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Ion PGM Template OT2 200 Kit User Guide (Pub. no

1. s Description 121 Supplier Cat number Quantity lon PGM Controls Kit v2 Life Technologies 4482010 1 Benchtop absorbent paper or mat MLS Bleach MLS Ethernet cable MLS 1 1 8 inch L wrench hex wrench or equivalent tool MLS 1 Heat block to be set at 75 C MLS 1 11 We have demonstrated this protocol using this specific material Substitution may adversely affect system performance 21 For materials needed for optional quality control of ISPs by the Qubit 2 0 Fluorometer see Appendix A Quality control of lon PGM Template OT2 200 lon Sphere Particles 12 lon PGM Template OT2 200 Kit User Guide Equipment use Prerequisites Contamination Chapter 1 Product information a Equipmentuse The Ion OneTouch 2 System is an automated system used for the generation of template positive lon PGM Template OneTouch 2 200 Ion Sphere Particles for semiconductor sequencing The manufacturer is not liable for any damage or injury that results from use of this manual by unauthorized or untrained parties This guide uses conventions and terminology that assume a working knowledge of the Microsoft Windows operating system the Internet and Internet based browsers The Ion OneTouch 2 System is For Research Use Only Not for use in diagnostic procedures f CAUTION A primary source of contamination is DNA fragments from previous sample proce
2. MII Order 39 PRC Management Methods for Controlling Pollution by Electronic Information Products Chemical safety WARNING GENERAL CHEMICAL HANDLING To minimize hazards ensure laboratory personnel read and practice the general safety guidelines for chemical usage storage and waste provided below and consult the relevant SDS for specific precautions and instructions Read and understand the Safety Data Sheets SDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials To obtain SDSs see the Documentation and Support section in this document Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the SDS Handle chemical wastes in a fume hood Ensure use of primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage
3. lon PGM Template OT2 200 Kit User Guide Appendix A Quality control of lon PGM Template OT2 200 lon Sphere y Particles Quality control using the Qubit 2 0 Fluorometer Templated lon Sphere Particle evaluation 1 Open the saved Qubit 2 0 Easy Calculator Microsoft Excel Spreadsheet file containing the Calibration Factor specifically calculated for the Qubit 2 0 Fluorometer used 2 Enter the raw RFU values from Alexa Fluor 488 and Alexa Fluor 647 Calibration Standards measurements in the appropriate fields for both the ISPs containing samples red cells and negative control sample purple cells IMPORTANT The Alexa Fluor 488 value must be gt 100 counts to produce a valid Templated ISPs value If the Alexa Fluor 488 value is lt 100 counts see the Qubit 2 0 Fluorometer quality control on page 73 Fluorophore Acceptable RFU Range Alexa Fluor 488 gt 100 counts no upper limit Samples with lt 100 counts usually correlate with no or very few ISPs in the assay Alexa Fluor 647 Any value with the condition that the Alexa Fluor 488 RFU value is gt 100 counts 3 In the appropriate field blue cells enter the lot specific conversion factor for unenriched Ion PGM Template OT2 200 Ion Sphere Particles available at http ioncommunity lifetechnologies com docs DOC 1420 Log in is required 4 The Percent Templated ISPs calculates automatically and
4. Run fails Various Retrieve the log files IMPORTANT Do notturn off or power cycle the instrument until the log files are downloaded see Download the log files from the lon OneTouch 2 Instrument on page 109 If a run fails contact Technical Support Precipitate in the lon PGM Storage of thawed Reagent 1 Ensure that the solution is fully thawed Template O12 200 Reagent Mix lt 2 C 2 Vortex the solution for 30 seconds then leave Mix after vortexing the tube at room temperature for 15 minutes 3 Vortex the solution again at maximum speed for 1 minute 4 Centrifuge the tube for 30 seconds 5 Visually inspect the solution at the bottom of the tube and verify that there is no residual precipitate If precipitate is visible then repeat steps 1 4 Note If precipitate is still visible then contact Technical Support 6 Keep the Reagent Mix at room temperature during use 7 After use store the solution at 2 C 8 C 54 lon PGM Template OT2 200 Kit User Guide Troubleshooting lon OneTouch ES sz Observation Possible cause Recommended action Excessive oil in the waste tray Various 1 Remove the oil waste tray then appropriately dispose of the oil Reinsert the tray into the slot then push the tray back fully into the instrument To further troubleshoot excessive oil from the instrument contact Technical Support lon OneTouch E
5. b1 b2 c Replace the retainer d Replace the 2 screws finger tighten e Reconnect the tubing Decontaminate the lon OneTouch ES Before returning the instrument for service decontaminate it according to the procedure below Materials and e Disposable rubber gloves equipment e Safety glasses required e Lab coat e Bleach e Water e Paper towels IMPORTANT This procedure does not guarantee total decontamination of the Ion OneTouch ES Decontaminate 1 Wear disposable rubber gloves safety glasses and a lab coat the instrument i 2 Use a cleaning pad wetted with a solution of 1 part chlorine bleach in 9 parts water 10 bleach solution to clean all outside surfaces of the Ion OneTouch ES Avoid getting bleach solution inside the chassis 3 Dry the surfaces of the Ion OneTouch ES with paper towels or other disposable wipes 4 Use cotton swabs to clean and dry areas that are difficult to reach lon PGM Template OT2 200 Kit User Guide 93 94 TM Appendix B Set up calibrate and maintain the lon OneTouch ES Decontaminate the lon OneTouch ES 5 Properly dispose of used cleaning materials to ensure that no one becomes exposed to contaminants lon PGM Template OT2 200 Kit User Guide Supplemental procedures Unpack and install the lon OneTouch 2 System 0 0 0000s eee 95 Install a firmware update 6 eee eens 96 Initialize the lon OneTouch
6. 000 e cece eee eee eee 88 lon OneTouch ES back panel layout 00 2 2 cece ees 88 Lubricate the syringe annually 0 00 ccc eects 89 Decontaminate the lon OneTouch ES eeceeeeeeeeeeeeeeeeeeeeeeees 93 Materials and equipment required 000 eee eee 93 Decontaminate the instrument 0 0 eee eee eens 93 APPENDIXC Supplemental procedures cceeeeeeeeeeees 95 Unpack and install the lon OneTouch 2 Systemes eck cone Sete eels eee wie 95 Unpack and install the lon OneTouch 2 Instrument 0 0eeeeeeeeeeeees 95 Unpack and install the lon OneTouch ES oo 4 coated oan sa cece nrnna 96 Install a firmware update 0 0 annene nannan eee eee 96 Install a firmware update with a USB flash drive 00 0c e ee eee eee 96 Alternative method Install a firmware update using an Ethernet connection 99 Initialize the lon OneTouch 2 Instrument 0 0 ccc cesses eeeeeeeeeeeees 100 Set up the lon OneTouch 2 Instrument ceeeeeeeeeeeeeeeeeeeeeeeees 100 Download the log files from the lon OneTouch 2 Instrument 00eeeee cues 109 lon PGM Template OT2 200 Kit User Guide 5 Contents lon OneTouch 2 Instrument op ration Suapan eh a a meee age EAS 110 Decontaminate the lon OneTouch 2 Instrument ceeeeeceeeeeeeeeeeeees 111 Materials and equipment required 000 cee eee eee 111 Decontami
7. 2 0 14 December 2012 Improved protocols Upgrade to Torrent Server Software v3 4 1 9 November 2012 New lon OneTouch 2 Instrument Simpler installation instructions New lon PGM Template OT2 200 Kit lon PGM Template OT2 200 Kit User Guide al LN x F KoV x z Product information z Aa ar l SSO Kit contents and storage conditions lon PGM IMPORTANT Use only the Ion PGM Template OT2 200 Kit Cat no 4480974 with Template OT2 200 this user guide and with the Ion OneTouch 2 System Do not use the kit with the Ion Kit OneTouch System Do not mix reactions or disposables including plates solutions and kit reagents from other template preparation kits The Ion PGM Template OT2 200 Kit includes the reagents required for preparing 10 reactions of template positive lon PGM Template OT2 200 Ion Sphere Particles ISPs on the Ion OneTouch 2 System IMPORTANT The shelf life of the lon PGM Template OT2 200 Reagent Mix and Ion PGM Template OT2 200 PCR Reagent B is 6 months from the date of initial thawing or to the date on the label whichever date is earlier Box Components Cap Quantity Volume Storage color lon PGM lon PGM OneTouch Plus Reaction Filter 10 15 C to 30 C Template OT2 Assembly reaction Reactions 200 filters and Kit Part no tubes 4481106 lon PGM lon OneTouch
8. IMPORTANT Do not lift the instrument by the metal handle used to access the Ion OneTouch 2 Amplification Plate 2 Ensure that the power switch is turned OFF then plug the 3 prong adapter of the power cord into the instrument 3 Plug the other end of the power cord into an electrical outlet of the appropriate voltage TM IMPORTANT The Ion OneTouch 2 Instrument draws 6 amps of current Do not exceed the circuit breaker limit for current If necessary plug multiple instruments into different circuits lon PGM Template OT2 200 Kit User Guide 95 Appendix C Supplemental procedures Install a firmware update 4 Turn the power switch ON Initial start up takes 3 minutes You may hear sounds from the instrument This is normal IMPORTANT Leave ON the Ion OneTouch 2 Instrument If the instrument is turned off critical log files are lost If you need to turn OFF the instrument or if a run fails download the log files see Download the log files from the Ion OneTouch 2 Instrument on page 109 Unpack and install 1 Unpack and install the lon OneTouch ES Instrument in a location different from the lon OneTouch the location used to prepare the amplification solution Refer to the Unpack and ES Install the Ion OneTouch 2 System Product Insert Pub no 4481875 IMPORTANT Do not lift the instrument by holding the syringe located at the back of the instrument 2 Install the AC line voltage
9. Note If you are using the lon PGM Hi Q Sequencing Kit select PGM Ion PGM Template OT2 200 Kit for Hi Q Hesg PGM lon PGM Template OT2 200 Kit for Hi Q PGM lon PGM Template OT2 200 Kit PGM lon PGM Template OT2 200 Kit for Hi O PGM lon PGM Template OT2 400 Kit Proton lon PI Template OT2 200 Kit se e Proton lon PI Template OT2 200 Kit v2 Proton lon PI Template OT2 200 Kit v2 with XT Proton lon PI Template OT2 200 Kit v3 Proton lon Proton Template OT2 Kit 0 00 gm O 02PSI 4 Touch Next 34 lon PGM Template OT2 200 Kit User Guide T Chapter 3 Prepare template positive lon PGM Template O72 200 lon ie Sphere Particles P4 Run the lon OneTouch 2 Instrument 5 Touch Assisted or Expert Protocol Proton Assisted Expert E 1 56 0 00 PSI E 24C e Assisted run Complete each task then touch Next After you touch Next on the last task you see a progress bar and the run begins After the runs starts you hear clicks from the instrument This is normal e Expert run Empty the waste container and oil waste tray if necessary then touch Next You see a progress bar and the run begins without the list of task screens IMPORTANT To cancel a run touch Abort then touch Yes to confirm cancellation If there is a high pressure event on the instrument the instrument aborts the run automatically and you do not have to touch Abort Aft
10. 2 Instrument 00 0c cece eee eee 100 Download the log files from the Ion OneTouch 2 Instrument 109 lon OneTouch 2 Instrument Operations ic0 sciaiseiiceerioreanvens 110 Decontaminate the Ion OneTouch 2 Instrument 0 0 0 005 111 Unpack and install the lon OneTouch 2 System TM For detailed instructions on site preparation and installation of the Ion OneTouch 2 Instrument and Ion OneTouch ES Instrument refer to e Site preparation and installation requirements Ion Personal Genome Machine System Site Preparation Guide for use with the Ion Personal Genome Machine Sequencer Ion Torrent Server and Ion OneTouch 2 System Pub no MANO0007516 e Unpacking and installation instructions See Unpack and install the Ion OneTouch 2 Instrument on page 95 and Unpack and install the Ion OneTouch ES on page 96 Unpack and install 1 Unpack and install the lon OneTouch 2 Instrument in a location different from the lon the location used to prepare the amplification solution Refer to the Unpack and OneTouch 2 Install the Ion OneTouch 2 System Product Insert Pub no 4481875 and the Ion Personal Genome Machine System Site Preparation Guide for use with the Ion Personal Genome Machine Sequencer Ion Torrent Server and Ion OneTouch 2 System Remove the instrument by laying the shipping box sideways on a table then sliding out the instrument Instrument
11. Chapter 3 Prepare template positive lon PGM Template OT2 200 lon ie Provided in the lon PGM Template OT2 Supplies 200 Kit Part no 4480981 lon OneTouch 2 Cleaning Adapter single use Provided in the lon PGM Template OT2 Solutions 200 Kit Part no 4481105 lon OneTouch Oil Other Materials and Equipment Kimwipes disposable wipes e 50 mL conical tube e Tube rack for 50 mL conical tube Sphere Particles Maintain the lon OneTouch 2 Instrument Note To ensure continued safe operation visually inspect the rotor assembly and casing periodically to ensure there are no signs of cracks or other physical damage see Ion OneTouch 2 Instrument layout on page 18 TM 1 Determine the appropriate reagents to use for maintaining the Ion OneTouch 2 Instrument Are you switching from the lon PGM Template OT2 200 Kit to another kit Then Yes Refer to the Demonstrated Protocol How to Set Up the lon One Touch 2 Instrument When Switching Between the lon PGM System and lon Proton System Pub no MAN0007719 Use the reagents from the appropriate kit to maintain the lon OneTouch 2 Instrument Proceed to step 2 Continue to use the reagents provided in the lon PGM Template OT2 200 Kit lon PGM Template OT2 200 Kit User Guide 39 Chapter 3 Prepare template positive lon PGM Template OT2 200 lon Sphere Particles Maintain the lon
12. Install the disposable injector 0 00 c eee eee eee 22 Install the lon OneTouch Oil 00cc cee eee eee eee eee eeeeeeeeneeeeenaes 23 Install the lon PGM OT2 Recovery Solution 0 0 00 ccc cece cece cee eeeee 24 Empty the Waste Container 0 00 eee eee ees 24 Prepare and install the amplification solution 0 000 e eee eee eee eee 25 Materials required for this procedure 0 200 cece eee eee eee eee 25 Prepare the amplification solution 0 00 c eee eee 25 Fill the lon PGM OneTouch Plus Reaction Filter Assembly 2000000 28 Install the filled lon PGM OneTouch Plus Reaction Filter Assembly on the lon OneTouch 2Instrument 000 cece eens 31 Run the lon OneTouch 2 Instrument ccccceeeeeeeeeeeeeeeeeeenuneeees 32 Recover the template positive lon PGM Template OT2 200 lon Sphere Particles 36 Materials required for this procedure snssansasennnnnn nurnerr eee eee 36 Recover the template positive lon PGM Template OT2 200 lon Sphere Particles nccc ac atuswedaates a ienaa a Ta E E E EE E E a A 36 Maintain the lon OneTouch 2 Instrument ccceeeeeeeeceeueeeeeeeeuenes 38 Materials required for this procedure 0 0 cece eee eee eee eee 39 Clean the lon OneTouch 2 Instrument 00cccceceeeeeeeeeeeeueneeeeeeas 39 CHAPTER4 Enrich the template positive lon PGM Template OT2 200 lon Sphe
13. Reagent Tubes 2 tubes Template OT2 a Supplies 200 lon OneTouch Recovery Routers 10 routers Kit Part no m 4480981 lon OneTouch Recovery Tubes 20 tubes lon OneTouch Sipper Tubes 2 tubes lon OneTouch 2 Amplification Plates 10 plates lon OneTouch ES Supplies 1 bag lon OneTouch 2 Cleaning Adapters 10 adapters lon PGM Template OT2 200 Kit User Guide 9 ad Chapter 1 Product information A Kit contents and storage conditions Box Components Cap Quantity Volume Storage color lon PGM lon PGM Template OT2 200 Reagent Mix Violet 5 tubes 1000 pL 30 C to 10 C Template OT2 each 2 C to 8 C after Reagents 200 IMPORTANT The lon PGM Template thaw Kit Part no OT2 200 Reagent Mix is shipped at 30 C to 4481107 10 C Immediately before use thaw tube s as needed After use store the thawed lon PGM Template OT2 200 Reagent Mix at 2 C to 8 C lon PGM Template OT2 200 Enzyme Mix Brown 1 tube 500 uL 30 C to 10 C lon PGM Template OT2 200 lon Sphere Black 1 tube 1000 pL Particles lon PGM lon PGM Template OT2 200 PCR ReagentB Blue 2 tubes 1500 pL 15 C to 30 C Template OT2 each Solutions 200 IMPORTANT The lon PGM Template Kit Part no OT2 200 PCR Reagent B is shipped at 15 C 4481105 to 30 C Store the lon PGM Template OT2 200 PCR Reagent B at room temperature
14. Add each component then pipet the amplification solution up and down to mix Order Reagent Cap color Volume 1 Nuclease free Water 25 uL 2 lon PGM Template OT2 200 Reagent Mix Violet 500 uL 3 lon PGM Template OT2 200 PCR Reagent B Blue 300 uL 4 lon PGM Template OT2 200 Enzyme Mix Brown 50 uL 5 Diluted library not stock library lt 25 uL Total 900 uL 4 Vortex the solution prepared in step 3 at maximum speed for 5 seconds then centrifuge the solution for 2 seconds 5 Prepare the lon PGM Template OT2 200 Ion Sphere Particles a Vortex the ISPs at maximum speed for 1 minute to resuspend the particles b Centrifuge the ISPs for 2 seconds c Pipet the ISPs up and down to mix d Immediately proceed to the next step lon PGM Template OT2 200 Kit User Guide 27 Chapter 3 Prepare template positive lon PGM Template OT2 200 lon Sphere Particles Prepare and install the amplification solution 6 Add the ISPs to the amplification solution Order Reagent Cap Color Volume 1 Amplification solution without ISPs from 900 uL step 3 of this procedure 2 lon PGM Template OT2 200 lon Sphere Black 100 uL Particles Total 1000 uL 7 Vortex the complete amplification solution prepared in step 6 at maximum speed for 5 seconds IMPORTANT Start the run on the Ion OneTouch 2 Instrument lt 15 minutes after pr
15. DIV 0 mowo a wo Conversion factor can be found on the lon Community website www iontorrent com qubit_files and is Templated Kit LOT specific Legend Green cells Raw RFU values of Alexa Fluor 488 and Alexa Fluor 647 Calibration Standards supplied in the lon Sphere Quality Control Kit Red cells Raw RFU values measured in Section 3 Measure the lon Sphere Particles ISPs 200 sample Purple cells Raw RFU values measured for negative control in Section 3 Measure the lon Sphere Particles ISPs 200 sample Blue cells Template kit LOT specific conversion factor IMPORTANT For each Qubit 2 0 Fluorometer used save a separate Qubit 2 0 Easy Calculator Microsoft Excel Spreadsheet file containing the Calibration Factor specifically calculated for that particular instrument Measure the This section describes the procedure for determining the percent templated ISPs for templated unenriched Ion Sphere Particles unenriched sample Sample preparation 1 From the Ion Sphere Quality Control Kit thaw the Ion Probes tube Annealing Buffer and Quality Control Wash Buffer 2 For unenriched Ion PGM Template OT2 200 Ion Sphere Particles if not 100 uL adjust sample volume to 100 uL with Ion OneTouch Wash Solution from the Ion PGM Template OT2 Solutions 200 Kit Part no 4481105 and transfer 2 uL to a 0 2 mL PCR tube 3 Add Ion Probes to sample s a If
