strips using strip caps 14 Agitate the PCR plate at room temperature 15 25 C for 5 10 min at 1400 rpm Sepharose beads sediment quickly Capturing of beads must take place immediately following agitation During this step prepare the PyroMark Q24 Vacuum Workstation for sample preparation see Appendix A page 53 15 Add 25 ul of PyroMark Annealing Buffer to each well of a PyroMark Q24 Plate Keep one of the PyroMark Q24 Plate Holders supplied with t
44. n Setup with the PyroMark CpG Software see the PyroMark CpG Software Online Help Things to do before starting Bm Follow the instructions in PyroMark Q96 ID User Manual to install the PyroMark Q96 ID EB The dilution buffer provided with the PyroMark Control Oligo needs to be diluted before use Prepare 1x dilution buffer by mixing 200 pl of 10x dilution buffer with 1800 ul of high purity water M Place the PyroMark Q96 Sample Prep Thermoplate Low on a heating block at 80 C for use in step 30 BE Allow all required reagents and solutions to reach room temperature 15 25 C before starting Procedure 1 Set up a simplex entry for the PyroMark Control Oligo by using the PyroMark Q96 ID Software alternatively an assay in the PyroMark CpG Software using the parameters below 2 Type the following sequence in Sequence to Analyze TAYGGTITGCA 3 Manually enter the following Dispensation Order ACGTTATCGTTGC 28 PyroMark Control Oligo Handbook 07 2009 For more information on how to create an Assay Setup file see the PyroMark Q96 ID Software Online Help or the PyroMark CpG Software Online Help Note For the CpG assay ignore the warning about sequence direction that is shown when the sequence to analyze and dispensation order is added Note For a thorough control of the instrument sample preparation and reagents additional blanks are included in the dispensing order TAC 0 A C G T T A T G T T B C Figure 12
45. n open the instrument lid 41 Open the dispensing unit and remove the dispensing tip holder and the PyroMark Q96 HS Plate PyroMark Control Oligo Handbook 07 2009 41 42 Close the dispensing unit and the instrument lid see PyroMark Q96 MD User Manual or PyroMark CpG Software Online Help 43 Discard the PyroMark Q96 Plate and clean the tips in the PyroMark Q96 Dispensing Tip Holder see the PyroMark Gold Q96 Reagents Handbook 44 Open the run in the PyroMark Q96 MD Software and analyze all wells The peak pattern should look like the one in Figure 21 T 46 296 C 53 896 2100 2000 1900 1700 1600 1500 1300 pe t ks CE E s c G T T G T T G c 5 10 Figure 21 Pyrogram trace from PyroMark MD Software AQ mode 45 Confirm the proper installation of the system and use of the reagents by evaluating the quality assessment quantification results background and difference in peak height with and without sample preparation PyroMark Q96 MD Software Select all wells by holding down the Ctrl or Shift key and click in the wells to mark them Selected wells appear with a square around them Click the arrow next to the Peak Heights Button on the Peak Heights Tab Select Export from the drop down list and save the data Open this file in Microsoft Excel Delimited and calculate the mean single peak height and background for each well see below PyroMark CpG Software Select Peak Heights from the Reports
46. n those expressly stated QV expo uo NS The purchaser and user of the product 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 Product and or its 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 Technical 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
47. nd allocate the created entry assay to appropriate wells Choose instrument parameters supplied by QIAGEN according to the reagents and cartridge that will be used for the run We recommend using 16 wells 8 wells for samples prepared using the PyroMark Q96 Vacuum Workstation and 8 samples added directly to PyroMark Q96 HS Plate Note A well is color coded according to the assay type loaded to the well For more information on how to create a Run Setup file in PyroMark Q96 MD Software see the PyroMark Q96 MD Software Online Help For more information on how to create a Run Setup file in PyroMark CpG Software see the PyroMark CpG Software Online Help PyroMark Control Oligo Handbook 07 2009 37 6 7 Save the Run Setup Print a list of required volumes of enzyme mix substrate mix and nucleotides and the plate setup Note To generate a list of required volumes in the PyroMark Q96 MD Software click View and Run in the browser area To generate a list of required volumes in the PyroMark CpG Software open the run file and select Volume Information from the Tools menu Prepare a master mix for DNA immobilization according to Table 8 Prepare a volume 10 greater than that required for the total number of reactions to be performed Note Gently shake the bottle containing Streptavidin Sepharose High Performance until it is a homogeneous solution Table 8 Master mix for DNA immobilization Component Volume sampl