16. a If the pipette tip is not positioned in line with the slot in the calibration shelf adjust the thumb screw at the back of the Tip Arm turn counterclockwise to move the tip forward or clockwise to move the tip back see the right following photograph until the tip is in line with the notch in the calibration shelf black outline and encircled in red in the left following photograph Tip in line with notch Fingers on thumbscrew b Adjust the height of the pipette tip in relation to the calibration shelf Press the Y minus button repeatedly until the tip touches the shelf Press the minus button 8 more times to lower the tip further Pressing the Y minus additional times button accounts for different tip lengths and variations during tip installation Note It is better to have the ASP aspiration height be too low than too high 86 lon PGM Template OT2 200 Kit User Guide Perform horizontal axis calibration TM Appendix B Set up calibrate and maintain the lon OneTouch ES FSS Calibrate the lon OneTouch ES Press Start Stop then wait for the Tip Arm to stop moving and for P1 to display Press Select Calibrate for 3 seconds until the instrument beeps 2 times and CAL is displayed Note The instrument will cycle through several values before CAL is displayed Press Horiz Adjust Instrument displays FLA then press Start Stop Place an empty 8 well strip in the sl
17. on page 25 a Set a P1000 pipette to 1000 uL and attach a new 1000 uL tip to the pipette b Vortex the amplification solution at maximum speed for a full 5 seconds then centrifuge the solution for 2 seconds Immediately proceed to the next step c Pipet the amplification solution up and down to mix then fill the tip with 1000 uL of the amplification solution lon PGM Template OT2 200 Kit User Guide 29 Chapter 3 Prepare template positive lon PGM Template OT2 200 lon Sphere Particles Prepare and install the amplification solution d Insert the tip firmly into the sample port so that the tip is perpendicular to the Ion PGM OneTouch Plus Reaction Filter Assembly and fully inserted into the sample port to form a tight seal e Slowly pipet the entire 1000 uL of the amplification solution through the f sample port Keep the plunger of the pipette depressed to avoid aspirating solution from the Ion PGM OneTouch Plus Reaction Filter Assembly With the plunger still depressed remove the tip from the sample port then appropriately discard the tip If necessary gently dab a Kimwipes disposable wipe around the ports to remove any liquid 5 Add Ion OneTouch Reaction Oil 27 mL size through the sample port a b 30 Set a P1000 pipette to 1000 uL and attach a new 1000 uL tip to the pipette Fill the tip with 1000 uL of Reaction Oil Insert the tip firmly into the sample port so that
18. Do notstore lon PGM Template OT2 200 PCR Reagent B at 2 C to 8 C lon OneTouch Oil 1bottle 450mL lon OneTouch Reaction Oil 1 bottle 27 mL Nuclease free Water 1 tube 15 mL lon PGM OT2 Recovery Solution 1 bottle 350 mL lon OneTouch Wash Solution 1 bottle 25 mL MyOne Beads Wash Solution Green 2 vials 1400 uL each Neutralization Solution Red 1 vial 100 uL Tween Solution 1 bottle 6 mL 11 This protocol has been validated using this specific material Substitution may adversely affect performance 21 lon OneTouch ES Supplies include 12 Eppendorf LoRetention Dualfilter 300 uL PCR pipette tips Fisher Cat no 02 717 342 and loose 8 well strips The ES supplies may be located at the bottom of the box 3l Each lon OneTouch 2 Cleaning Adapter is used for one cleaning only 10 lon PGM Template OT2 200 Kit User Guide Chapter 1 Product information Required materials and equipment not provided lon PGM The Ion PGM Enrichment Beads Cat no 4478525 are required for enriching 10 Enrichment Beads reactions of template positive Ion PGM Template OneTouch 2 200 Ion Sphere Particles ISPs on the Ion OneTouch 2 System Box Components Cap color Quantity Volume Storage lon PGM Dynabeads MyOne Streptavidin C1 Blue 1 bottle 150 uL 2 C to 8 C Enrichment Beads lon PGM System Beads 1l Life Technologies has validated this protocol usin
19. Logging into the lon Community is required Remove the existing program file qbt and two upgrade files bin from the USB drive if any Transfer the three downloaded files to the USB drive provided with the Qubit 2 0 Fluorometer recommended or another compatible USB drive Note Files on the USB drive must be in the root directory and cannot be ina folder Upgrade the firmware from the previous versions Power on the Qubit 2 0 Fluorometer by plugging in the unit 2 Insert the USB drive containing the program qbt and upgrade bin files into the USB port on the instrument In the upper right corner of the main menu touch V2 00 or V3 00 depending on the version If the image of the USB drive in the update screen displays a green dot touch Update to upgrade the instrument firmware The firmware update lasts approximately 2 minutes during which the Qubit 2 0 Fluorometer screen flashes Note If the image displays a red dot the Qubit 2 0 Fluorometer cannot detect the USB drive Ensure the USB drive is in place securely If the problem persists remove the USB drive and reinsert alee a Ae C 143un 2011 2 00PM v2 00 38 C 143un 2011 2 00PM v200 38 To update To update 1 Download new firmware from our website 1 Download new firmware from our website www invitrogen com qubit to your USB drive il Updating 2 Insert your USB drive into the Qubit firmware 3
20. Press Update button h ja Update jz Update Home Standards Sample Data Home Standards Sample Data lon PGM Template OT2 200 Kit User Guide TM 1 Appendix A Quality control of lon PGM Template OT2 200 lon Sphere Particles Quality control using the Qubit 2 0 Fluorometer 5 When the Qubit 2 0 Fluorometer displays the main screen confirm that V3 10 is displayed in the upper right corner which confirms that the upgrade was successful After the firmware has been updated the following screen will be displayed Confirm that V3 10 is displayed in the upper right corner Touch Yes to permanently upload the Ion Sphere QC program to the instrument 0 19 April 2012 2 00PM v3 1C od Do you wish to upload i Yes No Home Standards Sample Data 6 Confirm the Ion Sphere QC program qbt file is functional by checking the following screens a In the main menu the Ion selection option is present 19 April 2012 2 00 PM v3 10 Choose Your Assay Protein Ion Standards Sample Data lon PGM Template OT2 200 Kit User Guide 63 TM 1 Appendix A Quality control of lon PGM Template OT2 200 lon Sphere Particles Quality control using the Qubit 2 0 Fluorometer Upgrade for firmware V3 10 64 b After touching Ion MM AF 488 GEM and AF 647 GM selection options appear on the screen Note Touching AF 488 or AF 647 enters the respective measu
21. Record the value Note The data retained on the Qubit 2 0 Fluorometer can be transferred to a USB drive See the Optional Data Transfer to USB Drive on page 70 for details If more than one sample is being processed all samples can be read with the AF 488 setting before moving on to the AF 647 setting 5 Touch Home touch Ion and then touch AF 647 Insert the sample into the Qubit 2 0 Fluorometer close the lid and touch Read Note The lettering on the Read 2 selection option changes from white to red when reading a sample The lettering changes back to white when the reading of the sample is finished in approximately 5 seconds 6 Record the value IMPORTANT Ensure that you read the negative control Quality Control Wash Buffer only in both the Alexa Fluor 488 and Alexa Fluor 647 settings and record the RFU values Optional Data Transfer to USB Drive 1 Ensure that the USB drive is inserted in the instrument 2 In the main menu touch Data at the bottom right corner of the screen 3 In the data screen touch USB drive then wait for the instrument to download the data to the USB drive Note The download creates a csv file that can be opened on your computer using any spreadsheet software such as Microsoft Excel software 19 April 2012 2 00 PM v3 10 Name Date 1 1 00 25 PM 14 Jun 2011 Cah Rename Home Standards Sample
22. carefully remove and discard the supernatant without disturbing the pellet of Dynabeads MyOne Streptavidin C1 Beads 5 Add 130 uL of MyOne Beads Wash Solution to the Dynabeads MyOne Streptavidin C1 Beads Note You add the resuspended Dynabeads MyOne Streptavidin C1 Beads in the 130 uL MyOne Beads Wash Solution to Well 2 of the 8 well strip 6 Remove the tube from the magnet vortex the tube for 30 seconds and centrifuge the tube for 2 seconds 46 lon PGM Template OT2 200 Kit User Guide Fill the 8 well strip 1 T Chapter 4 Enrich the template positive lon PGM Template OT2 200 lon Sphere Particles D Prepare reagents then fill the 8 well strip Ensure that the template positive ISPs from the Ion OneTouch 2 Instrument are in 100 uL of Ion PGM OT2 Recovery Solution and are in Well 1 of the 8 well strip see Recover the template positive Ion PGM Template OT2 200 Ion Sphere Particles on page 36 Well 1 with the ISPs is on the left Square shaped tab Rounded tab Note If the template positive ISPs were stored at 2 C to 8 C centrifuge the ISPs at 15 500 x g for 2 5 minutes then carefully remove all but 100 uL of supernatant With a new tip pipet up and down to resuspend the ISPs Transfer the suspension from the tube into Well 1 of the 8 well strip If you have not already assessed the quality of the unenriched template positive ISPs use one of the following method
23. during the run close to the front bottom edge of the well IMPORTANT If you use more than one Ion OneTouch ES do not switch Trays or Tip Arms between instruments Each Tray and Tip Arm is calibrated with a particular instrument To track the Tray and Tip Arm each component has a printed label with the matching serial number of the instrument 1 Set up the Ion OneTouch ES see Set up the Ion OneTouch ES on page 75 2 Install a new tip 3 Restore the factory default settings a Power OFF the instrument and wait 3 seconds b While holding down Vert Adjust power ON the instrument 4 Put the instrument into calibration mode a Power the instrument OFF b While holding down Select Calibrate power the instrument ON Keep holding down Select Calibrate until P1 is displayed c Press Select Calibrate for 3 seconds until the instrument beeps 2 times and CAL is displayed Note The instrument will cycle through several values before CAL is displayed 5 Press Vert Adjust The instrument displays ASP Aspirate or z bottom position 6 Press Start Stop The Tip Arm lowers to bring the tip near the notch in the calibration shelf on the left side of the Tray lon PGM Template OT2 200 Kit User Guide 85 Appendix B Set up calibrate and maintain the lon OneTouch ES Calibrate the lon OneTouch ES 7 Adjust if needed the position of the bottom of the pipette tip
24. immediately after the main menu displays 4 Repeat steps 1 3 as needed Disposable injector remains e Reagent build up 1 Gently pull from the top of the disposable in down position in the lon e New part injector until the disposable injector just OneTouch DL Injector Hub returns to the up position in the Injector Hub see Install the disposable injector on page 22 2 Briefly press then release the spring loaded top of the Injector Hub 5 10 times at the point indicated by the arrow see figures 4a 4c on page 26 You should hear a click 3 If the Injector Hub remains in the down position repeat step 2 once up to 10 more clicks Note If the Injector Hub still does not move freely and click up into place then contact Technical Support lon PGM Template OT2 200 Kit User Guide 53 Troubleshooting lon OneTouch 2 Instrument Observation Possible cause Recommended action Centrifuge lid does not open e Power failure 1 Slide a 1 8 inch L wrench hex wrench or equivalent tool into the right hole and along the top edge of the centrifuge hinge e Software crash 2 Push the tool into the hole until there is a slight compression of the tool against the instrument and the centrifuge lid unlocks and opens 3 Remove the tool from the hole then open the lid 4 If necessary troubleshoot the lid lock then use the instrument normally Do not force the lid open
25. 8 well strip as follows see the figure following step 4 Well number Reagent to dispense in well Well 10 Entire template positive ISP sample 100 pL prepared in step 1 of this procedure U Well 2 130 uL of Dynabeads MyOne Streptavidin C1 Beads resuspended in MyOne Beads Wash Solution prepared in Wash and resuspend the Dynabeads MyOne Streptavidin C1 Beads on page 46 B Well 3 300 uL of lon OneTouch Wash Solution W Well 4 300 uL of lon OneTouch Wash Solution W Well 5 300 uL of lon OneTouch Wash Solution W Well 6 Empty Well 7 300 uL of freshly prepared Melt Off Solution prepared in Prepare Melt Off Solution on page 46 M Well 8 Empty 1 Well closest to the square shaped tab 4 Confirm that the square shaped tab is on the left then insert the filled 8 well strip with the 8 well strip pushed all the way to the right end of the slot of the Tray Install 0 2 mL PCR tube Install 8 well strip with square shaped tab on the left and strip in hole of Tip Loader pushed to the right end of the slot CaP FONE ge 4 amp 48 lon PGM Template OT2 200 Kit User Guide Chapter 4 Enrich the template positive lon PGM Template OT2 200 lon Sphere Particles Prepare the lon OneTouch ES Prepare the lon OneTouch ES 1 Load anew tip in the Tip Arm a Place a new tip in the Tip Loader Remove the Tip Arm from the cradle and align th
26. Kit Cat no 4480974 with this user guide and with the Ion OneTouch 2 System Do not use the kit with the Ion OneTouch System System Do not mix reactions or disposables including plates solutions and kit reagents from other template preparation kits lon PGM Template OT2 200 Kit User Guide 7 About this guide Revision history Revision history Revision Date Description of change A 0 18 August 2014 Users recommended to upgrade firmware after updating to Torrent Server Software v4 2 Support added for lon PGM Hi Q Sequencing Kit users Library dilution table in Prepare and install the amplification solution updated to 100 pM for all listed libraries Version numbering changed to alphanumeric format and reset to A 0 in conformance with internal document control procedures 5 0 14 October 2013 Users recommended to upgrade firmware after updating to Torrent Server Software v4 0 Library dilution table added to Prepare and install the amplification solution on page 29 Discontinuation of use of the lon OneTouch Lid 4 0 21 May 2013 Upgrade firmware after updating to Torrent Server Software v3 6 Minor reorganization of topics for greater ease of use Minor changes and corrections to text and figures made 3 0 13 March 2013 Updated screen captures and procedures associated with v3 4 2 firmware upgrade including Final Spin functionality
27. Matertals required armsa etwas in etn he ene Bee RE ewig Paes Cee 60 Upgrade the Qubit 2 0 Fluorometer firmware and software 61 Upgrade for firmware V2 00 or V3 00 2 00 cece eee 61 Upgrade for firmware V3 10 2 0 c eee eee 64 Calculate the Qubit 2 0 Fluorometer calibration factor 00 66 Measure the templated unenriched sample 002 0c rnrn eee eee eee 68 Qubit 2 0 Fluorometer quality control seai ieoa i ence ee eee ei eee te ee eae tare eee 73 Quality control using the Guava easyCyte 5 Flow Cytometer 00000eeee 73 Demonstrated protocol Quality control using Attune Acoustic Focusing Cytometer 74 APPENDIX B_ Set up calibrate and maintain the lon OneTouch ES orrat ease a ae ao ea NO 75 Set up the lon OneTouch ES oann anaana nanan ea enaces seen osteetaiea es on 75 Materials required for this procedure 00202 eee e eee eee eee eee 75 Set up the instrument 202 22 59 ecg BS eee ene Paws Lenn wee saint aa E i eee 76 Perform a residual volume test 2 0 cece eee eee eee teens 80 Calibrate the lon OneTouch ES sieasdieciciseriss vkieebbiensind Caneidaneaaioids 84 Perform vertical calibration 0 c eee teens 85 Perform horizontal axis calibration 0 000 ee eee eee eee 87 Maintain the lon OneTouch ES cece see eeeeeeeeee cnet eeeeeeeeeees 88 Materials and equipment required
28. OneTouch 2 Instrument 2 Check the level of Ion OneTouch Oil in the Reagent Tube a Ensure that the left Reagent Tube has 220 mL of Oil b If the Reagent Tube has lt 20 mL of Oil pour Oil into the Reagent Tube until it is half full 3 Remove and appropriately discard the used Ion PGM OneTouch Plus Reaction Filter Assembly Remove the assembly from the instrument by grasping the filter Note The Reaction Tube is filled with Ion OneTouch Oil 4 Keep the Ion OneTouch 2 Amplification Plate in the heat block 5 Place a 50 mL conical tube in a tube rack then place the tube rack with the tube to the right of the instrument see photograph in step 10 6 Obtain an Ion OneTouch 2 Cleaning Adapter 40 lon PGM Template OT2 200 Kit User Guide Chapter 3 Prepare template positive lon PGM Template OT2 200 lon Sphere Particles Maintain the lon OneTouch 2 Instrument 7 Firmly insert the 3 ports of the single use Cleaning Adapter into the three holes see Figure 7a on the top stage of the Ion OneTouch 2 Instrument see Figure 7b The tab protruding from the outer edge of the Cleaning Adapter fits into the front notch of the stage 7a 7b Notch 8 Remove the disposable injector from the Ion OneTouch DL Injector Hub a Place one hand on the centrifuge lid b With the other hand firmly grip the rigid plastic connector at the top of the disposable injector lon PGM Template OT2 20
29. at your facility or sending the instrument for repair maintenance trade in disposal or termination of a loan decontamination forms may be requested from customer service Before using any cleaning or decontamination methods except those recommended by the manufacturer users should confirm with the manufacturer that the proposed method will not damage the equipment Safety and electromagnetic compatibility EMC standards The instrument design and manufacture complies with the standards and requirements for safety and electromagnetic compatibility as noted in the following table 116 lon PGM Template OT2 200 Kit User Guide Safety EMC Appendix E Safety Safety and electromagnetic compatibility EMC standards Reference Description EU Directive European Union Low Voltage Directive 2006 95 EC IEC 61010 1 Safety requirements for electrical equipment for measurement EN 61010 1 control and laboratory use Part 1 General requirements UL 61010 1 CSA C22 2 No 61010 1 IEC 61010 2 010 EN 61010 2 010 Safety requirements for electrical equipment for measurement control and laboratory use Part 2 010 Particular requirements for laboratory equipment for the heating of materials IEC EN 61010 2 020 Safety requirements for electrical equipment for measurement control and laboratory use Part 2 020 Particular requirements for laboratory centrifuges IEC 61010 2 081 EN 61010 2 081 Safety r
30. disposable tubing to install the disposable injector see Install the disposable injector on page 22 lon PGM Template OT2 200 Kit User Guide 21 Chapter 3 Prepare template positive lon PGM Template OT2 200 lon Sphere Particles Set up the lon OneTouch 2 Instrument Install the Note Before use inspect the long metal shaft of the disposable injector Some d ispo sable disposable injectors may be slightly bent which is normal If you have questions injector about the disposable injector contact Technical Support 1 Place one hand on the centrifuge lid Place the other hand at the top of the disposable injector and insert the disposable injector straight into the port of the Ion OneTouch DL Injector Hub A CAUTION PHYSICAL INJURY HAZARD The pointed end of the disposable injector can puncture your skin Keep your hand away from the point of the disposable injector Note The color of the disposable injector may vary 2 Keep your hand on the centrifuge lid then push the disposable injector through the port until the disposable injector just stops at the base of the router 3 Release the disposable injector 22 lon PGM Template OT2 200 Kit User Guide Install the lon OneTouch Oil Chapter 3 Prepare template positive lon PGM Template OT2 200 lon Sphere Particles Set up the lon OneTouch 2 Instrument 4 Confirm automatic placement of the disposable injector above the router
31. e Nuclease free Water Provided in the lon PGM Template OT2 Reagents 200 Kit Part no 4481107 e lon PGM Template OT2 200 Reagent Mix e lon PGM Template OT2 200 Enzyme Mix e lon PGM Template OT2 200 lon Sphere Particles Provided in the lon PGM Template OT2 Reactions 200 Kit Part no 4481106 e lon PGM OneTouch Plus Reaction Filter Assembly Optional Provided in the lon PGM Controls Kit v2 Part no 4482010 e col DH10B Control 200 Library Other Materials and Equipment e Diluted library e Microcentrifuge e 1 5 mL Eppendorf LoBind Tubes e Tube rack for 15 mL conical tube e Pipettes e Vortexer e Heat block set at 75 C IMPORTANT Use only the lon PGM Template OT2 200 Kit Cat no 4480974 with this user guide and with the Ion OneTouch 2 Instrument Do not use the kit with the Ion OneTouch System Do not mix reactions or disposables including plates solutions and kit reagents from other template preparation kits IMPORTANT We recommend preparing the amplification solution in a room dedicated to pre PCR activities or in a controlled pre PCR hood lon PGM Template OT2 200 Kit User Guide 25 TM 5 Chapter 3 Prepare template positive lon PGM Template OT2 200 lon f Sphere Particles Prepare and install the amplification solution 1 Prepare the reagents as follows Reagents Preparation lon PGM Template OT2 1 Allow the reagent mix t
32. for your system see Maintain the Ion OneTouch 2 Instrument on page 38 1 Ensure that the centrifuge lid of the lon OneTouch 2 Instrument is closed IMPORTANT If you raise the centrifuge lid do not hit the disposable injector against the instrument You can damage the disposable injector If you damage the disposable injector appropriately dispose of the injector amplification plate and tubing Use a new disposable injector and Ion OneTouch 2 Amplification Plate 32 lon PGM Template OT2 200 Kit User Guide Chapter 3 Prepare template positive lon PGM Template OT2 200 lon 2 On the home screen touch Run 4 1 65 lon PGM Template OT2 200 Kit User Guide Sphere Particles Run the lon OneTouch 2 Instrument Open Li 0 00 PSI 4 ISEN Wy a a 2 CO O a 33 Pai Chapter 3 Prepare template positive lon PGM Template OT2 200 lon f Sphere Particles Run the lon OneTouch 2 Instrument 3 Touch the drop down menu then select PGM Ion PGM Template OT2 200 Kit Heeg PGM lon PGM Template OT2 200 Kit PGM lon PGM Template OT2 200 Kit PGM lon PGM Template OT2 200 Kit for Hi Q PGM lon PGM Template OT2 400 Kit Proton lon PI Template OT2 200 Kit c iis Proton lon PI Template OT2 200 Kit v2 Proton lon PI Template OT2 200 Kit v2 with XT Proton lon PI Template OT2 200 Kit v3 Proton lon Proton Template OT2 Kit 0 00 gm O 02PSI E
33. fuse module refer to the Unpack and Install the Ion OneTouch 2 System Product Insert 3 Ensure that the power switch is OFF then plug the female end no pins of the power cord into the instrument 4 Plug the multi pin end of the instrument power cord into the surge protector or line conditioner 5 Plug the surge protector or line conditioner into an electrical outlet IMPORTANT The voltage of the electrical outlet must match the voltage of the surge protector and the selected voltage of the installed AC line voltage fuse TM module in the Ion OneTouch ES 6 Ensure that the Ion OneTouch ES is in a room at an operating temperature of 15 C to 25 C 60 F to 77 F 7 Turn the power switch ON 8 Set up the Ion OneTouch ES see Set up the instrument on page 76 then perform the residual volume test see Perform a residual volume test on page 80 TM Note Leave ON the Ion OneTouch ES 9 Set up and run the Ion OneTouch 2 System see Chapter 2 Getting started Install a firmware update Install a firmware IMPORTANT Reformatting the USB flash drive erases data on the drive update with a USB flash drive 1 Ensure that the USB flash drive is FAT32 formatted according to the operating system where the USB flash drive is installed Macintosh operating system a Insert a USB flash drive into a USB port 96 lon PGM Template OT2 200 Kit User Guide Appendix C Sup
34. is displayed for each sample Qubit Calibration Factor Calculation Previously calculated calibration factor Different for each Qubit 2 0 Fluorometer Calibration Standard Alexa Fluor 488 Calibration Standard _ g Alexa Fluor 647 Calibration Standard MAM Enter raw RFU values for negative control sample here Values will be the same for all samples measured at the same time Percent Templated ISPs Enter raw RFU values for the ISPs containing samples here Calibration Standard RawRFU Value Background RFU Negpfive Control Tube Sample ID Conversion Factor Percent Templated ISPs a ovo O es ovo mwao v N Automatically calculated Percent Template ISPs value Conversion factor can be found on the lon Community website www iontorrent com qubit_files and is Templated Kit LOT specific Enter template kit LOT specific value here Legend Green cells Raw RFU values of Alexa Fluor 488 and Alexa Fluor 647 Calibration Standards supplied in the lon Sphere Quality Control Kit Red cells Raw RFU values measured in Section 3 Measure the lon Sphere Particles ISPs 200 sample Purple cells Raw RFU values measured for negative control in Section 3 Measure the lon Sphere Particles ISPs 200 sample Blue cells Template kit LOT specific conversion factor Acceptance criteri
35. light for long periods of time and direct sunlight 2 Vortex well to mix and pulse spin the tube to remove any liquid trapped in the cap 3 Transfer 200 uL of each standard into two separate Qubit assay tubes Pulse spin to bring all the liquid to the bottom of the tube lon PGM Template OT2 200 Kit User Guide TM 1 Appendix A Quality control of lon PGM Template OT2 200 lon Sphere Particles Quality control using the Qubit 2 0 Fluorometer Calibration standard measurement IMPORTANT Prior to using the Qubit 2 0 Fluorometer ensure that the instrument is running Firmware V3 10 and the Ion_PluginV310_AF qbt file has been permanently uploaded to the instrument See section Upgrade the Qubit 2 0 Fluorometer firmware and software on page 61 for more information about managing Qubit 2 0 Fluorometer firmware versions 1 Touch Ion to access Alexa Fluor 488 and Alexa Fluor 647 measurement options 2 Touch AF 488 and insert the Alexa Fluor 488 Calibration Standard reagent into the Qubit 2 0 Fluorometer close the lid and touch Read Note The lettering on the Read E selection option changes from white to red when reading a sample The lettering changes back to white when the reading of the sample is finished in approximately 5 seconds 3 Record the RFU value and remove sample from Qubit 2 0 Fluorometer 4 Touch Home touch Ion and then touch AF 647 Insert the Alexa Fluor 647 Calibrat
36. on ice before use Unpack and install the lon OneTouch 2 Instrument and lon OneTouch ES TM For detailed instructions on site preparation and installation of the Ion OneTouch 2 Instrument and Ion OneTouch FS refer to e Site preparation and installation requirements Refer to the Ion Personal Genome Machine System Site Preparation Guide for use with Ion Personal Genome Machine Sequencer Ion Torrent Server and Ion OneTouch 2 System Pub no MANO0007516 e Unpacking and installation instructions See Unpack and install the Ion OneTouch 2 System on page 95 14 lon PGM Template OT2 200 Kit User Guide Chapter 2 Getting started aie it Check the firmware Check the firmware TM Firmware updates to the software controlling the Ion OneTouch 2 Instrument are periodically released The firmware updates are available through the Torrent Server To check the firmware version on the home screen touch Options then touch Info To update the firmware use the USB flash drive or use the Ethernet connection see Install a firmware update with a USB flash drive on page 105 IMPORTANT Ensure that the latest firmware is installed on the Ion OneTouch 2 Instrument To get the latest firmware upgrade to Torrent Server Software v4 2 or later then use a USB flash drive or an Ethernet connection to upgrade your instrument The latest firmware for the Ion OneTouch 2 Instrument is required to perform th
37. on the Ion OneTouch 2 Instrument appropriately dispose of all components as directed Complete the remaining tasks to set up for a sample run see Perform a verification run on page 15 Download the log files from the lon OneTouch 2 Instrument Log files capture important information regarding instrument operation Should the Ion OneTouch 2 Instrument fail downloaded log files are used to troubleshoot the instrument IMPORTANT Until the log files are downloaded leave ON the Ion OneTouch 2 Instrument If the instrument is turned off critical log files are lost If you need to turn OFF the instrument or if a run fails download the log files 1 2 Ensure that the Ion OneTouch 2 Instrument is not performing a run Format a USB flash drive if necessary see Install a firmware update with a USB flash drive on page 96 Confirm that a formatted USB flash drive has no data files on it Insert the USB flash drive into the USB connection located on the back of the Ion OneTouch 2 Instrument Power switch on off Ethernet connection USB connection Power socket lon PGM Template OT2 200 Kit User Guide 109 Appendix C Supplemental procedures lon OneTouch 2 Instrument operations 4 On the home screen touch Options then touch Export Log A LEIS g Export Log Upgrade Final Spin 4 1 64 0 04 PSI E 415C The Export Log screen asks if you would like t
38. specifications in all material respects Where a valid and timely claim in respect of breach of Ion Torrent warranty is submitted to Life Technologies Life Technologies may at its discretion replace repair or modify the Ion Torrent instrument Any agreed replacement shall be at 1 1 like kind basis at no cost to the buyer For Ion Torrent chips or reagents reasonably determined by Life Technologies to be defective independent of user error shall be replaced by Life Technologies on a 1 1 like kind basis at no cost to buyer provided that such defective Ion Torrent chips or reagents were used by buyer prior to their expiration date or if there is no expiration date the Ion Torrent chips or reagents were used within six 6 months of receipt and the defect was promptly reported with appropriate detail to Life Technologies technical support NO OTHER WARRANTIES SHALL BE APPLICABLE TO ION TORRENT PRODUCTS WHETHER OR NOT ANY FURTHER WARRANTY DOCUMENTATION MAY BE INCLUDED IN THE SHIPMENT WITH THE EXCEPTION OF THIRD PARTY WARRANTIES WITH RESPECT TO THIRD PARTY PRODUCT ANY THIRD PARTY PRODUCTS ARE NOT COVERED BY THIS SECTION AND ANY WARRANTIES FOR THIRD PARTY PRODUCTS ARE PROVIDED BY THE ORIGINAL MANUFACTURER OF THE THIRD PARTY PRODUCT Warranties are made only to buyer purchasing the Ion Torrent Product directly from Life Technologies are not transferable and do not extend to the benefit of any other person or entity unless o
39. the tip is perpendicular to the Ion PGM OneTouch Plus Reaction Filter Assembly and fully inserted into the sample port to form a tight seal Slowly pipet the 1000 uL of the Reaction Oil through the sample port then keep the plunger of the pipette depressed With the plunger depressed remove the tip from the sample port then appropriately discard the tip Set the P1000 pipette to 500 uL and attach a new 1000 uL tip to the pipette Fill the tip with 500 uL of Reaction Oil lon PGM Template OT2 200 Kit User Guide Install the filled lon PGM OneTouch Plus Reaction Filter Assembly on the lon OneTouch 2 Instrument Chapter 3 Prepare template positive lon PGM Template OT2 200 lon Sphere Particles Prepare and install the amplification solution h Insert the tip firmly into the sample port so that the tip is perpendicular to the Ion PGM OneTouch Plus Reaction Filter Assembly and fully inserted into the sample port to form a tight seal i Slowly pipet 500 uL of the Reaction Oil through the sample port then keep the plunger of the pipette depressed j With the plunger still depressed remove the tip from the sample port then appropriately discard the tip k If necessary gently dab a Kimwipes disposable wipe around the ports to remove any liquid 1 Invert the lon PGM OneTouch Plus Reaction Filter Assembly IMPORTANT Follow the next steps exactly to minimize contact of the short tu
40. transfer the entire sample to a Qubit assay tube IMPORTANT Ensure that you measure the volumes accurately To generate a negative control add 200 uL of Quality Control Wash Buffer to a fresh Qubit assay tube Read the sample s using the Qubit 2 0 Fluorometer as described below Sample measurement IMPORTANT Prior to using the Qubit 2 0 Fluorometer ensure that the instrument is running Firmware V3 10 and the Ion_PluginV310_AF qbt file has been permanently uploaded to the instrument See section Upgrade the Qubit 2 0 Fluorometer firmware and software on page 61 for more information about managing Qubit 2 0 Fluorometer firmware versions Power on the Qubit 2 0 Fluorometer 2 Touch Ion to access Alexa Fluor 488 and Alexa Fluor 647 measurement options lon PGM Template OT2 200 Kit User Guide 69 TM Ml Appendix A Quality control of lon PGM Template OT2 200 lon Sphere Particles Quality control using the Qubit 2 0 Fluorometer 3 Touch AF 488 and insert the sample into the Qubit 2 0 Fluorometer close the lid and touch Read Note The lettering on the Read BE selection option changes from white to red when reading a sample The lettering changes back to white when the reading of the sample is finished in approximately 5 seconds Note If more than one sample is being processed all samples can be read with the AF 488 setting before moving on to the AF 647 setting 4
41. 0 Kit User Guide 41 Chapter 3 Prepare template positive lon PGM Template OT2 200 lon Sphere Particles Maintain the lon OneTouch 2 Instrument c Slowly and steadily withdraw the disposable injector straight from the port of the Injector Hub Ay CAUTION PHYSICAL INJURY HAZARD The pointed end of the disposable injector can puncture your skin Keep your hand away from the point of the disposable injector 9 Gently pull the disposable tubing downwards on the both sides of the pinch valve until the disposable tubing is out of the valve 10 Place the used disposable injector into the empty 50 mL conical tube in the tube rack The conical tube will be used for waste 42 lon PGM Template OT2 200 Kit User Guide 11 12 13 14 15 16 17 T Chapter 3 Prepare template positive lon PGM Template OT2 200 lon Sphere Particles DS Maintain the lon OneTouch 2 Instrument CO ow Ea On the home screen of the instrument touch Clean n Open Lid 4 1 65 we 0 00 PSI 4 IENS Complete each task displayed on the screen then touch Next After you touch Next on the last task you see a progress bar and the cleaning begins Ensure that the task in bold displays at the end of the cleaning run Remove plate injector conical tube and waste Note Keep the used Cleaning Adapter on the instrument between runs Appropriately dispose of the waste in the 50 mL conical tube R
42. 2 mL PCR tube has gt 200 uL of solution containing the enriched ISPs After a successful run on the instrument the sample is in 230 uL of Melt Off Solution Ion OneTouch Wash Solution and Neutralization Solution If the tube has lt lt 200 uL of solution containing the enriched ISPs contact Technical Support 8 Remove the used tip While you are standing above the Tip Arm and with the Tip Arm in its cradle twist the tip counterclockwise and pull it downward to remove and discard the tip IMPORTANT Improper removal of tips can loosen the metal tip adapter fitting on the Tip Arm and affect instrument operation 9 Remove and discard the used 8 well strip Sequence or store the template positive ISPs e Proceed to sequencing using the lon PGM Sequencing 200 Kit v2 Cat no 4482006 or the Ion PGM Hi Q Sequencing Kit Cat no A25592 Refer to the Ion PGM Sequencing 200 Kit v2 User Guide Pub no MAN0007273 or the Ion PGM Hi Q Sequencing Kit User Guide Pub no MAN0009816 or e Store the enriched ISPs at 2 C to 8 C for up to 3 days lon PGM Template OT2 200 Kit User Guide 51 TM Chapter 4 Enrich the template positive lon PGM Template OT2 200 lon fe Sphere Particles Perform lon Sphere Particles quality control Perform lon Sphere Particles quality control You can determine the enrichment efficiency using one of the following methods Quality assessment by Then O
43. 3035 1 e Qubit 2 0 Easy Calculator Microsoft Excel Spreadsheet file containing the instrument specific Calibration Factor see Calculate the Qubit 2 0 Fluorometer calibration factor on page 66 e Ion Sphere Quality Control Kit Cat no 4468656 e Unenriched Ion Sphere Particles e GeneAmp PCR System 9700 thermal cycler Cat no N8050200 or equivalent lon PGM Template OT2 200 Kit User Guide TM 1 Appendix A Quality control of lon PGM Template OT2 200 lon Sphere Particles Quality control using the Qubit 2 0 Fluorometer Upgrade the This section provides information about upgrading the firmware and the software of Qubit 2 0 the Qubit 2 0 Fluorometer The following illustration summarizes the upgrade path Fluorometer for each firmware version firmware and Qubit 2 0 Fluorometer Qubit 2 0 Fluorometer software Firmware v2 00 or v3 00 Firmware v3 10 To a USB drive download the program and upgrade files To a USB drive download the lon_PluginV310_AF qbt V3 10 program file Qubit_FW_MainCPU bin and lon_PluginV310_AF qbt V3 10 Qubit_FW_UsbHost bin v v Power on the Qubit 2 0 Power on the Qubit 2 0 Fluorometer and connect the Fluorometer and connect the USB drive USB drive vy v When prompted touch Yes to Touch V2 00 or V3 00 upload the lon Sphere Quality Control Assay for V3 10 v Touch Update v When prompted touch Yes to upload the lon Sphere Quality Control Assay for V3 10 Up