48. ntain 70 ethanol 1 PyroMark Denaturation Solution 2 PyroMark Wash Buffer 3 and high purity water 4 Refill the troughs to approximately these levels whenever needed Start the vacuum pump Apply vacuum to the tool by opening the vacuum switch Wash the filter probes by lowering the probes into high purity water Parking position Let approximately 180 ml of water flush through the filter probes i e empty the trough Ensure that the water is being transferred to the waste container If not ensure that the tubing is connected properly and there is no leakage 6 Refill Parking Position with 180 ml of high purity water SU depo P9 54 PyroMark Control Oligo Handbook 07 2009 7 Switch off the vacuum switch on the vacuum workstation Off and place the tool in the Parking P position Appendix C Emptying the Waste Container and Troughs WARNING Hazardous chemicals The PyroMark Denaturation Solution used with the PyroMark Q24 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 instrument operators are not exposed to hazardous levels of toxic substances chemical or biological as defined in the applicable Material Safety Data Sheets MSDs or OSHA ACGIH or COSHH documents Venting
49. oMark Control Oligo Component Volume Concentration PyroMark Control Oligo 10 ul 20 uM 1x Dilution buffer 90 ul First serial dilution 100 pl 2 uM First serial dilution from above 30 ul 2 uM 1x Dilution buffer 1470 ul Final dilution 1500 pl 0 04 uM Make sure that the 10x dilution buffer supplied with the PyroMark Control Oligo is diluted with high purity water before use See Things to do before starting page 10 PyroMark Control Oligo Handbook 07 2009 11 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 26 27 Add 25 ul of the diluted 0 04 uM PyroMark Control Oligo to each well of a PyroMark Q24 Plate Heat the PyroMark Q24 Plate with the PyroMark Oligo at 80 C for 2 min using a heating block and the prewarmed PyroMark Q24 Plate Holder Remove the PyroMark Q24 Plate from the plate holder and let the samples cool to room temperature 15 25 C for at least 5 min Load the PyroMark Q24 Cartridge with the appropriate volumes of PyroMark Gold Q24 Reagents as given in the Pre Run Information report from step 8 The Pre Run Information report found in the Tools menu at run setup see the PyroMark Q24 Software User Guide provides information about the volume of nucleotides enzyme mixture and substrate mixture needed for the assay Open the cartridge gate and insert the filled PyroMark Q24 Cartridge with the label facin
50. oMark Control Oligo 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 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 Control Oligo is tested against predetermined specifications to ensure consistent product quality 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 qiagen com support MSDS aspx where you can find view and print the MSDS for each QIAGEN kit and kit component 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 PyroM
51. oMark Control Oligo to 8 wells of a 96 well PCR plate or strips 11 Seal the PCR plate or strips using strip caps 12 Agitate the PCR plate at room temperature 15 25 C for 5 10 min at 1400 rpm During this step prepare the PyroMark Q96 Vacuum Workstation for sample preparation see Appendix B page 54 Note Sepharose beads sediment quickly Capturing of beads must take place immediately following agitation 13 Add 40 pl of PyroMark Annealing Buffer to the wells of the PyroMark Q96 Plate Low that will be used with the immobilized PyroMark Control Oligo to be processed with the PyroMark Q96 Vacuum Workstation Add 40 ul of the diluted 0 05 uM PyroMark Control Oligo to 8 additional wells according to the Run Setup Since the oligonucleotide is self primed no sequencing primer is required The beads are released into PyroMark Annealing Buffer 14 Place the PCR plate or strips and the PyroMark Q96 Plate Low on the worktable of the PyroMark Q96 Vacuum Workstation see Figure 14 Ensure that the plate is in the same orientation as when samples were loaded Figure 14 Placement of PCR plate or strips and PyroMark Q96 Plate Low on the PyroMark Q96 Vacuum Workstation The marked positions contain 70 ethanol 1 PyroMark Denaturation Solution 2 PyroMark Wash Buffer 3 and high purity water 4 15 Apply vacuum to the tool by opening the vacuum switch on the vacuum workstation PyroMark Control Oligo Handbook 07 2