44. After emptying a waste container seal it with the cap provided Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure that the waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply 118 lon PGM Template OT2 200 Kit User Guide Appendix E Safety Biological hazard safety Biological hazard safety AX WARNING BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious diseases All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Safety equipment also may include items for personal protection such as gloves coats gowns shoe covers boots respirators face shields safety glasses or goggles Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially biohazardous materials Follow all applicable local state provincial and or national regulations The following references provide general guidelines when handling biological samples in laboratory environment U S Department of Hea
45. Arm is not resting on top of the thumb screw causing the Tip Arm to tilt forward The following photograph shows the correct position of the Tip Arm ai j Q Note For the residual volume test you do not need to put a 0 2 mL PCR into the hole of the Tip Loader lon PGM Template OT2 200 Kit User Guide 81 TM Appendix B Set up calibrate and maintain the lon OneTouch ES Perform a residual volume test 3 Load an 8 well strip on the Ion OneTouch ES a Load 80 uL water or Ion OneTouch Wash solution into the second well Well 2 from the square tabbed end of the 8 well strip Square shaped tab Rounded tab b Load the 8 well strip into the slot of the Tray so that the square tabbed end is to the left and the 8 well strip is pushed all the way to the right until it touches the end of the slot IMPORTANT Before running the residual volume test carefully read and familiarize yourself with step 4 of this procedure 4 Run the residual volume test During the test confirm that the tip is centered between the sides of the wells when moving in or out of a well a Turn the instrument ON b Wait for the instrument to initialize The screen displays rdy The Tip Arm performs a series of movements and returns to the home position 5 seconds c Press Start Stop d Wait for the instrument to aspirate the solution from Well 2 and completely remove the tip from Well 2 then manually push th
46. Briefly press then release the spring loaded top of the Injector Hub at the point indicated by the arrows in figures 4a 4c You should hear a click 4a up position 4b down position 4c up position IMPORTANT If the Injector Hub remains in the down position see Troubleshooting on page 53 IMPORTANT If you raise the centrifuge lid do not hit the disposable injector against the instrument You can damage the disposable injector If you damage the disposable injector appropriately dispose of the injector amplification plate and tubing Use a new disposable injector and Ion OneTouch 2 Amplification Plate Fill the appropriate Ion OneTouch Reagent Tube with Ion OneTouch Oil on the left front port A If you are Then Using a new Template 1 Use anew Reagent Tube from the kit Kit 2 Discard the used Reagent Tube and Sipper Tube Appropriately discard the residual Oil 3 Use fresh gloves to attach the Luer Lok end of a new lon OneTouch Sipper Tube to the eftfront port Do not let the Sipper Tube touch any surface 4 Invert the lon OneTouch Oil bottle 450 mL size 3 times to mix then fill the Reagent Tube Aa Afull with Oil Minimize bubbles 5 Insert the filled Reagent Tube into the eftfront port and screw the Reagent Tube firmly into place one quarter turn on the instrument Refilling the Reagent 1 Remove the Reagent Tube from the instrument Tube between runs 2 In
47. Do not lift the lid by the tubing attached to the lon OneTouch DL Injector Hub 2 Insert a on Ion OneTouch Recovery Tube into each of the slots in the centrifuge lon PGM Template OT2 200 Kit User Guide 101 SSE Appendix C Supplemental procedures Initialize the lon OneTouch 2 Instrument 3 Pinch the sides of the on Ion OneTouch Recovery Router then push the Recovery Router down into the center slot of the centrifuge so that the Recovery Router is seated flat and secure in the center of the rotor Recovery Tube Recovery Router Recovery Tube Note The Recovery Router is intentionally offset from the Recovery Tubes 4 Close the lid of the centrifuge Install the lon OneTouch 2 Amplification Plate 1 If there is a used Ion OneTouch 2 Cleaning Adapter on the instrument remove and appropriately discard it Note The Cleaning Adapter may be filled with Ion OneTouch Oil 2 Push the handle back to open the heat block Ay CAUTION Hot Surface Use care when working around this area to avoid being burned by hot components WARNING Safety Hazard Do not use the instrument with flammable or explosive materials Use only the materials specified for use with the instrument to ensure safety 102 lon PGM Template OT2 200 Kit User Guide Appendix C Supplemental procedures gt Initialize the lon OneTouch 2 Instrument 3 Obtain an Ion OneTouch 2 Amplification Plate The disposable
48. ORY EXPRESS OR IMPLIED ALL OF WHICH OTHER WARRANTIES ARE EXPRESSLY DISCLAIMED INCLUDING WITHOUT LIMITATION ANY IMPLIED WARRANTY OF MERCHANTABILITY FITNESS FOR lon PGM Template OT2 200 Kit User Guide Appendix D Instrument warranty A PARTICULAR PURPOSE NON INFRINGEMENT OR REGARDING RESULTS OBTAINED THROUGH THE USE OF ANY PRODUCT INCLUDING WITHOUT LIMITATION ANY CLAIM OF INACCURATE INVALID OR INCOMPLETE RESULTS WHETHER ARISING FROM A STATUTE OR OTHERWISE IN LAW OR FROM A COURSE OF PERFORMANCE DEALING OR USAGE OF TRADE lon PGM Template OT2 200 Kit User Guide 113 Safety VAN WARNING GENERAL SAFETY Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document Before using an instrument or device read and understand the safety information provided in the user documentation provided by the manufacturer of the instrument or device Before handling chemicals read and understand all applicable Safety Data Sheets SDSs and use appropriate personal protective equipment gloves gowns eye protection etc To obtain SDSs see the Documentation and Support section in this document Symbols on this instrument 114 Symbols may be found on the instrument to warn agains
49. S Observation Possible cause Recommended action Excessive foaming e Improperly calibrated or inadequate volume in one or more wells e Loose fitting e Cracked pipette tip 1 Use the recommended volumes for all wells 2 Ensure that fittings are tight especially at the elbow fitting and the pipette tip is not cracked 3 If necessary perform the residual volume check If the residual volume check fails then calibrate the instrument Brown pellet in centrifuged tube of enriched ISPs Residual Dynabeads MyOne Streptavidin C1 Beads 1 Pipet the suspension with the brown pellet up and down 10 times to resuspend the pellet 2 Place the 0 2 mL PCR tube against a magnet such as a DynaMag 2 magnet for 4 minutes 3 Transfer the supernatant with the enriched ISPs to a new 0 2 mL PCR tube without disturbing the pellet of Dynabeads MyOne Streptavidin C1 Beads 4 Sequence or store the enriched ISPs lon PGM Template OT2 200 Kit User Guide 55 Troubleshooting E on OneTouch ES Observation Possible cause Recommended action E12 E22 or E23 error displays during the run or during calibration Calibration values are out of range Power OFF the instrument and wait 3 seconds While holding down Vert Adjust power ON the instrument This step restores the factory default settings Recalibrate the vertical axis see Perform vertical
50. UPS must be rated for 1500 W output or higher The 1500 VA unit from APC provides several minutes of backup power for the Ion OneTouch 2 Instrument the Ion OneTouch ES lon PGM Template OT2 200 Kit User Guide 11 ad Chapter 1 Product information hae Additional materials required for lon OneTouch ES AU Recommended materials for the lon OneTouch 2 System Instrument the lon PGM Sequencer and the Torrent Server Use a surge protector or line conditioner as needed see Unpack and install the Ion OneTouch 2 System on page 95 The following additional materials are required for use and maintenance of the Ion OneTouch ES Instrument y Description Supplier Cat no Quantity DynaMag 2 magnet Life 12321D 1 Technologies 0 2 mL PCR tubes Axygen PCR 02 L C 1000 case BioExpress T 3035 1 120 strips of 8 1M NaOH Major Laboratory Supplier Xiameter PMX 200 Silicone Fluid 2 Neely PMX200 1250 1 95 lbs Industries OPT 11 We have demonstrated this protocol using this specific material Substitution may adversely affect system performance 21 Material required for periodic maintenance of the lon OneTouch ES Recommended materials for the lon OneTouch 2 System The Ion OneTouch 2 System uses common molecular biology equipment supplies and reagents MLS Fisher Scientific http www fisherscientific com or other major laboratory supplier
51. USER GUIDE ion torrent 6 AOXO oy life technologies lon PGM Template OT2 200 Kit for use with the lon OneTouch 2 System Catalog Number 4480974 Publication Number MAN0007220 Revision A 0 For Research Use Only Not for use in diagnostic procedures technologies The information in this guide is subject to change without notice DISCLAIMER LIFE TECHNOLOGIES CORPORATION AND OR ITS AFFILIATE S DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR MPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT TO THE EXTENT ALLOWED BY LAW IN NO EVENT SHALL LIFE TECHNOLOGIES AND OR ITS AFFILIATE S BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF Important Licensing Information This product may be covered by one or more Limited Use Label Licenses By use of this product you accept the terms and conditions of all applicable Limited Use Label Licenses Trademarks All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified Bioanalyzer is a trademark of Agilent Technologies Inc Agencourt and AMPure are registered trademarks of Beckman Coulter Inc Cyis a registered trademark of GE Healthcar
52. a eens Eiaa iE E E yp apa ate ooa EE E eg eval EEE ai 13 Prerequisites cece chk wend ove eee eee a by Sa REE eee Noe Ree he ee Rees 13 ContaMiNnatiOn so 0 65 2e ae wecnibeawee eh a a cae epee adeno 13 Instrument clearances 020 c eee eee e tenes 13 mM CHAPTER2 Getting started c cece cece ee cence eee eee e eens 14 WORKFLOW scaciid ou dees een dep ies dbiahe pie adewac ick eee ee ead Guns ahs ae 14 Procedural guidelines tuens aa ea aiai tee eee eee 14 Unpack and install the lon OneTouch 2 Instrument and lon OneTouch ES 14 Check the firmware sence yee el eh cag Rind oe ba nee pine haa ed Ree ee ee 15 Initialize the lon OneTouch 2 Instrument cceeeeeeeeeceeeeeeeeeeenes 15 Perform a verification run 2 0 2 cee teen ee 15 m CHAPTER3 Prepare template positive lon PGM Template OT2 200 lon Sphere Particles ssesecusicosetasnanedateasatadseseetacsanb eases 17 Work tlow muriti coeur Cel thee ct a be ee Batches a E E E a eiie 17 Set up the lon OneTouch 2 Instrument ceeceeccseeeeeeeeeeeeueeeeeeeeas 17 lon OneTouch 2 Instrument laYOUt 2 selb eda eee beeen eke ee EE baad 18 Materials required for this procedure 00 eee e cece ee eee eee eee 18 Install the lon OneTouch Recovery Tubes and lon OneTouch Recovery Router 19 Install the lon OneTouch 2 Amplification Plate 0 000 eee eee eee 19 lon PGM Template OT2 200 Kit User Guide 3 Contents
53. a for unenriched lon PGM Template OT2 200 lon Sphere Particles The optimal amount of library corresponds to the library dilution point that gives Percent Templated ISPs between 10 30 lon PGM Template OT2 200 Kit User Guide 71 72 TM Appendix A Quality control of lon PGM Template OT2 200 lon Sphere Particles Quality control using the Qubit 2 0 Fluorometer Samples that fall within the recommended range generally produce the most data however samples that fall outside of the recommended range can still meet the throughput specifications on the Ion chips The recommended optimal range is not intended to be a pass fail criteria The range provides guidance for the quality of the sample Note If the results are outside the desired Percent Templated ISPs range then increase or decrease the library input appropriately Percent Templated ISPs Description lt 10 Sample contains an insufficient number of templated ISPs to achieve optimal loading density on the lon Chip 10 30 Optimal amount of library gt 30 Sample will yield multi templated ISPs mixed reads lon PGM Template OT2 200 Kit User Guide Qubit 2 0 Fluorometer quality control TM 1 Appendix A Quality control of lon PGM Template OT2 200 lon Sphere Particles Qubit 2 0 Fluorometer quality control The following table contains a troubleshooting information for the unenriched Ion Sphere Quality Contro
54. are enriched ISPs see Set up the Ion OneTouch 2 Instrument on page 17 4 If you have a Qubit 2 0 System determine the percent template positive ISPs see Appendix A Quality control of lon PGM Template OT2 200 Ion Sphere Particles If you do not have a Qubit 2 0 System proceed to step 5 lon PGM Template OT2 200 Kit User Guide 15 y Lirias 16 Chapter 2 Getting started Perform a verification run 5 Sequence the control library on the lon PGM System using the Ion 314 Chip v2 Cat no 4482261 Set the lon PGM Sequencer to 500 flows Load the sample then analyze the results Note Use the lon PGM Sequencing 200 Kit v2 Cat no 4482006 Refer to the Ion PGM Sequencing 200 Kit v2 User Guide Pub no MAN0007273 6 Review the run report from the Torrent browser and confirm successful sequencing results with the control library The AQ20 result of the template positive ISPs must be 220 Mb with the Ion 314 Chip v2 Note If the AQ20 result is lt 20 Mb with the Ion 314 Chip v2 sequence the control library again and measure AQ20 If the AQ20 result is still not satisfactory contact Technical Support 7 The instrument is ready for use lon PGM Template OT2 200 Kit User Guide Workflow Prepare template positive lon PGM Template OT2 200 lon Sphere Particles Set up the lon OneTouch 2 Instrument on page 17 h Prepare and install the amplification solut
55. bing in the Reaction Tube with the amplification solution a Keep the lon PGM OneTouch Plus Reaction Filter Assembly in the tube rack orient the assembly so that the sample port is on your left Sample port b Lift straight out the lon PGM OneTouch Plus Reaction Filter Assembly from the tube rack With the short tubing in the Reaction Tube on the left rotate the assembly to your right until the Reaction Tube is inverted and the 3 ports of the Reaction Plus Filter face down IMPORTANT Correct rotation of the lon PGM OneTouch Plus Reaction Filter Assembly ensures minimal exposure of the short tubing in the Reaction Tube with the aqueous phase lon PGM Template OT2 200 Kit User Guide 31 TM ce Chapter 3 Prepare template positive lon PGM Template OT2 200 lon 2 Sphere Particles Run the lon OneTouch 2 Instrument 2 Insert the 3 ports of the Reaction Plus Filter into the three holes see figure 2a on the top stage of the Ion OneTouch 2 Instrument see figure 2b so that the Ion PGM OneTouch Plus Reaction Filter Assembly is firmly seated on the instrument The tab protruding from the outer edge of the Reaction Plus Filter fits into the front notch of the stage 2a 2b Tab Notch Note After inserting the lon PGM OneTouch Plus Reaction Filter Assembly bubbles may shoot up into the Reaction Tube Run the lon OneTouch 2 Instrument Note Ensure that you have the correct firmware update
56. bout customer training lon contact information Web site lifetechnologies com iontorrent Ion community ioncommunity lifetechnologies com Support email ionsupport lifetech com Phone numbers In North America 1 87 SEQUENCE 1 877 378 3623 Outside of North America 1 203 458 8552 120 lon PGM Template OT2 200 Kit User Guide For support visit lifetechnologies com support or email techsupportfdlifetech com lifetechnologies com 5 August 2014 technologies
57. bration on page 85 Tip grinds into base of instrument and Code 1999 displays e Unit not calibrated properly e Vertical calibration setting too low or out of range Erase the memory on the instrument Hold down the vertical adjust button while powering ON the instrument The instrument beeps several times Recalibrate the instrument see Calibrate the lon OneTouch ES on page 84 Perform a residual volume test see Perform a residual volume test on page 80 Tip is hitting the top of tray at start of run Tray is not properly seated in the instrument Check for debris between the tray and the instrument then reinstall the tray Press down firmly to ensure that tray is fully seated in the instrument Error displays Various 1 2 Power the instrument OFF then ON If the error continues to display erase the memory on the instrument Hold down the vertical adjust button while powering ON the instrument The instrument beeps several times Recalibrate the instrument see Calibrate the lon OneTouch ES on page 84 Perform residual volume check see Perform a residual volume test on page 80 Instrument does not aspirate or dispense liquids Loose fitting s Ensure that the Luer Lok connections at the elbow on the Tip Arm and at the tubing on the rear syringe pump are finger tight Ensure that the metal tip adapter fitting on the Tip Arm is f
58. calibration on page 85 Note The default setting for the vertical axis is 310 If the setting is lt 310 the instrument will likely display an error because the Tip Arm position is too high a Press the W minus button to lower the Tip Arm until the tip touches the shelf b Press the W minus button amp more times Typical vertical axis settings are 340 370 Recalibrate the horizontal axis see Perform horizontal axis calibration on page 87 Press the g amp plus button to move the Tip Arm to the right until the tip touches the left tab of the strip Note The default setting for the horizontal axis is 625 Typical horizontal axis settings are 640 670 AC line voltage module installed incorrectly Determine the voltage of the electrical outlet to plug in the lon OneTouch ES Align the arrow by the correct voltage on the AC line voltage module with the adjacent white arrow in the lower right corner of the fuse socket If the AC line voltage module is installed incorrectly 1 Gently remove the module with your fingernail or a small flathead screwdriver Rotate the module so that the correct voltage on the module is aligned and adjacent to the white arrow in the lower right hand corner of the fuse socket Insert the AC line voltage module into the fuse socket 56 lon PGM Template OT2 200 Kit User Guide Troubleshooting El lon OneTouch ES Observatio
59. d clicks open then remove and discard the Ion OneTouch Recovery Router 3 Carefully remove both Ion OneTouch Recovery Tubes from the instrument and put the two Recovery Tubes in a tube rack You may see some cloudiness in the tube which is normal 4 Remove excess lon PGM OT2 Recovery Solution from the ISPs a Use a pipette to remove all but 50 uL of the Recovery Solution from each Recovery Tube Withdraw the supernatant from the surface and on the opposite side from the pellet Remove any white flocculent material Do not disturb the ISP pellet Recovery Tube Location of ISP pellet b With a new tip and using the same tip for both tubes resuspend the ISPs in the remaining lon PGM OT2 Recovery Solution Pipet the pellet up and down until each pellet disperses in the solution STOPPING POINT Transfer the suspensions from both tubes into a new 1 5 mL Eppendorf LoBind Tube for a total of 100 uL Add 1 mL of Ion OneTouch Wash Solution to the 100 uL ISP suspension Store the ISPs at 2 C to 8 C for up to 3 days If the template positive ISPs were stored at 2 C to 8 C centrifuge the ISPs at 15 500 x g for 2 5 minutes then carefully remove all but 100 uL of supernatant With a new tip pipet up and down to resuspend the ISPs Proceed to step 5 5 Obtain an 8 well strip from the Ion OneTouch ES Supplies Kit Ensure that the square shaped tab of an 8 well strip is on the left Square shaped tab Rounde
60. d tab PTE 6 Transfer the suspensions from both tubes 1 tube if stored into Well 1 of the 8 well strip for a total of 100 uL of ISP suspension in the well lon PGM Template OT2 200 Kit User Guide 37 TM 5 Chapter 3 Prepare template positive lon PGM Template OT2 200 lon ES Sphere Particles Maintain the lon OneTouch 2 Instrument 7 Retain an aliquot of the unenriched Ion PGM Template OT2 200 Kit from Well 1 for quality assessment Assess the quality of the unenriched template positive ISPs using one of the following methods Quality assessment by Then Qubit 2 0 Fluorometer _ Transfer a 2 0 uL aliquot of the unenriched ISPs to a 0 2 mL PCR Tube then see Quality control using the Qubit 2 0 Fluorometer on page 60 Optional Guava Transfer a 1 0 uL aliquot of the unenriched ISPs to a easyCyte 5 Flow 1 5 mL Eppendorf LoBind Tube Refer to the on Cytometer Sphere Particles ISPs Quality Assessment Using the Guava easyCyte 5 Flow Cytometer User Bulletin Pub no 4470082 available on the lon Community website http ioncommunity lifetechnologies com Demonstrated protocol Transfer a 1 0 uL aliquot of the unenriched ISPs to a Quality assessment by 1 5 mL microcentrifuge tube Put the sample on ice the Applied Biosystems then refer to Demonstrated Protocol lon Sphere Attune Acoustic Particles ISPs Quality Assessment using the Applied Focusing Cytometer B
61. dinances for proper disposal provision and contact customer service for information about responsible disposal options Ne pas liminer ce produit avec les d chets usuels non soumis au tri s lectif CAUTION Pour minimiser les cons quences n gatives sur l environnement a la suite de l limination de d chets lectroniques ne pas liminer ce d chet lectronique avec les d chets usuels non soumis au tri s lectif Se conformer aux ordonnances locales sur les d chets municipaux pour les dispositions d limination et communiquer avec le service a la client le pour des renseignements sur les options d limination responsable Safety information for instruments not manufactured by Thermo Fisher Scientific Some of the accessories provided as part of the instrument system are not designed or built by Thermo Fisher Scientific Consult the manufacturer s documentation for the information needed for the safe use of these products Instrument safety General LN CAUTION Do not remove instrument protective covers If you remove the protective instrument panels or disable interlock devices you may be exposed to serious hazards including but not limited to severe electrical shock laser exposure crushing or chemical exposure Physical injury A servicing CAUTION Moving Parts Moving parts can crush pinch and cut Keep hands clear of moving parts while operating the instrument Disconn
62. dule so that the correct voltage on the module is aligned and adjacent to the white arrow in the lower right hand corner of the fuse socket 3 Insert the AC line voltage module into the fuse socket Instrument is not at the recommended operating temperature Ensure that the lon OneTouch ES is at an operating temperature of 60 F to 77 F 15 C to 25 C Solution overflowing during run Overloaded reagent volumes Repeat with reagent volumes described in enrichment procedure Tip is causing 8 well strip to lift Tip is not aligned vertically out of Tray slot during run Perform vertical calibration procedure see Perform vertical calibration on page 85 Percent enrichment is lt 50 as Multiple causes measured by the PGM System Contact Technical Support lon PGM Template OT2 200 Kit User Guide 57 Troubleshooting E on OneTouch ES Observation Possible cause Recommended action One of the following e Strip lifts up during strip push e Strip lifts up when tip is raised from well e Immediately after strip push the strip is not in contact with the magnet Instrument is not calibrated properly Perform horizontal position calibration see Perform vertical calibration on page 85 Perform vertical calibration see Perform horizontal axis calibration on page 87 Perform horizontal position calibration see page Perform vertical cali
63. e 8 well strip to the left so that Well 4 is positioned directly under the Tip Arm e Wait for the instrument to dispense the tip contents into Well 4 f Press Start Stop to stop the test run then press Start Stop again to return the Tip Arm to the home position g Place a P10 pipette at the front bottom of Well 2 aspirate the entire residual water or Ion OneTouch Wash Solution from the well then estimate the residual volume 82 lon PGM Template OT2 200 Kit User Guide TM ETD Appendix B Set up calibrate and maintain the lon OneTouch ES Perform a residual volume test 5 Remove the used tip With the Tip Arm in its cradle and while standing above the Tip Arm twist the tip counterclockwise and pull it downward to remove and discard the tip IMPORTANT Improper removal of tips can loosen the metal tip adapter fitting on the Tip Arm and affect instrument operation 6 Remove and discard the used 8 well strip lon PGM Template OT2 200 Kit User Guide 83 Appendix B Set up calibrate and maintain the lon OneTouch ES Calibrate the lon OneTouch ES 7 After performing the residual volume test take one or more of the following actions Observation Possible cause Recommended actions Residual volume in Well 2 is lt 5 uL Proceed to Prepare reagents then fill the 8 well strip on page 46 Residual volume in Well 2 is gt 5 uL IMPORTANT The volume is measured from th
64. e Sipper Tube touch any surfaces 4 Invert the Recovery Solution 3 times to mix then fill the Reagent Tube a quarter full with Recovery Solution Minimize bubbles 5 Insert the filled Reagent Tube into the right front port and screw the Reagent Tube firmly into place one quarter turn on the instrument Refilling the 1 Remove the Reagent Tube from the instrument Reagent Tube 2 Invert the bottle of Recovery Solution 3 times between runs 3 Add more Recovery Solution to the solution in the Reagent Tube until the tube is a quarter full Minimize bubbles 4 Insert the filled Reagent Tube into the right front port and screw the Reagent Tube firmly into place one quarter turn on the instrument Empty the Waste 1 Pull the external tubing from the port of the Waste Container Container ie 2 Empty the Waste Container into the appropriate receptacle 3 Push the tubing onto the port of the empty Waste Container 24 lon PGM Template OT2 200 Kit User Guide T Chapter 3 Prepare template positive lon PGM Template OT2 200 lon Pay Sphere Particles Prepare and install the amplification solution Prepare and install the amplification solution Materials required for this procedure Prepare the amplification solution Provided in the lon PGM Template 0T2 Solutions 200 Kit Part no 4481105 e lon PGM Template OT2 200 PCR Reagent B e lon OneTouch Reaction Oil 27 mL size
65. e UK Limited Guava is a registered trademark and easyCyte is a trademark of Guava Technologies Inc Kimwipes is a registered trademark of Kimberly Clark Corporation Eppendorf and Eppendorf LoBind are registered trademarks of Eppendorf AG Luer Lok is a registered trademark of Becton Dickinson and Company Xiameter is a registered trademark of Dow Corning Corporation Microsoft Windows and Excel are registered trademarks of Microsoft Corporation Macintosh is a registered trademark of Apple Inc Tween is a registered trademark of Uniqema Americas LLC 2014 Thermo Fisher Scientific Inc All rights reserved Contents About this GUIDE x cise cree scccecvarwseesseiaw ein ake ete entice dice ete wae ds 7 PUPRPOSC sisii taeda eee aaerha Go ee Peete Se ad Re eee ead 7 REVISION MIStONY sci occ sdanddndenc eee dade eeu inde dee eee E kano nace nae 8 Mm CHAPTER1 Product information ccceeeeeeeeeeeeeeeeees 9 Kit contents and storage conditions 02 2 cece eens 9 lon PGM Template OT2 200 Kit 0 20 ees 9 lon PGM Enrichment Beads 2 0000 eee eee eee eee eee 11 Required materials and equipment not provided AT ea a a a teen 11 Required materials for the lon OneTouch 2 System EE EA E E EEA EEE 11 Additional materials required for lon OneTouch ES 02 eee eeeee 12 Recommended materials for the lon OneTouch 2 Systemi Sind si dSecbasenarenarrtivas ana 12 EQUIPMENE USE oie ccc occ nisin ee Se
66. e all risk when using these protocols and recognize that support for Life Technologies Demonstrated Protocols occurs through community discussion All customers are encouraged to discuss and contribute via the Ion Community lon PGM Template OT2 200 Kit User Guide Set up calibrate and maintain the lon OneTouch ES Set up the Ion OneTouch ES 4isianciewvie eee ectareneituwienceeiaes 75 Perform a residual volume test 00 c cece eee eee Epi erie kS 80 Calibrate the Ion OneTouch ES ananunua eevee ve sale eave ndaneuews 84 Maintain the Ion OneTouch ES 00 cece cece eee eee ene eees 88 Decontaminate the Ion OneTouch ES 00 cece ee teen eee eens 93 IMPORTANT Ensure that the fuse module for the lon OneTouch ES is properly installed according to 110 120 V or 240 V line voltages Refer to Unpack and Install the Ion OneTouch 2 System Product Insert Pub no 4481875 Set up the lon OneTouch ES Before the first ISP enrichment run set up the instrument Materials Provided in the lon PGM Template OT2 required for this Solutions 200 Kit Part no 4481105 procedure e Nuclease free Water Provided in the lon PGM Template OT2 Supplies 200 Kit Part no 4480981 e 8 well strip e Eppendorf LoRetention Dualfilter Tips P300 Provided in the lon OneTouch 2 System Part no 4474779 e Corning Brand 96 Well Strip Ejector Thermo Fisher Cat no 07 200 22 un
67. e boot eed book niet hee Rae ene a E arae a a ies 120 lon contact information 22 c eee eee eee nee eee 120 lon PGM Template OT2 200 Kit User Guide About this guide AN CAUTION ABBREVIATED SAFETY ALERTS Hazard symbols and hazard types specified in procedures may be abbreviated in this document For the complete safety information see the Safety appendix in this document IMPORTANT Before using this product read and understand the information in the Safety Appendix in this document Purpose TM This user guide describes how to use the Ion OneTouch 2 System to prepare enriched template positive Ion PGM Template OT2 200 Ion Sphere Particles with 200 base pair average insert libraries on the Ion Personal Genome Machine PGM System The Ion OneTouch 2 System includes the Ion OneTouch 2 Instrument and the Ion OneTouch ES Instrument The user guide is organized as follows e Prepare template positive ISPs containing clonally amplified DNA using the Ion PGM Template OT2 200 Kit for up to 200 base read libraries with the Ion OneTouch 2 Instrument see Chapter 3 Prepare template positive lon PGM Template OT2 200 Ion Sphere Particles e Enrich the template positive ISPs with the lon OneTouch ES see Chapter 4 Enrich the template positive lon PGM Template OT2 200 Ion Sphere Particles TM TM IMPORTANT Use only the lon PGM Template OT2 200
68. e bottom of the well not from the sides The tip height is too high during aspiration Restore defaults then calibrate the instrument see Calibrate the lon OneTouch ES on page 84 Aspiration is irregular This instrument out of calibration Restore defaults then calibrate the instrument see Calibrate the lon OneTouch ES on page 84 The 8 well strip lifts as the tip rises to the top of the well The tip is angled too far forward or the tip height is set too low The 8 well strip lifts as the tip rises to the top of the well Verify that the tip is vertical and positioned directly over the notch in the calibration shelf If the tip is positioned correctly Restore defaults then calibrate the instrument see Calibrate the lon OneTouch ES on page 84 Calibrate the lon OneTouch ES Perform horizontal and vertical calibrations so that during operation the tip is optimally positioned in the well of the 8 well strip 84 lon PGM Template OT2 200 Kit User Guide Perform vertical calibration TM Appendix B Set up calibrate and maintain the lon OneTouch ES Calibrate the lon OneTouch ES Note that the 8 well strip is always tilted at a fixed 10 degree angle in the slot The pipette tip is vertical When the tip is aligned properly during calibration so that it is in line with the notch in the calibration shelf the tip touches the bottom of the well
69. e bubbles d Install the Reagent Tube on the instrument 2 Insert the filled Reagent Tube into the left front port and screw the Reagent Tube firmly into place one quarter turn on the instrument Install the lon PGM OT2 Recovery Solution IMPORTANT Use only the Ion PGM OT2 Recovery Solution in the Ion OneTouch kit that you are currently using Do not use a different recovery solution from another kit 1 Ensure that the Recovery Solution is clear If it is clear proceed to the next step If the Recovery Solution is not clear heat the bottle of Recovery Solution in a 30 C bath until the Recovery Solution is clear 106 lon PGM Template OT2 200 Kit User Guide Appendix C Supplemental procedures P2H Initialize the lon OneTouch 2 Instrument E0 gt 2 Fill the appropriate lon OneTouch Reagent Tube with lon PGM OT2 Recovery Solution on the right front port a Use anew Reagent Tube from the kit b Use fresh gloves to attach the Luer Lok end of a new Ion OneTouch Sipper Tube to the right front port Do not let the Sipper Tube touch any surfaces Minimize bubbles c Invert the Recovery Solution bottle 3 times then fill the Reagent Tube a quarter full with Recovery Solution d Install the filled Reagent Tube on the instrument 3 Insert the filled Reagent Tube into the right front port and screw the Reagent Tube firmly into place one quarter turn on the instrument Install the lon O
70. e cleaning procedure with the Ion OneTouch 2 Cleaning Adapter refer to the Options screen on the instrument display To update the firmware to the appropriate version see Install a firmware update on page 96 Initialize the lon OneTouch 2 Instrument IMPORTANT Before operating the Ion OneTouch 2 Instrument for the first time you must perform the one time initialization on the instrument Initialization primes the pumps and tubing lines for reliable operation Perform initialization at any time before the first run For detailed instructions on initialization of the Ion OneTouch 2 Instrument see Initialize the Ion OneTouch 2 Instrument on page 100 Perform a verification run To ensure optimal use of the lon OneTouch 2 System we recommend first preparing and enriching template positive lon PGM Template OT2 200 Ion Sphere Particles ISPs on the system with a control library 1 Obtain the E coli DH10B Control Library bag from the Ion PGM Controls Kit v2 Cat no 4482010 and use the E coli DH10B Control 200 Library 2 In an Eppendorf LoBind Tube dilute 1 uL of control library into 260 uL of Nuclease free Water Use 25 uL of the dilution in the amplification solution see Prepare the amplification solution on page 25 3 Follow the operating instructions to set up and use the Ion OneTouch 2 Instrument to prepare template positive ISPs and to use the lon OneTouch ES to prep
71. e metal fitting of the Tip Arm with the tip Keeping the fitting on the Tip Arm vertical firmly press the Tip Arm down onto the new tip until the Tip Arm meets the Tip Loader Hold the Tip Arm to the Tip Loader for 1 second to ensure proper installation of the tip Lift the Tip Arm straight up to pull the installed tip from the Tip Loader tube b Return the Tip Arm to the cradle Tilt the Tip Arm back see figure b1 Align the pins with the round notches in the cradle see figure b2 then lower the Tip Arm into position see figure b3 Move the Tip Arm forward into the working position b1 b2 b3 lon PGM Template OT2 200 Kit User Guide 49 TM Chapter 4 Enrich the template positive lon PGM Template OT2 200 lon S Sphere Particles Perform the run 2 Ensure that the back bottom end of the Tip Arm is not resting on top of the thumb screw causing the Tip Arm to tilt forward This is the correct position of the Tip Arm Install a new opened 0 2 mL PCR tube into the hole of the Tip Loader 3 Add 10 uL of Neutralization Solution to a new 0 2 mL PCR tube 4 Insert the opened 0 2 mL PCR tube with the Neutralization Solution into the hole in the base of the Tip Loader as shown in the preceding photograph Perform the run 1 Confirm that a new tip and opened 0 2 mL PCR tube with the Neutralization Solution have been loaded and that the 8 well strip is correctly loaded Ensure that Well 1 ISP sample
72. ect power before lon PGM Template OT2 200 Kit User Guide 115 Appendix E Safety Safety and electromagnetic compatibility EMC standards Electrical WARNING Ensure appropriate electrical supply For safe operation of the instrument Plug the system into a properly grounded receptacle with adequate current capacity Ensure the electrical supply is of suitable voltage Never operate the instrument with the ground disconnected Grounding continuity is required for safe operation of the instrument WARNING Power Supply Line Cords Use properly configured and approved line cords for the power supply in your facility A N WARNING Disconnecting Power To fully disconnect power either detach or unplug the power cord positioning the instrument such that the power cord is accessible Cleaning and CAUTION Cleaning and Decontamination Use only the cleaning and decontamination decontamination methods specified in the manufacturer s user documentation It is the responsibility of the operator or other responsible person to ensure the following requirements are met No decontamination or cleaning agents are used that could cause a HAZARD as a result of a reaction with parts of the equipment or with material contained in the equipment The instrument is properly decontaminated a if hazardous material is spilled onto or into the equipment and or b prior to having the instrument serviced
73. emove and appropriately dispose of the used Amplification Plate disposable injector and tubing from the instrument a Push the handle to open the heat block b Remove the disposable tubing from the Ion OneTouch DL Catch c Gently pull back the Amplification Plate from the inlet and outlet holes of the instrument d Remove the Amplification Plate from the heat block and appropriately dispose of the used Amplification Plate injector and tubing e Leave the heat block open CAUTION Hot Surface Use care when working around this area to avoid being burned by hot components On the instrument display touch Open Lid wait until the lid clicks open then open the centrifuge lid Wipe the residue from the centrifuge lid with dry Kimwipes disposable wipers and close the centrifuge lid Touch Next to return to the home screen on the instrument lon PGM Template OT2 200 Kit User Guide 43 I Y OX AVA x Z a gt O OP Eo Cy K E al E7 Workflow Materials required Enrich the template positive lon PGM Template OT2 200 Ion Sphere Particles Perform the residual volume test on the lon OneTouch ES on page 45 vy Prepare reagents then fill the 8 well strip on page 46 vy Prepare the lon OneTouch ES on page 49 y Perform the run on page 50 y Sequence or store the template positive ISPs on page 51 y Perform lon Sphere Particles quality control o