52. ocked Dispensing Unit as indicated by a missing presequencing signal and no peaks in the Pyrogram Clean the Dispensing Unit and check that it is working properly Discard the Dispensing Unit according to federal state and local environmental regulations for disposal of laboratory waste Follow the instructions supplied with the reagents Follow the instruction in the protocols for preparing the PyroMark Control Oligo Make sure to dilute the PyroMark Control Oligo in the dilution buffer as described in the protocols Make sure that the 10x dilution buffer provided is first diluted to 1x using high purity water PyroMark Control Oligo Handbook 07 2009 Comments and suggestions j Contaminated sample Change buffers Only use buffers that are leads to unusually high supplied by QIAGEN or QIAGEN authorized consumption of substrate distributors mixture noted as a high DD Use the zoom in function to check if any peaks presequencing signal have been generated select a section of Pyrogram with the left mouse button Very high peaks PyroMark Control Oligo Follow the instruction in the protocols for not correctly prepared preparing the PyroMark Control Oligo Make sure to dilute the PyroMark Control Oligo in the dilution buffer as described in the protocols Make sure that the 10x dilution buffer provided is first diluted to 1x using high purity water Background Comments and suggestions High background a The s
53. om temperature 15 25 C before starting Procedure 1 Set up a simplex entry for the PyroMark Control Oligo by using the PyroMark Q96 MD Software alternatively set up an assay in the PyroMark CpG Software using the parameters below 2 Type the following sequence in Sequence to Analyze TAYGGTITGCA 3 Manually enter the following Dispensation Order ACGTTATCGTTGC 36 PyroMark Control Oligo Handbook 07 2009 For more information on how to create an Assay Setup file see the PyroMark Q96 MD Software Online Help or the PyroMark CpG Software Online Help Note For the CpG assay ignore the warning about sequence direction that is shown when the sequence to analyze and dispensation order is added For a thorough control of the instrument sample preparation and reagents additional blanks are included in the dispensing order TAC Figure 17 Histogram from PyroMark Q96 MD Software Nucleotide additions 1 2 3 5 and 11 are blank dispensations and serve as negative controls The seventh and the eighth dispensations analyze the variable position wobbled degenerated base Figure 18 Histogram from PyroMark CpG Software Nucleotide additions 1 2 3 5 and 11 are blank dispensations and serve as negative controls The seventh and the eighth dispensations analyze the variable position wobbled degenerated base 4 Save the entry PyroMark Q96 MD Software assay PyroMark CpG Software 5 Open a new run a
54. on the tool Off and place the tool in the Parking P position Turn off the vacuum pump At the end of a working day liquid waste and remaining solutions should be discarded and the PyroMark Q24 Vacuum Workstation should be checked for dust and spillage see Appendix C page 55 Heat the PyroMark Q24 Plate with the samples at 80 C for 2 min using a heating block and the prewarmed PyroMark Q24 Plate Holder Remove the PyroMark Q24 Plate from the plate holder and let the samples cool to room temperature 15 25 C for at least 5 min Load the PyroMark Q24 Cartridge with the appropriate volumes of PyroMark Gold Q24 Reagents as given in the Pre Run Information report from step 8 The Pre Run Information report found in the Tools menu at run setup see the PyroMark Q24 Software User Guide provides information about the volume of nucleotides enzyme mixture and substrate mixture needed for the assay PyroMark Control Oligo Handbook 07 2009 25 33 Open the cartridge gate and insert the filled PyroMark Q24 Cartridge with the label facing out Push the cartridge in fully and then push it down 34 Ensure the cartridge is properly inserted and close the gate Refer to the PyroMark Q24 User Manual for more information 35 Open the plate holding frame and place the plate on the heating block 36 Close the plate holding frame and the instrument lid 37 Insert the USB memory stick containing the run file into the USB p