74. eparing the amplification solution 8 Proceed immediately to Fill the Ion PGM OneTouch Plus Reaction Filter Assembly Fill the lon PGM IMPORTANT We recommend filling the Ion PGM OneTouch Plus Reaction Filter OneTouch Plus Assembly in a room dedicated to pre PCR activities or in a controlled pre PCR hood Reaction Filter Do not use a reaction filter assembly from any other template preparation kit Assembly 1 Obtain an Ion PGM OneTouch Plus Reaction Filter Assembly from the Ion PGM Template OT2 200 Kit Sample port lon PGM OneTouch Plus Reaction Filter Short tubing from sample port to lon OneTouch Reaction Tube lon OneTouch Reaction Tube 28 lon PGM Template OT2 200 Kit User Guide Chapter 3 Prepare template positive lon PGM Template OT2 200 lon Sphere Particles Prepare and install the amplification solution 2 Place the lon PGM OneTouch Plus Reaction Filter Assembly into a tube rack so that the 3 ports of the lon PGM OneTouch Plus Reaction Filter Assembly face up 3 Locate the sample port on the lon PGM OneTouch Plus Reaction Filter Assembly The short tubing in the Reaction Tube is connected to the sample port Sample port Sample port Note The color of the short tubing attached to the sample port may vary 4 Add the amplification solution through the sample port to prepare the amplification solution see Prepare the amplification solution
75. equirements for electrical equipment for measurement control and laboratory use Part 2 081 Particular requirements for automatic and semi automatic laboratory equipment for analysis and other purposes IEC 60825 1 2007 Safety of laser products Part 1 Equipment classification and requirements EN 60825 1 2007 Radiation Safety of Laser Products Equipment Classification Requirements and User s Guide Reference Description Directive European Union EMC Directive 2004 108 EC EN 61326 1 Electrical Equipment for Measurement Control and Laboratory Use EMC Requirements Part 1 General Requirements FCC Part 18 47 CFR U S Standard Industrial Scientific and Medical Equipment AS NZS 2064 Limits and Methods of Measurement of Electromagnetic Disturbance Characteristics of Industrial Scientific and Medical ISM Radiofrequency Equipment ICES 001 Issue 3 Industrial Scientific and Medical ISM Radio Frequency Generators lon PGM Template OT2 200 Kit User Guide 117 Appendix E Safety Chemical safety Environmental Reference Description design Directive 2012 19 EU European Union WEEE Directive Waste electrical and electronic equipment Directive 2011 65 EU European Union RoHS Directive Restriction of hazardous substances in electrical and electronic equipment Directive 2006 66 EC European Union Battery Directive
76. er a run is aborted follow these steps in this order Download the log files for troubleshooting by Technical Support see Download the log files from the Ion OneTouch 2 Instrument on page 109 Turn OFF the instrument If necessary retain all consumables on the instrument for troubleshooting After successful troubleshooting appropriately dispose of all used consumables and turn ON the instrument Set up the instrument with new kit components see Set up the Ion OneTouch 2 Instrument on page 17 Prepare a new amplification solution see Prepare and install the amplification solution on page 25 Start a new run 6 Remove the samples lt 16 hours after starting the run If you touched Next on the Centrifuge screen to centrifuge samples at the end of the run proceed immediately to Recover the template positive lon PGM Template OT2 200 Ion Sphere Particles on page 36 lon PGM Template OT2 200 Kit User Guide 35 TM Chapter 3 Prepare template positive lon PGM Template OT2 200 lon Sphere Particles Recover the template positive lon PGM Template OT2 200 lon Sphere Parti cles Recover the template positive lon PGM Template OT2 200 lon Sphere Particles J zo 4 X E Materials Provided in the lon PGM Template OT2 required for this Solutions 200 Kit Part no 4481105 procedure lon OneTouch Wash Solution Other Materials and Equipment e 1 5 mL E
77. for this 200 Kit Part no 4480981 procedure e 2 lon OneTouch Reagent Tubes e lon OneTouch Recovery Router e 2lon OneTouch Recovery Tubes e lon OneTouch 2 Amplification Plate e 21lon OneTouch Sipper Tubes Provided in the lon PGM Template OT2 Solutions 200 Kit Part no 4481105 e lon OneTouch Oil e lon PGM OT2 Recovery Solution Note We have validated this protocol using only the material specified Substitution may adversely affect performance and safety IMPORTANT Use only the lon PGM Template OT2 200 Kit Cat no 4480974 with this user guide and with the Ion OneTouch 2 System Do not use the kit with the Ion OneTouch System Do not mix reactions or disposables including plates solutions and kit reagents from other template preparation kits 18 lon PGM Template OT2 200 Kit User Guide Install the lon OneTouch Recovery Tubes and lon OneTouch Recovery Router Install the lon OneTouch 2 Amplification Plate Chapter 3 Prepare template positive lon PGM Template OT2 200 lon Sphere Particles Set up the lon OneTouch 2 Instrument On the instrument display touch Open Lid wait until the lid clicks open then lift and hold the side of the centrifuge lid IMPORTANT Do not lift the lid by the tubing attached to the Ion OneTouch DL Injector Hub Do not force the lid open Insert a Ion OneTouch Recovery Tube into each of the slots in the centrifuge P
78. g this specific material Substitution may adversely affect performance Required materials and equipment not provided Required The Ion OneTouch 2 System uses common molecular biology equipment supplies materials for the and reagents MLS Fisher Scientific http www fisherscientific com or other major lon OneTouch 2 laboratory supplier System J Description Supplier Cat no Quantity lon OneTouch 2 System Life 4474779 1 The system includes Tecnnologies e lon OneTouch 2 Instrument e lon OneTouch ES e AC Power Supply and Cords e Installation Kit e lon OneTouch ES GeneAmp PCR System 9700 thermal cycler or Life N8050200 Base 1 equivalent Technologies 4314443 Block 1 5 mL Eppendorf LoBind Tubes Eppendorf 022431021 250 box Microcentrifuge 2 MLS 1 Pipettes P2 P20 P200 P1000 and appropriate low MLS 1 each retention tips Vortexer with a rubber platform MLS 1 Tube rack to fit 15 mL conical tube MLS 1 Tube rack for 50 mL conical tube MLS 1 Heat block set to 75 C MLS 1 11 We have demonstrated this protocol using this specific material Substitution may adversely affect system performance 21 Must fit standard 1 5 and 0 2 mL microcentrifuge tubes must generate 15 500 x g Note We recommend using an uninterruptable power supply UPS for laboratories that experience frequent power outages or line voltage fluctuations The
79. grade for Note The previous program files qbt are not compatible with the new V3 10 firmware V2 00 or firmware The new program file lon_PluginV310_AF qbt is not compatible with V3 00 V3 00 firmware Materials required for update Qubit 2 0 Fluorometer Part no Q32866 e USB drive included with Qubit 2 0 Fluorometer e Program qbt and upgrade bin files Jon_PluginV310_AF qbt V3 10 Qubit_FW_MainCPU bin V3 10 Qubit_FW_UsbHost bin Guidelines for downloading files We recommend that you use a Windows OS based computer to download and transfer the Qubit 2 0 Fluorometer files to the USB drive Note Do not use a Macintosh computer to transfer the files to the USB drive if the serial number of your Qubit 2 0 Fluorometer is lower than 1104004846 lon PGM Template OT2 200 Kit User Guide 61 62 TM 1 Appendix A Quality control of lon PGM Template OT2 200 lon Sphere Particles Quality control using the Qubit 2 0 Fluorometer We also recommend that you use the USB drive provided with Qubit 2 0 Fluorometer for file transfer Other compatible USB drives may also be used Visit http ioncommunity lifetechnologies com community products pgm to see a list of approved USB drives Logging into the lon Community is required 1 Download the program file qbt and two upgrade files bin fromhttp ioncommunity lifetechnologies com community products pgm onto a Windows based PC desktop
80. h the disposable injector through the port until the disposable injector just stops at the base of the router lon PGM Template OT2 200 Kit User Guide 105 Appendix C Supplemental procedures Initialize the lon OneTouch 2 Instrument 3 Release the disposable injector 4 Confirm automatic placement of the disposable injector above the router Briefly press then release the spring loaded top of the Injector Hub at the point indicated by the arrows in figures 4a 4c You should hear a click 4a up position 4b down position 4c up position IMPORTANT If the Injector Hub remains in the down position see Troubleshooting on page 53 IMPORTANT If you raise the centrifuge lid do not hit the disposable injector against the instrument You can damage the disposable injector If you damage the disposable injector appropriately dispose of the injector amplification plate and tubing Use a new disposable injector and Ion OneTouch 2 Amplification Plate Install the lon OneTouch Oil 1 Fill the appropriate Ion OneTouch Reagent Tube with Ion OneTouch Oil on the left front port Jk a Use anew Reagent Tube from the kit b Use fresh gloves to attach the Luer Lok end of a new Ion OneTouch Sipper Tube to the left front port Do not let the Sipper Tube touch any surface c Invert the on Ion OneTouch Oil bottle 450 mL size 3 times to mix then fill the Reagent Tube half full with Oil Minimiz
81. inch the sides of the lon OneTouch Recovery Router then push the Recovery Router down into the center slot of the centrifuge so that the Recovery Router is seated flat and secure in the center of the rotor Recovery Tube Recovery Router Recovery Tube Note The Recovery Router is intentionally offset from the Recovery Tubes Close the lid of the centrifuge If there is a used Ion OneTouch 2 Cleaning Adapter on the instrument remove and appropriately discard it Note The Cleaning Adapter may be filled with Ion OneTouch Oil Push the handle back to open the heat block CAUTION Hot Surface Use care when working around this area to avoid being burned by hot components WARNING Safety Hazard Do not use the instrument with flammable or explosive materials Use only the materials specified for use with the instrument to ensure safety lon PGM Template OT2 200 Kit User Guide 19 Chapter 3 Prepare template positive lon PGM Template OT2 200 lon Sphere Particles Set up the lon OneTouch 2 Instrument 3 Obtain an Ion OneTouch 2 Amplification Plate The disposable tubing and disposable injector are attached to the Amplification Plate An Amplification Plate supplied in the lon PGM Template OT2 200 Kit has ion on the tab Note Before use inspect the plate port of the Amplification Plate to ensure that it is straight and perpendicular to the plate Do not disconnect tubing fr
82. inch valve a Align the disposable tubing with the slot that runs along the bottom of the pinch valve see figure 8a b Gently pull the disposable tubing upwards on the both sides of the pinch valve see figure 8b until the disposable tubing is in the slot and secured in the round notch on each side of the pinch valve see figure 8c 8a 8b 8c c If necessary adjust the disposable tubing along the notches of the open pinch valve so that there is sufficient length of disposable tubing to install the disposable injector see Install the disposable injector on page 105 104 lon PGM Template OT2 200 Kit User Guide Appendix C Supplemental procedures gt Initialize the lon OneTouch 2 Instrument Install the disposable injector Note Before use inspect the long metal shaft of the disposable injector Some disposable injectors may be slightly bent which is normal If you have questions about the disposable injector contact Technical Support 1 Place one hand on the centrifuge lid Place the other hand at the top of the disposable injector and insert the disposable injector straight into the port of the Ion OneTouch DL Injector Hub Ay CAUTION PHYSICAL INJURY HAZARD The pointed end of the disposable injector can puncture your skin Keep your hand away from the point of the disposable injector Note The color of the disposable injector may vary 2 Keep your hand on the centrifuge lid then pus
83. ing final centrifugation to pellet the ISPs in the lon OneTouch Recovery Tubes Decontaminate the lon OneTouch 2 Instrument Before returning the instrument for service decontaminate it according to the procedure below Materials and e Disposable rubber gloves equipment e Safety glasses required e Lab coat e Bleach e Water e Paper towels IMPORTANT This procedure does not guarantee total decontamination of the Ion OneTouch 2 Instrument Decontaminate 1 Wear disposable rubber gloves safety glasses and a lab coat the instrument 2 Use a cleaning pad wetted with a solution of 1 part chlorine bleach in 9 parts water 10 bleach solution to clean all outside surfaces of the Ion OneTouch 2 Instrument Use care to avoid getting bleach solution inside the chassis 3 Dry the surfaces of the lon OneTouch 2 Instrument with paper towels or other disposable wipes 4 Use cotton swabs to clean and dry areas that are difficult to reach 5 Properly dispose of used cleaning materials to ensure that no one becomes exposed to contaminants lon PGM Template OT2 200 Kit User Guide 111 112 Instrument warranty For new Ion Torrent instruments Life Technologies warrants to and only to buyer for twelve 12 months from the date of shipping that the Ion Torrent software and Ion Torrent instruments are free from defects in material and workmanship and conform to Life Technologies published
84. inger tight IMPORTANT After any adjustments to the metal tip adapter recalibrate the lon OneTouch ES 58 lon PGM Template OT2 200 Kit User Guide Quality control of lon PGM Template OT2 200 lon Sphere Particles Quality Control assay sase crne raro e eee enn nee a 59 Quality control using the Qubit 2 0 Fluorometer 00000 e eee eee 60 Qubit 2 0 Fluorometer quality control s s 0 0c eee e eee eee eee 73 Quality control using the Guava easyCyte 5 Flow Cytometer 73 Demonstrated protocol Quality control using Attune Acoustic Focusing CytOmietEt sa saisai aaa nena E R KTR E E Fine E ea ace a RS 74 Quality Control assay The Ion Sphere Quality Control assay on the Qubit 2 0 Fluorometer labels the Ion Sphere Particles ISPs 200 with two different fluorophores Alexa Fluor 488 and Alexa Fluor 647 The probe labeled Alexa Fluor 488 anneals to primer B sites or all of the ISPs present The probe labeled Alexa Fluor 647 anneals to primer A sites or only the ISPs with extended templates The ratio of the Alexa Fluor 647 fluorescence templated ISPs to the Alexa Fluor 488 fluorescence all ISPs present yields the templated ISPs The following is the schematic of the probes labeled Alexa Fluor 488 and Alexa Fluor 647 annealed to an ISP Note The drawing is not to scale B primer A Adapter Sequence lon PGM Template OT2 200 Kit Use
85. instrument specific calibration factor IMPORTANT You must upgrade the Qubit 2 0 Fluorometer firmware and software prior to performing the following procedure See Upgrade the Qubit 2 0 Fluorometer firmware and software on page 61 for more information Each Qubit 2 0 Fluorometer has a unique Calibration Factor that must be calculated and applied to all Percent Templated ISPs calculations Note It is only necessary to calculate the calibration factor once for a particular instrument unless a problem is suspected Materials required e Qubit 2 0 Easy Calculator Microsoft Excel Spreadsheet Logging into the Ion Community is required e Qubit 2 0 Fluorometer Part no Q32866 with V3 10 firmware e USB drive containing the qbt file e Qubit Assay Tubes Part no Q32856 e Ion Sphere Quality Control Kit Part no 4468656 Download Qubit 2 0 Easy Calculator Download the Qubit 2 0 Fluorometer Easy Calculator Microsoft Excel Spreadsheet file from http ioncommunity lifetechnologies com community products pgm Logging into the lon Community is required and save the file to the computer used for Qubit 2 0 Fluorometer data analysis Calibration standard preparation 1 From the Ion Sphere Quality Control Kit thaw the Alexa Fluor 488 and Alexa Fluor 647 Calibration Standard reagents Note Both the Alexa Fluor 488 and Alexa Fluor 647 molecules are photosensitive so avoid exposure to
86. ion on page 25 y Run the lon OneTouch 2 Instrument on page 32 h Recover the template positive lon PGM Template OT2 200 lon Sphere Particles on page 36 v Maintain the lon OneTouch 2 Instrument on page 38 Set up the lon OneTouch 2 Instrument Note If this is the first use of the instrument perform the one time initialization procedure see Initialize the lon OneTouch 2 Instrument on page 100 Perform initialization at any time before the first run If this is the first run after initialization proceed to Prepare and install the amplification solution on page 25 The instrument consumables are already installed and ready for the run lon PGM Template OT2 200 Kit User Guide 17 Chapter 3 Prepare template positive lon PGM Template OT2 200 lon Sphere Particles Set up the lon OneTouch 2 Instrument lon OneTouch 2 lon PGM OneTouch Plus Instrument layout Reaction Filter Assembly Instrument display Clamp handle to access the Amplification Plate in the heat block lon OneTouch DL Injector Hub Built in centrifuge to install the Recovery Tubes and Recovery Router Waste Container Oil waste tray Pinch valve to hold lon OneTouch Oil A disposable tubing lon PGM OT2 Recovery Solution Note The photograph does not show the disposable tubing Materials Provided in the lon PGM Template OT2 Supplies required
87. ion Standard into the Qubit 2 0 Fluorometer close the lid and touch Read Note The lettering on the Read BE selection option changes from white to red when reading a sample The lettering changes back to white when the reading of the sample is finished in approximately 5 seconds 5 Record the RFU value and remove sample from Qubit 2 0 Fluorometer Calibration Factor calculation 1 In the Qubit 2 0 Easy Calculator enter each recorded RFU value in the appropriately labeled green cell to display the Calibration Factor specific for the Qubit 2 0 Fluorometer lon PGM Template OT2 200 Kit User Guide 67 TM i E Appendix A Quality control of lon PGM Template OT2 200 lon Sphere Particles Quality control using the Qubit 2 0 Fluorometer 2 Save a copy of the Qubit 2 0 Easy Calculator containing the Calibration Factor for use as a template for future Percent Templated ISPs calculations Note Affix a sticker with the instrument specific Calibration Factor to the Qubit 2 0 Fluorometer Qubit Calibration Factor Calculation Calibration Standard Calibration Factor Alexa Fluor 488 Calibration Standard Alexa Fluor 647 Calibration Standard DIV O Templated Signal Ratio Calibration Standard Raw RFU Value Background RFU Negative Control Tube Sample ID Alexa Fluor 488 Alexa Fluor 647 Alexa Fluor 488 Alexa Fluor 647 Conversion Factor Percent Templated ISPs j
88. iosystems Attune Acoustic Focusing Cytometer User Bulletin Pub no 4477181 available on the lon Community website http ioncommunity lifetechnologies com 11 Life Technologies Demonstrated Protocols have been successfully demonstrated by Life Technologies research and development but not formally validated 8 Enrich the template positive ISPs see Chapter 4 Enrich the template positive Ion PGM Template OT2 200 Ion Sphere Particles IMPORTANT Do not store the recovered template positive ISPs at 30 C to 10 C Do not store the ISPs in lon PGM OT2 Recovery Solution see step 4 of this procedure Maintain the lon OneTouch 2 Instrument IMPORTANT Follow the cleaning procedure in this section to clean the Ion OneTouch 2 Instrument with the Ion OneTouch 2 Cleaning Adapter Always perform the cleaning procedure after every run Do not skip this procedure The cleaning procedure is performed according to the steps displayed on the instrument after removing the Recovery Tubes TM Note To set up the Ion OneTouch 2 Instrument when switching between sequencing platforms refer to the Demonstrated Protocol How to Set Up the Ion OneTouch 2 Instrument When Switching Between the Ion PGM System and the Ion Proton System Pub no MAN0007719 38 lon PGM Template OT2 200 Kit User Guide Materials required for this procedure Clean the lon OneTouch 2 Instrument T
89. is the left most well and that the 8 well strip is pushed to the far right position within the slot 2 Pipet the contents of Well 2 up and down to resuspend the beads before starting the run Do not introduce bubbles into the solution 3 If necessary turn ON the Ion OneTouch ES and wait for the instrument to initialize The screen displays rdy The Tip Arm performs a series of initialization movements and returns to the home position 5 seconds 4 Press Start Stop The screen displays run during the run The run takes 35 minutes Note If necessary to stop a run press Start Stop The instrument completes the current step then stops the run and displays End Press Start Stop again to return the Tip Arm to the home position It is not possible to restart where you left off after stopping a run 5 At the end of the run the instrument displays End and beeps every 60 seconds Press the Start Stop button to silence this alarm and reset the Ion OneTouch ES for the next run The instrument can be left on between runs 6 Immediately after the run securely close and remove the PCR tube containing the enriched ISPs 50 lon PGM Template OT2 200 Kit User Guide T Chapter 4 Enrich the template positive lon PGM Template OT2 200 lon Sphere Particles Sequence or store the template positive ISPs 7 Mix the contents of the PCR tube by gently inverting the tube 5 times Note Ensure that the 0
90. l 8 is gt 5 0 uL Routine use and residual volume in Well 1 Operate the instrument without performing and Well 8 is lt 5 0 uL the residual volume test Proceed to Prepare reagents then fill the 8 well strip on page 46 lon PGM Template OT2 200 Kit User Guide 45 TM Chapter 4 Enrich the template positive lon PGM Template OT2 200 lon fa Sphere Particles Prepare reagents then fill the 8 well strip Prepare reagents then fill the 8 well strip Prepare Melt Off Prepare fresh Melt Off Solution by combining the components in the following order solukon Order Component Volume 1 Tween Solution 280 uL 2 1 M NaOH 40 uL Total 320 uL IMPORTANT Prepare Melt Off Solution as needed but appropriately dispose of the solution after 1 day The final composition of the Melt Off Solution is 125 mM NaOH and 0 1 Tween 20 detergent Wash and 1 Vortex the tube for 30 seconds to thoroughly resuspend the beads then resuspend the centrifuge the tube of Dynabeads MyOne Streptavidin C1 Beads for 2 seconds Dynabea ds 2 Open the tube then use a new tip to pipet up and down the dark pellet of beads MyOne oes until the pellet disperses Immediately proceed to the next step Streptavidin C1 Beads 3 Transfer 13 uL of Dynabeads MyOne Streptavidin C1 Beads to a new 1 5 mL Eppendorf LoBind Tube 4 Place the tube on a magnet such as a DynaMag 2 magnet for 2 minutes then
91. l assay on the Qubit 2 0 Fluorometer Qubit 2 0 lon PGM System Fluorometer y Possible cause Recommended action observation observation lt 10 Lower loading Too little library input into e Increase library input to target 20 Templated Lower enriched template preparation 25 templated ISPs ISPs or Lower key signal l Lower throughput e Continue with sequencing expect lower throughput gt 30 Increased number of Too much library input into Decrease library input to target 20 Templated filtered reads template preparation 25 templated ISPs ISPs r but lt 70 e Continue with sequencing expect lower throughput gt 70 Increased primer Adapter dimer e Check Bioanalyzer traces for Templated dimer filtered reads contaminating library more adapter dimer peak Amplicon library ISPs Lower throughput likely in short amplicon lon or lon AmpliSeq library peak around AmpliSeq or miRNA 70 bp miRNA library peak around libraries 60bp e Re purify Agencourt library using AMPure XP Kit clean up steps as outlined in the appropriate user guides Low loading lon OneTouch 2 Troubleshoot with Technical Support or a Low enriched Lower throughput High filtered reads Instrument underperformance Field Application Scientist Quality control using the Guava easyCyte 5 Flow Cytometer The Guava easyCyte 5 Flow Cytometer can be used for quality assessment of unenriched and e
92. less 8 well strips are supplied loose and out of the frame lon PGM Template OT2 200 Kit User Guide 75 OF D Appendix B Set up calibrate and maintain the lon OneTouch ES Set up the lon OneTouch ES Other Materials and Equipment e lon OneTouch ES e Elbow fitting for lon OneTouch ES e 0 2 mL PCR tubes e Pipettes e Vortexer e Optional Felt tipped pen Set up the 1 Locate the Tray Tip Arm and Tip Loader of the Ion OneTouch ES instrument 76 2 Install the tray a Wipe the instrument and the bottom of the Tray with a damp lab wipe to remove any packaging debris b Place the Tray on the Ion OneTouch ES with the calibration shelf on the left as shown in the following photograph c Push the Tray down firmly so that the Tray fits snugly in the clamps then confirm that the Tray is level IMPORTANT For proper operation of the lon OneTouch ES the tray must be firmly and uniformly seated in the cutout for the tray lon PGM Template OT2 200 Kit User Guide Appendix B Set up calibrate and maintain the lon OneTouch ES pa Set up the lon OneTouch ES 3 Install the supplied elbow fitting and place the Tip Arm in the cradle a Insert the male end of the elbow fitting into the female connector on the Tip Arm see figure a1 then finger tighten the lock ring on the elbow fitting see figure a2 al a2 b Connect the tubing to the elbow fitting on the top of the Tip A
93. lth and Human Services Biosafety in Microbiological and Biomedical Laboratories BMBL 5th Edition HHS Publication No CDC 21 1112 Revised December 2009 found at www cdc gov biosafety publications bmb15 BMBL pdf World Health Organization Laboratory Biosafety Manual 3rd Edition WHO CDS CSR LYO 2004 11 found at www who int csr resources publications biosafety Biosafety7 pdf lon PGM Template OT2 200 Kit User Guide 119 Documentation and support Obtaining SDSs Safety Data Sheets SDSs are available from www lifetechnologies com support Note For SDSs of chemicals from third party manufacturers contact the chemical manufacturer Obtaining Certificates of Analysis The Certificate of Analysis provides detailed quality control and product qualification information for each product Certificates of Analysis are available on our website Go to www lifetechnologies com support and search for the Certificate of Analysis by product lot number which is printed on the box Obtaining support For the latest services and support information for all locations go to www lifetechnologies com support At the website you can e Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities e Search for user documents SDSs vector maps and sequences application notes formulations handbooks certificates of analysis citations and other product support documents e Obtain information a
94. n Possible cause Recommended action E12 or E22 error displays when e Fuse installed incorrectly 1 Ensure that the fuse module is installed the unit is initializing e Unit below operating temp correctly and that the unit is at its e Bad program or calibration setting or e Tip Armis not moving recommended operating temperature 2 Reboot the instrument Power OFF the instrument wait 3 seconds then power ON the instrument 3 Ifthe error persists restore the factory defaults then re calibrate the instrument a Power OFF the instrument and wait 3 seconds b While holding down Vert Adjust power ON the instrument This step restores the factory default settings c Repeat 3a 3b as needed to restore the factory defaults d Calibrate the vertical and horizontal axes see Perform vertical calibration on page 85 pages and Perform horizontal axis calibration on page 87 Either of the following AC line voltage module e E12orE22errordisplays Stalled incorrectly e Tip Arm does not move or moves slightly 1 Determine the voltage of the electrical outlet to plug in the lon OneTouch ES 2 Align the arrow by the correct voltage on the AC line voltage module with the adjacent white arrow in the lower right corner of the fuse socket If the AC line voltage module is installed incorrectly 1 Gently remove the module with your fingernail or a small flathead screwdriver 2 Rotate the mo
95. n page 52 Provided in the lon PGM Template OT2 Solutions 200 Kit Part no 4481105 lon OneTouch Wash Solution MyOne Beads Wash Solution Tween Solution Neutralization Solution Nuclease free Water Provided in the lon PGM Template OT2 Supplies 200 Kit Part no 4480981 44 8 well strip Eppendorf LoRetention Dualfilter Tips P300 lon PGM Template OT2 200 Kit User Guide T Chapter 4 Enrich the template positive lon PGM Template OT2 200 lon Sphere Particles C Perform the residual volume test on the lon OneTouch ES Provided in the lon PGM Enrichment Beads Cat no 4478525 Dynabeads MyOne Streptavidin C1 Beads Other Materials and Equipment e 1 5 mL Eppendorf LoBind Tubes e 0 2 mL PCR tubes e 1MNaQOH e Pipettes e Vortexer e DynaMag 2 magnet e Microcentrifuge Perform the residual volume test on the lon OneTouch ES IMPORTANT Ensure that the AC line voltage module is installed correctly into the Ion OneTouch ES Instrument Refer to the Unpack and Install the Ion OneTouch 2 System Product Insert Pub no 4481875 TM TM Ensure that the Ion OneTouch FES is set up see Set up the Ion OneTouch ES on page 75 If the condition is Then First use of the instrument and during Perform a residual volume test see monthly maintenance Perform a residual volume test on page 80 Routine use and residual volume in Well 1 and Wel
96. nate the instrument 0020 c eee eee 111 APPENDIX D_ Instrument warranty cceeceeeeeeeeeeeees 112 APPENDIX E Satety vicaciccace tincture bateaaeedee pat sesexcepecadsae 114 Symbols on this instrument 0 00 cece cece cece eee eee teen eee ee eeeees 114 Safety information for instruments not manufactured by Thermo Fisher Scientific 115 Instrument Satety esceas is td aieadhn ad mikes hk Sede hein elie wee a E EE a ai 115 General a cscs cartels heart eb E ded anki Sees eiyaden aiemia hws aiadath Lybbdy Ad 115 Physical njury jc4 ceretskie bee ated ede ial rote e a A EE aaa aiae a aa ate 115 Flocea eoe te EAEE EE A A EE DAS ANEAN 116 Cleaning and decontamination 00 cc eee ee eee 116 Safety and electromagnetic compatibility EMC standards 00005 116 Safety araa tee massa Dau E Meese AS Seca ME Rew owls wad Remind eS E a A iets 117 EMG aaam eae a aoe wisn sada bes te lanes See Stele 117 Environmental design 02 0c cece eee eee eee eens 118 Che mical Safety 2 sc cccce ete axcateh acest sce Spee Sede dee See seach sane AE ea ete ePeie ate a ee 118 Biological hazard safety 2c cece ee eee eee 119 Documentation and support ce cece eee e eee eee eee eeeeeees 120 Obtaining SDSS serep n ace arhep ott Ta he sE ee pi dkat ai et EEEE E aS 120 Obtaining Certificates of Analysis 0 0 0 0 eee eee 120 Obtaining support 2 5 0 cca a
97. neTouch 2 Cleaning Adaptor 1 Obtain an Ion OneTouch 2 Cleaning Adapter 2 Firmly insert the 3 ports of the Cleaning Adapter into the three holes see Figure 2a on the top stage of the Ion OneTouch 2 Instrument see Figure 2b The tab protruding from the outer edge of the Cleaning Adapter fits into the front notch of the stage 2a 2b lon PGM Template OT2 200 Kit User Guide 107 Appendix C Supplemental procedures Initialize the lon OneTouch 2 Instrument Initialize the instrument 1 On the home screen touch Options i C sf 165 0 00 PSI 4 35 1 2 On the Options screen touch Initialize Initialization takes 10 minutes Continue to set up the lon OneTouch 2 Instrument 1 Remove and retain the Ion OneTouch 2 Cleaning Adapter You can reuse the Cleaning Adapter one time for the instrument maintenance protocol after the first run but you must appropriately dispose of the used Cleaning Adapter after maintenance see Maintain the lon OneTouch 2 Instrument on page 38 108 lon PGM Template OT2 200 Kit User Guide 2 GE Appendix C Supplemental procedures PS Download the log files from the lon OneTouch 2 Instrument Keep all of the disposable components that were used for initialization in place including the Ion OneTouch 2 Amplification Plate disposable line disposable injector lon OneTouch Router and Ion OneTouch Recovery Tubes IMPORTANT After the first run
98. nriched Ion PGM Template OT2 200 Ion Sphere Particles generated for up to 200 base read sequencing on the Ion PGM System For details refer to the Ion Sphere Particles ISPs Quality Assessment Using the Guava easyCyte 5 Flow Cytometer User Bulletin Pub no 4470082 available on the lon Community website ioncommunity lifetechnologies com lon PGM Template OT2 200 Kit User Guide 73 TM 1 Appendix A Quality control of lon PGM Template OT2 200 lon Sphere Particles Demonstrated protocol Quality control using Attune Acoustic Focusing Cytom eter Demonstrated protocol Quality control using Attune Acoustic Focusing Cytometer 74 The Applied Biosystems Attune Acoustic Focusing Cytometer can be used for quality assessment of unenriched and enriched Ion PGM Template OT2 200 Ion Sphere Particles generated for up to 200 base read sequencing on the Ion PGM Sequencer For details refer to the Demonstrated Protocol Ion Sphere Particles ISPs Quality Assessment using the Applied Biosystems Attune Acoustic Focusing Cytometer User Bulletin Pub no 4477181 available on the lon Community website ioncommunity lifetechnologies com IMPORTANT Life Technologies Demonstrated Protocols have been successfully demonstrated by Life Technologies research and development but not formally validated There are no technical specifications for Life Technologies demonstrated protocols Users assum
99. o come to room temperature 200 Reagent Mix before use 2 Vortex the solution for 30 seconds then centrifuge the solution for 2 seconds Note Visually inspect the solution at the bottom of the tube and verify that there is no residual precipitate If precipitate is visible refer to Troubleshooting on page 53 3 Keep the reagent mix at room temperature during use Store thawed reagent mix at 2 C to 8 C lon PGM Template OT2 1 Vortex the reagent for 1 minute then centrifuge the 200 PCR Reagent B solution for 2 seconds 2 Inspect the reagent e Ifthe solution is clear then prepare the amplification solution Keep Reagent B at room temperature e Ifthe solution is cloudyor has crystals or has been accidentally stored at 2 C to 8 C heat the reagent for 1 minute in a heat block set at 75 C Vortex the reagent for 1 minute then centrifuge the solution for 2 seconds 3 Inspect the reagent If the reagent is e Cloudy or has crystals repeat steps 1 2 until the reagent is clear then equilibrate the reagent to room temperature and prepare the amplification solution e Clear Equilibrate the reagent to room temperature then prepare the amplification solution Store Reagent B at room temperature IMPORTANT Do not use the reagent if it is cloudy or has crystals lon PGM Template OT2 1 Centrifuge the enzyme for 2 seconds 200 Enzyme Mix 2 Place on ice lon PGM Template OT2 Place the suspension at room temperatu
100. o export the log files 5 On the Export log screen touch Yes The log files export to the USB flash drive and the status update displays After export the Options screen displays 6 Remove the USB flash drive from the instrument then verify on a computer that the instrument downloaded the log files to the flash drive If the files are e Downloaded Proceed to the next step e Not downloaded Repeat steps 3 6 until the log files are downloaded 7 Save the log files to a computer 8 Turn OFF the instrument The instrument deletes the log files If necessary contact Technical Support lon OneTouch 2 Instrument operations If the on screen commentis Then the instrument is Priming and Filling Recovery Solution Priming the filter and filling the collection tubes with lon PGM OT2 Recovery Solution Sample Injection Injecting sample and starting emulsification Amplification Amplifying the sample by PCR Filling Recovery Solution Completely filling the lon OneTouch Recovery Tubes with lon PGM OT2 Recovery Solution ISP Collection Breaking the emulsion and collecting the ISPs Wash Cycles Washing the instrument and draining the washes 110 lon PGM Template OT2 200 Kit User Guide Appendix C Supplemental procedures Decontaminate the lon OneTouch 2 Instrument If the on screen commentis Then the instrument is Final Spin Perform
101. oltage of the surge protector and the selected voltage of the installed AC line voltage fuse TM module in the Ion OneTouch ES 9 Turn the power switch ON Optional Leave the instrument ON indefinitely lon PGM Template OT2 200 Kit User Guide 79 TM Appendix B Set up calibrate and maintain the lon OneTouch ES Perform a residual volume test Perform a residual volume test 1 Set up the Ion OneTouch ES see Set up the Ion OneTouch ES on page 75 2 Install a new tip in the Tip Arm a Place anew tip in the Tip Loader Remove the Tip Arm from the cradle and align the metal fitting of the Tip Arm with the tip Keeping the fitting on the Tip Arm vertical firmly press the Tip Arm down onto the new tip until the Tip Arm meets the Tip Loader Hold the Tip Arm to the Tip Loader for 1 second to ensure proper installation of the tip Lift the Tip Arm straight up to pull the installed tip from the Tip Loader tube 80 lon PGM Template OT2 200 Kit User Guide TM Appendix B Set up calibrate and maintain the lon OneTouch ES Perform a residual volume test b Return the Tip Arm to the cradle Tilt the Tip Arm back see figure b1 Align the pins with the round notches in the cradle see figure b2 then lower the Tip Arm into position see figure b3 Move the Tip Arm forward into the working position b1 b2 b3 gt in gt ag N Note Ensure that the back bottom end of the Tip