55. ort at the front of the instrument Do not remove the USB memory stick before the run is finished 38 Select Run in the main menu using the and screen buttons and press OK 39 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 40 When the run file is selected press Select to start the run 41 When the run is finished and the instrument confirms that the run file has been saved to the USB memory stick press Close 42 Remove the USB memory stick 43 Open the instrument lid 44 Open the cartridge gate and remove the PyroMark Q24 Cartridge by lifting it up and pulling it out 45 Close the gate 46 Open the plate holding frame and remove the PyroMark Q24 Plate from the heating block 47 Close the plate holding frame and the instrument lid 48 Discard the PyroMark Q24 Plate and clean the PyroMark Q24 Cartridge see the PyroMark Gold Q24 Reagents Handbook 49 Open the run in the PyroMark Q24 Software and analyze all wells The peak pattern should look like the one in Figure 11 C 5396 T 4796 L L n L 4 2L n n 1 1 1 E S A C G T T A T Cc G T T G C 5 10 Figure 11 Pyrogram of PyroMark Q24 Troubleshooting Run 26 PyroMark Control Oligo Handbook 07 2009 50 Confirm the proper installation of the system and use of the reagents by evaluating the quality assessment quantific
56. pensation the Assay Setup order PyroMark Control Oligo Handbook 07 2009 47 Comments and suggestions c Buffers or reagents Follow the instructions supplied with the incorrectly diluted or reagents Include an empty well containing incorrectly stored only PyroMark Annealing Buffer in your run to check if background peaks are coming from the nucleotides d Dispensation error seen Clean or replace the Dispensing Unit If the for example as split problem remains contact QIAGEN Technical peaks Services for contact information see back cover or visit www qiagen com e Blocked Dispensing Unit Nucleotides are not dispensed correctly due to a blocked needle in the Dispensing Unit Clean the Dispensing Unit and check that it is working properly f Damaged Dispensing Unit Discard the Dispensing Unit according to federal state and local environmental regulations for disposal of laboratory waste g Annealing time too long Carry out annealing for the correct time and at the temperatures described in the protocols High background a The storage conditions for Check the storage conditions and the one or more reagent did expiration date of the reagents and use new not comply with the reagents if necessary instructions given in Storage page 4 b Reagents have expired Check the storage conditions and the expiration date of the reagents and use new reagents if necessary 48 PyroMark Control Oligo Handbook 07 200
57. plates in each package 96 well sequencing reaction plate for use with PyroMark Q96 MD 100 plates in each package Cartridges for dispensing nucleotides and reagents For use with PyroMark Q24 PyroMark Q96 MD and PyroMark Q96 ID t For use with PyroMark Q24 Vacuum Workstation and PyroMark Q96 Vacuum Workstation PyroMark Control Oligo Handbook 07 2009 Cat no 979203 971802 979009 979006 979007 979008 979201 979302 972804 979002 979101 979004 57 Ordering Information Product Contents Cat no PyroMark Q96 HS Reusable tips 4 in each package for 979102 Reagent Tip 4 dispensing reagents RDTs for use with PyroMark Q96 MD PyroMark Q96 HS Reusable tips 8 in each package for 979103 Nucleotide Tip 8 dispensing nucleotides NDTs for use with PyroMark Q96 MD Related products PyroMark Q24 Sequence based detection platform for 9001514 Instrument Pyrosequencing of 24 samples in parallel PyroMark Q24 Application software for PyroMark Q24 9019062 Software PyroMark Q24 Vacuum Workstation for preparing single Varies Workstation stranded DNA from 24 samples PyroMark Q24 For performance check of system 979204 Validation Oligo PyroMark Q96 ID Sequence based detection platform for 9001525 Instrument Pyrosequencing of 96 samples in parallel PyroMark Q96 ID Application software for PyroMark Q96 9019083 Software ID PyroMark Q96 MD Sequence based detection platform for 9001526
58. rantees the performance of all products in the manner described in 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 4 PyroMark Control Oligo Handbook 07 2009 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 giagen 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 Pyr
59. ress Back When the run file is selected press Select to start the run When the run is finished and the instrument confirms that the run file has been saved to the USB memory stick press Close Remove the USB memory stick Open the instrument lid Open the cartridge gate and remove the PyroMark Q24 Cartridge by lifting it up and pulling it out Close the gate Open the plate holding frame and remove the PyroMark Q24 Plate from the heating block Close the plate holding frame and the instrument lid Discard the PyroMark Q24 Plate and clean the PyroMark Q24 Cartridge see the PyroMark Gold Q24 Reagents Handbook Open the run in the PyroMark Q24 Software and analyze all wells The peak pattern for Run 2 should look like the one in Figure 7 C 54 T 46 wa Figure 7 Pyrogram of Run 2 Confirm the proper installation of the system and use of the reagents by evaluating the quality assessment quantification results single peak heights and background To obtain peak height values select Export Peak Heights from the Tools menu Save the data in a suitable format csv or tsv Open this file in Microsoft Excel Delimited and calculate the mean single peak height and background for each well as described below m Perform a quality assessment All wells should give Passed quality shown as a blue bar in the bottom field of the well when looking at the overv
60. strument and PyroMark Q96 Vacuum Workstation follow Protocol Troubleshooting Procedure for the PyroMark Q96 ID Instrument and PyroMark Q96 Vacuum Workstation page 28 A Pyrosequencing reaction is performed with 8 wells containing the PyroMark Control Oligo and 8 wells containing the PyroMark Control Oligo prepared on the PyroMark Q96 Vacuum Workstation Troubleshooting the PyroMark Q96 MD Instrument and PyroMark Q96 Vacuum Workstation To perform a troubleshooting of the PyroMark Q96 MD Instrument and PyroMark Q96 Vacuum Workstation follow Protocol Troubleshooting Procedure for the Instrument and PyroMark Q96 Vacuum Workstation page 36 A Pyrosequencing reaction is performed with 8 wells containing the PyroMark Control Oligo and 8 wells containing the PyroMark Control Oligo prepared on the PyroMark Q96 Vacuum Workstation PyroMark Control Oligo Handbook 07 2009 7 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 For use with the PyroMark Q24 System Bm PyroMark Q24 Instrument cat no 9001514 PyroMark Q24 Software cat no 9019062 PyroMark Q24 Plate 100 cat no 979201 PyroMark Q24 Cartridge 3 cat no 979202 PyroMark Q24 Vacuum Workstation cat no 9001518 220V 9001516 110V 9001519 1
61. t and PyroMark Q96 Vacuum Workstation 36 Troubleshooting Guide 44 Appendix A Preparation of the PyroMark Q24 Vacuum Workstation 53 Appendix B Preparation of the PyroMark Q96 Vacuum Workstation 54 Appendix C Emptying the Waste Container and Troughs 55 References 56 Ordering Information 57 PyroMark Control Oligo Handbook 07 2009 3 Kit Contents PyroMark Control Oligo Catalog no 979203 Control Oligo 20 UM 50 ul 10x Dilution Buffer 2x 1 7 ml Handbook 1 Storage The PyroMark Control Oligo should be stored at 20 C upon arrival Repeated thawing and freezing gt 5 x per year should be avoided The PyroMark Control Oligo is stable until the expiration date when stored under these conditions Product Use Limitations PyroMark Control Oligo is intended for molecular biology applications This product is neither intended for the diagnosis prevention or treatment of a disease nor has it 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 Product Warranty and Satisfaction Guarantee QIAGEN gua
62. t lid 35 Open the process chamber lid 36 Open the plate holding frame 37 Place the PyroMark Q96 Plate Low on the heating block 38 Close the plate holding frame and the process chamber lid 39 Open the dispensing unit cover release the latch then open the cover 40 Insert the filled dispensing cartridge with the label facing out 41 Close the dispensing unit cover Make sure that the latch snaps into its locked position 42 Close the instrument lid 43 Perform a run see PyroMark Q96 ID User Manual or the PyroMark CpG Software Online Help 44 After run open the instrument lid 45 Open the dispensing unit and remove the reagent cartridge by lifting it up and pulling it out 46 Close the dispensing unit PyroMark Control Oligo Handbook 07 2009 33 47 48 49 50 51 34 Open the process chamber lid and remove the PyroMark Q96 Plate Low from the heating block see PyroMark Q96 ID User Manual Close the process chamber and the instrument lid see PyroMark Q96 ID User Manual Discard the PyroMark Q96 Plate Low and clean the PyroMark Q96 Cartridge see the PyroMark Gold Q96 Reagents Handbook Open the run in the PyroMark Q96 ID Software PyroMark CpG Software and analyze all wells see PyroMark Q96 ID Software Online Help PyroMark CpG Software Online help The peak pattern should look like the one in Figure 16 T 48 196 51 996 5 10 Figure 16 Pyrogram o
63. the protocols Small or missing peaks a Insufficient amount of Make sure to dilute the PyroMark Control Oligo template for correctly and use the amounts specified in the immobilization protocols b Not enough enzyme or Fill the Dispensing Unit according to the substrate for all wells instructions in the Pre Run Information report PyroMark Control Oligo Handbook 07 2009 49 o h 50 Wells marked in the Run Setup do not agree with sample placement in the plate One or more of the nucleotide compartments in the Dispensing Unit not correctly filled with reagents or nucleotides Dispensation error seen for example as split peaks Blocked Dispensing Unit Damaged Dispensing Unit Buffers or reagents incorrectly diluted or incorrectly stored PyroMark Control Oligo not correctly prepared Comments and suggestions Check that you loaded the PyroMark Q24 or Q96 Plate correctly according to the Run Setup Make sure that sufficient reagents are added to the Dispensing Unit Follow the instructions for use supplied with the products Clean or replace the Dispensing Unit If the problem remains contact QIAGEN Technical Services for contact information see back cover or visit www giagen com Nucleotides are not dispensed correctly due to a blocked needle in the Dispensing Unit Clean the Dispensing Unit and check that it is working properly Enzymes or substrates are not dispensed correctly due to a bl
64. to verify proper installation of the PyroMark instruments Function of the PyroMark Q24 Instrument To verify correct function of the PyroMark Q24 Instrument follow Protocol Verifying the Function of the PyroMark Q24 Instrument page 10 The PyroMark Control Oligo is added directly to PyroMark Q24 Plate without prior preparation on the PyroMark Q24 Vacuum Workstation 6 PyroMark Control Oligo Handbook 07 2009 Function of the PyroMark Q24 Instrument and PyroMark Q24 Vacuum Workstation To verify correct function of the PyroMark Q24 Instrument and PyroMark Q24 Vacuum Workstation follow Protocol Verifying the Function of the PyroMark Q24 Instrument and PyroMark Q24 Vacuum Workstation page 14 The PyroMark Control Oligo is prepared using the PyroMark Q24 Vacuum Workstation before analysis on the PyroMark Q24 Troubleshooting the PyroMark Q24 Instrument and PyroMark Q24 Vacuum Workstation To perform a troubleshooting of the PyroMark Q24 Instrument and PyroMark Q24 Vacuum Workstation follow Protocol Troubleshooting Procedure for the PyroMark Q24 Instrument and PyroMark Q24 Vacuum Workstation page 21 A Pyrosequencing reaction is performed with 8 wells containing the PyroMark Control Oligo and 8 wells containing the PyroMark Control Oligo prepared on the PyroMark Q24 Vacuum Workstation Troubleshooting the PyroMark Q96 ID Instrument and PyroMark Vacuum Workstation To perform a troubleshooting of the PyroMark Q96 ID In
65. torage conditions Check the storage conditions and the expiration for one or more reagent date of the reagents and use new reagents if did not comply with the necessary instructions given in Storage page 4 b Reagents have expired Check the storage conditions and the expiration date of the reagents and use new reagents if necessary m N X sm PyroMark Control Oligo Handbook 07 2009 51 Difference in peak height with and without sample preparation Comments and suggestions Incorrect sample preparation a Liquid left in some wells Replace corresponding filter probe in the vacuum or tubes when capturing tool of the PyroMark Q24 or PyroMark Q96 the beads containing Vacuum Workstation See the sample preparation immobilized template to guidelines available at our Technical Support the filter probes 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 b Filter probes not Check the filter probes Add 80 ul of high purity working properly water to each well of a PCR plate Start the vacuum pump and apply vacuum by opening the vacuum switch On Lower the vacuum tool into the PCR plate and wait 10 s Check that all wells of the PCR plate are empty If not replace the failed filter probes and repeat the test c White debris
66. trate for all wells instructions in the Pre Run Information report c Wells marked in the Run Check that you loaded the PyroMark Plate Setup do not agree with correctly according to the Run Setup sample placement in the plate d One or more of the Make sure that sufficient reagents are added to nucleotide compartments the Dispensing Unit Follow the instructions for in the Dispensing Unit not use supplied with the products correctly filled with reagents or nucleotides e Dispensation error seen Clean or replace the Dispensing Unit If the for example as split problem remains contact QIAGEN Technical peaks Services for contact information see back cover or visit www giagen com f Blocked Dispensing Unit Nucleotides are not dispensed correctly due to a blocked needle in the Dispensing Unit Clean the Dispensing Unit and check that it is working properly Enzymes or substrates are not dispensed correctly due to a blocked Dispensing Unit as indicated by a missing presequencing signal and no peaks in the Pyrogram Clean the Dispensing Unit and check that it is working properly g Damaged Dispensing Unit Discard the Dispensing Unit according to federal state and local environmental regulations for disposal of laboratory waste h Buffers or reagents Follow the instructions supplied with the incorrectly diluted or reagents incorrectly stored 46 PyroMark Control Oligo Handbook 07 2009 Comments and suggestions
67. ugh containing high purity water trough 4 and agitate the tool for 10 s 32 PyroMark Control Oligo Handbook 07 2009 26 Wash the filter probes by lowering the probes into the second trough with high purity water parking position and applying vacuum Flush the probes with 180 ml high purity water 27 Raise the tool up and back beyond 90 vertical for 5 s to drain liquid from the filter probes see Figure 15 28 Turn off the vacuum switch on the vacuum workstation Off and place the tool in the Parking P position 29 Turn off the vacuum pump At the end of a working day liquid waste and remaining solutions should be discarded and the PyroMark Q96 Vacuum Workstation should be checked for dust and spillage see Appendix C page 55 30 Heat the PyroMark Q96 Plate Low with the samples at 80 C for 2 min using a heating block and the prewarmed PyroMark Q96 Sample Prep Thermoplate Low 31 Remove the PyroMark Q96 Plate from the thermo plate and let the samples cool to room temperature 15 25 C for at least 5 min 32 Load the PyroMark Q96 Cartridge with the appropriate volumes of PyroMark Gold Q96 Reagents Note To generate a list of required volumes in the PyroMark Q96 ID Software click View and Run in the browser area To generate a list of required volumes in the PyroMark CpG Software open the run file and select Volume Information from the Tools menv 33 Switch on the instrument 34 Open the instrumen
68. yroMark Control Oligo to 8 additional wells according to the Run Setup Note Since the oligonucleotide is self primed no sequencing primer is required The beads are released into PyroMark Annealing Buffer 14 Place the PCR plate or strips and the PyroMark Q96 HS Plate on the worktable of the PyroMark Q96 Vacuum Workstation see Figure 19 Note Ensure that the plate is in the same orientation as when samples were loaded Figure 19 Placement of PCR plate or strips and PyroMark Q96 HS Plate on the PyroMark Q96 Vacuum Workstation The marked positions contain 7096 ethanol 1 PyroMark Denaturation Solution 2 PyroMark Wash Buffer 3 and high purity water 4 15 Apply vacuum to the tool by opening the vacuum switch on the vacuum workstation PyroMark Control Oligo Handbook 07 2009 39 16 Wash the filter probes by lowering the probes into high purity water parking position Let approximately 180 ml of water flush through the filter probes i e empty the trough 17 Refill the parking position with 180 ml high purity water 18 Carefully lower the filter probes into the PCR plate or strips to capture the beads containing immobilized template Hold the probes in place for 15 s Take care when picking up the tool Note Sepharose 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 19 Transfer the tool to the trough conta

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