102. om the top plate port If you have questions about the plate contact Technical Support 4 Insert the Amplification Plate a Hold the disposable injector connected to the disposable tubing in one hand and the Amplification Plate in the other hand CAUTION PHYSICAL INJURY HAZARD The pointed end of the disposable injector can puncture your skin Keep your hand away from the point of the disposable injector b Insert the Amplification Plate into the heat block so that the single plate port aligns with the left hole of the lon OneTouch 2 Instrument 5 Pull the handle of the heat block to close the block The disposable tubing is under the handle 20 lon PGM Template OT2 200 Kit User Guide Chapter 3 Prepare template positive lon PGM Template OT2 200 lon Sphere Particles Set up the lon OneTouch 2 Instrument 6 Thread the disposable tubing through the Ion OneTouch DL Tubing Catch 7 Install the disposable tubing in the pinch valve a Align the disposable tubing with the slot that runs along the bottom of the pinch valve see figure 7a b Gently pull the disposable tubing upwards on the both sides of the pinch valve see figure 7b until the disposable tubing is in the slot and secured in the round notch on each side of the pinch valve see figure 7c 7a 7b 7c c If necessary adjust the disposable tubing along the notches of the open pinch valve so that there is sufficient length of
103. one Fluid SYRINGE ASSY SYRINGE VALVE amp VALVE COVER TUBING CONNECTION lon PGM Template OT2 200 Kit User Guide TM Appendix B Set up calibrate and maintain the lon OneTouch ES Maintain the lon OneTouch ES Lubricate the 1 Disassemble the syringe located on the back of the instrument syringe annually a Disconnect the tubing lon PGM Template OT2 200 Kit User Guide 89 90 Appendix B Set up calibrate and maintain the lon OneTouch ES Maintain the lon OneTouch ES b Remove the 2 screws lon PGM Template OT2 200 Kit User Guide Appendix B Set up calibrate and maintain the lon OneTouch ES oye Maintain the lon OneTouch ES CO c Remove the retainer lon PGM Template OT2 200 Kit User Guide 91 ro Appendix B Set up calibrate and maintain the lon OneTouch ES Maintain the lon OneTouch ES e Remove the plunger from the syringe body a f Apply a thin layer of Xiameter PMX 200 Silicone Fluid to the inside of the syringe body 2 Reassemble the syringe a Push the plunger all the way into the syringe body then pull back out approximately inch 92 lon PGM Template OT2 200 Kit User Guide A Appendix B Set up calibrate and maintain the lon OneTouch ES Decontaminate the lon OneTouch ES b Engage the plunger with its mate end see figure b1 then insert the valve into its docking position see figure b2
104. onnection located on the back of the Ion OneTouch 2 Instrument Power switch on off ion torrent be AOXO oy Life cane 4 Model len OneTouch Instrument IWS1005527 CE AANER ima This oyato is intanded tor reeperch une erty is eat to Be otaa fer theprantic cr Paraan Ape ak Seer a r a S arme ns C7 sortenser 20 2 Ecc Ql ase save 2 Ethernet connection ol alt Lau ht wy ive USB connection Power socket 7 On the Upgrade screen touch Yes If you touch No the Options screen displays 8 Wait 10 seconds for the screen to display the software status update e Ifthe status update displays proceed to step 9 e Ifthe Options screen displays then there are no firmware updates 98 lon PGM Template OT2 200 Kit User Guide Alternative method Install a firmware update using an Ethernet connection 10 11 12 Appendix C Supplemental procedures Install a firmware update Touch Options then touch Info to ensure that the new version of the firmware update is installed Turn the power switch OFF wait several seconds then remove the USB flash drive Turn the power switch ON Prepare for the next run see Set up the lon OneTouch 2 Instrument on page 17 Note Use a shielded Ethernet cable to connect to the Ion OneTouch 2 Instrument 1 2 Turn the power switch OFF Plug an Ethernet cable from the Ion OneTouch 2 Ins
105. orometer Appendix A Quality control of lon PGM 2 With the USB drive containing the Ion_PluginV310_AF qbt file inserted into the Qubit 2 0 Fluorometer power cycle the instrument by unplugging and plugging it back in Touch Yes to permanently upload the file to the instrument 19 April 2012 2 00PM v3 10 cted Do you wish to upload Yes No Home Standards Sample Data 3 Confirm the Ion Sphere QC program qbt file is functional by checking the following screens a In the main menu the Ion selection option is present Protein Ion Standards Sample Data b After touching Ion iM AF 488 GES and AF 647 GBM selection options appear on the screen Note Touching AF 488 or AF 647 enters the respective measurement channels C 19 April 2012 2 00 Pm v3 10 O 19 April 2012 2 00 PM v3 11 0 19 April 2012 2 00 PM v3 10 Choose Your Assay Bree AE pia AF 488 AF 647 Insert Assay Tube Insert Assay Tube Read Read Standards Standards Standards Sample Data lon PGM Template OT2 200 Kit User Guide 65 TM 1 Appendix A Quality control of lon PGM Template OT2 200 lon Sphere Particles Quality control using the Qubit 2 0 Fluorometer Calculate the Qubit 2 0 Fluorometer calibration factor 66 4 Proceed to Calculate the Qubit 2 0 Fluorometer calibration factor This section describes the procedure to determine the Qubit 2 0 Fluorometer
106. ot in the Tray with the square tab on the left Push the 8 well strip to the far left in the slot as possible Observe the position of the 8 well strip relative to the position of the tip When properly calibrated the 8 well strip is lt 1 mm of touching but not pushing on the tip To clearly see the relationship between the pipette tip calibration shelf and notch during calibration mark each of them with a felt tip pen Adjust the horizontal position of the Tip Arm so that the tip just touches the left tab of the 8 well strip when the 8 well strip is pushed to the far left of the slot in the Tray a Apply slight pressure to keep the 8 well strip to the far left b Press the plus button repeatedly until the tip touches the 8 well strip Each press of the plus key moves the Tip Arm to the right by 0 002 inches 50 um which may be difficult to detect Press Start Stop to save the setting and for P1 to display Power the instrument OFF wait gt 3 seconds then power the instrument ON to return to normal operating mode Perform a residual volume test see Perform a residual volume test on page 80 lon PGM Template OT2 200 Kit User Guide 87 TM Appendix B Set up calibrate and maintain the lon OneTouch ES Maintain the lon OneTouch ES Maintain the lon OneTouch ES Materials and equipment required lon OneTouch ES back panel layout 88 Xiameter PMX 200 Silic
107. plemental procedures Install a firmware update b Double click the Macintosh HDD icon c Navigate to the Applications folder then click Utilities Disk Utility d Double click the Disk Utility application e Highlight the hard drive the USB flash drive to format and click the Erase button f From the volume format select MS DOS FAT then enter a name for the USB flash drive g Click Erase h Confirm the FAT32 formatting Microsoft Windows operating system a Insert the USB flash drive into a USB port b Double click My Computer c Right click the USB flash drive icon then select Format d Click the drop down menu and select FAT32 then click Start e Confirm the FAT32 formatting 2 Copy the file containing the firmware update bz2 file to the top level directory of the USB flash drive 3 On the Ion OneTouch 2 Instrument turn the power switch ON 4 Ensure that the instrument is not performing a run and that the home screen displays ion torrent zg Open Lid lon PGM Template OT2 200 Kit User Guide 97 Appendix C Supplemental procedures Install a firmware update 5 On the home screen touch Options then touch Upgrade Initialize ard Upgrade ExportLog F Final Spin 4 1 64 0 04 PSI 4 415C Note The Upgrade screen asks if you would like to upgrade the firmware but immediately proceed to the next step 6 Insert the USB flash drive into the USB c
108. ppendorf LoBind Tubes e Pipettes e Vortexer e Microcentrifuge Recover the 1 At the end of the run follow the screen prompts to centrifuge the sample If you template positive removed the Reaction Tubes at the end of the run before the Ion OneTouch 2 lon PGM Instrument had spun the sample or have not processed the sample after 15 minutes centrifuge the sample on the instrument Template OT2 200 lon Sphere Particles a On the home screen of the instrument touch Open Lid wait until the lid clicks open then insert the two filled Ion OneTouch Recovery Tubes from the run in the centrifuge rotor Close the lid until it locks b Touch Options then touch Final Spin see figure below then follow the screen prompts touch Next on the next 2 screens until the centrifugation begins Centrifugation of the samples takes 10 minutes oe nee eal Initialize Upgrade ExportLog Final Spin 4 1 64 e 0 02 PSI 4 41 5 C c Immediately proceed to step 2 AS CAUTION ROTATION HAZARD Wait until rotation stops before opening Rotating parts can cause injury 36 lon PGM Template OT2 200 Kit User Guide Chapter 3 Prepare template positive lon PGM Template OT2 200 lon Sphere Particles Recover the template positive lon PGM Template OT2 200 lon Sphere Parti cles 2 Immediately after the centrifuge has stopped on the instrument display touch Open Lid wait until the li
109. processing one sample add 19 uL Annealing Buffer and 1 uL Ion Probes directly to the 0 2 mL PCR tube containing the ISPs and mix well by pipetting up and down b If processing more than one sample generate an Ion Probe Master Mix e 21 uL Annealing Buffer samples 1 uL Ion Probes samples total volume required e Add 20 uL of Ion Probe Master Mix to the 0 2 mL PCR tubes containing the ISPs then mix well by pipetting up and down 68 lon PGM Template OT2 200 Kit User Guide TM 1 Appendix A Quality control of lon PGM Template OT2 200 lon Sphere Particles Quality control using the Qubit 2 0 Fluorometer Load the tube s into a thermal cycler then perform the following protocol to anneal the Ion Probes Stage Temperature Time Hold 95 C 2 min Hold 37 C 2 min Remove unbound probes by washing the sample s three times with 200 uL of Quality Control Wash Buffer a Add 200 uL of Quality Control Wash Buffer to the 0 2 mL tube s b Vortex properly to mix and centrifuge at 15 500 x g for 1 5 minutes c Being careful not to disturb the pelleted ISPs remove the supernatant and leave behind 10 uL Note Compare to a 10 uL standard for reference d Repeat steps a through c two times for a total of three Quality Control Wash Buffer washes After the final wash add 190 uL of Quality Control Wash Buffer for a total volume of 200 uL mix by pipetting up and down five times and
110. ptional Guava easyCyte 5 Flow _ Transfer a 1 0 uL aliquot of the enriched ISPs to Cytometer a 1 5 mL Eppendorf LoBind Tube Refer to the lon Sphere Particles ISPs Quality Assessment Using the Guava easyCyte 5 Flow Cytometer User Bulletin Pub no 4470082 available on the lon Community website ioncommunity lifetechnologies com Demonstrated protocol Quality Transfer a 1 0 uL aliquot of the enriched ISPs to assessment by the Applied a 1 5 mL Eppendorf LoBind Tube Put the Biosystems Attune Acoustic sample on ice then refer to Demonstrated Focusing Cytometer Protocol lon Sphere Particles ISPs Quality Assessment using the Applied Bios ystems Attune Acoustic Focusing Cytometer User Bulletin Pub no 4477181 available on the lon Community website ioncommunity lifetechnologies com 11 Life Technologies Demonstrated Protocols have been successfully demonstrated by Life Technologies research and development but not formally validated 52 lon PGM Template OT2 200 Kit User Guide Troubleshooting lon OneTouch 2 Instrument Observation Possible cause Recommended action Firmware does not update or Firmware not updating on the 1 Power the instrument OFF then ON the status update screen instrument 2 Ensure that the USB flash drive is FAT32 does not display lt 10 seconds formatted and that the file is in the root directory 3 Remove then reinsert the USB flash drive
111. r Guide 59 TM 1 Appendix A Quality control of lon PGM Template OT2 200 lon Sphere Particles Quality control using the Qubit 2 0 Fluorometer Quality control using the Qubit 2 0 Fluorometer Materials required 60 IMPORTANT Effective 15 February 2012 the Alexa Fluor 488 and Alexa Fluor 647 Calibration Standards replace the spectrally similar FAM and Cy 5 dyes respectively previously used in the kit To update the existing plugin on the Qubit 2 0 Fluorometer with the new dye names install the new plugin file Ion_PluginV310_AF qbt Both sets of dye labeled calibration standards can be used in conjunction with firmware version 3 00 and previous versions of plugin files ion_plugin qbt or Ion_plugin_AF qbt We highly recommend that you install the new firmware version 3 10 and the new Ion plugin file on_PluginV310_AF for ease of use as these new versions allow you to operate the Ion plugin on the Qubit 2 0 Fluorometer without requiring the USB drive Log to be connected into the device at all times The selection options on the user interface of the instrument matches the new Alexa Fluor 488 and Alexa Fluor 647 Calibration Standards once the new plugin file is installed e Qubit 2 0 Fluorometer Cat no Q32866 with the V3 10 firmware and the Ion Sphere Quality Control assay e Qubit Assay Tubes Cat no Q32856 e PCR tubes 0 2 mL Axygen Part no PCR 02 L C or BioExpress Part no T
112. re Particles cccceceeeececeeceeeceuceeeeeess 44 Ana E E ant ad E E E wah ee dea E E E ES T TEE 44 Materials reg ired rigga poii cece eee Eoi eea a G a a aiaa a A 44 Perform the residual volume test on the lon OneTouch ES 0e0eeeeeeeeeee 45 Prepare reagents then fill the 8 well Strip 0 cece eee eee eee ees 46 Prepare Melt Off Solution 00 22 cece eee 46 Wash and resuspend the Dynabeads MyOne Streptavidin C1 Beads 46 Fill the 8 well Strip ogress eiei ee eee eee eee 47 Prepare the lon OneTouch ES ic usin dus tcc nebeehaatietewsigesstesnies2e st woun 49 Perform the run escrir eness innra eee CE Oe Cena Coca eed ee 50 Sequence or store the template positive ISPs 0 cee eee eee eee 51 Perform lon Sphere Particles quality control 0000 ce eee eee eee ees 52 ME GUDLESNOOUING 2f2 sides ideen Seanucdanantatenwadsseeteeeesster teen ddas 53 lon OneTouch 2 Instrument 2 ccc cece eee rreren erreneren rerne 53 lon OneTouch Goro g chee c rep tecaeseenr eevee ttrt rnrn rrr rrrrrrr renren ats 55 lon PGM Template OT2 200 Kit User Guide Contents APPENDIX A Quality control of lon PGM Template OT2 200 lon Sphere Poel HIGGS grant tee boa teenie enue a nen aeace 59 Quality Control assay s i eg sede ene ee nee aah OR eee Reged edge Ge eee eee 59 Quality control using the Qubit 2 0 Fluorometer ceeeeeeeee cesses eees 60
113. re 200 lon Sphere Particles IMPORTANT Use only lon PGM Template OT2 200 Ion Sphere Particles ISPs in the lon PGM Template OT2 200 Kit with the Ion OneTouch 2 System Do not use ISPs from other or previously used kits 2 Depending on your library type and concentration dilute the library as shown in the table below Use the library dilution within 48 hours of preparation 26 lon PGM Template OT2 200 Kit User Guide Chapter 3 Prepare template positive lon PGM Template OT2 200 lon Sphere Particles M HQ g es2s Prepare and install the amplification solution Note If you are troubleshooting the amplification process to 260 uL of Nuclease free Water add 1 uL of the E coli DH10B Control 200 Library from the Ion PGM Controls Kit v2 Cat no 4482010 lon AmpliSeq lon AmpliSeq gDNA lon Total DNA Library RNA Library Fragment or RNA Seq Amplicon Library Library Library 100 pM 100 pM 100 pM 100 pM concentration Volume of library 2 uL 4 uL 6 5 uL 5 uL Volume of 23 uL 21 uL 18 5 uL 20 uL Nuclease free Water Total volume of 25 uL 25 uL 25 uL 25 uL diluted library to add to the amplification solution a Vortex the diluted library for 5 seconds then centrifuge for 2 seconds b Place the diluted library on ice 3 Ina1 5 mL Eppendorf LoBind Tube at 15 C to 30 C add the following components in the designated order You add the ISPs in step 6 of this procedure
114. rement channels O ig aprizoiz 200m vai CO asap 2012 2 00 Pm vaio U Conie Choose Your Assay Aa EE AF 488 AF 647 Insert Assay Tube Insert Assay Tube M Back Read Standards Home Standards Standards Sample Data 7 Proceed to complete the instruction in Calculate the Qubit 2 0 Fluorometer calibration factor on page 66 If your Qubit 2 0 Fluorometer is equipped with V3 10 firmware you only need to upload the Ion Sphere Quality Control assay into your fluorometer S 19April2012 2 00PM v3 10 3 Firmware version IMPORTANT Do not re upgrade the firmware if the instrument is shipped with V3 10 version To upload the Ion Sphere Quality Control assay into your fluorometer use the following steps 1 Download the new Ion_PluginV310_AF qbt file from http ioncommunity lifetechnologies com community products pgm to your USB drive to activate the application to accept the new names Logging into the Ion Community is required Note We recommend that you use the USB drive provided with Qubit 2 0 Fluorometer for file transfer however other compatible USB drives may also be used Visit http ioncommunity lifetechnologies com community products pgim to see a list of approved USB drives Logging into the lon Community is required lon PGM Template OT2 200 Kit User Guide TM 1 Template OT2 200 lon Sphere Particles Quality control using the Qubit 2 0 Flu
115. rm lon PGM Template OT2 200 Kit User Guide 77 oA or Appendix B Set up calibrate and maintain the lon OneTouch ES Set up the lon OneTouch ES c Finger tighten the connectors at both ends of the elbow Do not twist or coil the tubing d Tilt the Tip Arm back Align the pins with the round notches in the cradle see figure d1 then lower the Tip Arm into position Move the Tip Arm forward into the working position see figure d2 d1 d2 Note Ensure that the back bottom end of the Tip Arm is not resting on top of the thumb screw causing the Tip Arm to tilt forward Do not adjust the angle of the arm unless you need to calibrate the instrument 78 lon PGM Template OT2 200 Kit User Guide Appendix B Set up calibrate and maintain the lon OneTouch ES Set up the lon OneTouch ES 4 Place the Tip Loader in the slot on the left side of the instrument deck 5 Ensure that the AC line voltage fuse module is installed in the proper orientation Refer to the Unpack and Install the Ion OneTouch 2 System Product Insert Pub no 4481875 6 Ensuring that the power switch is turned OFF plug the female end no pins of the power cord into the instrument 7 Plug the multi pin end of the instrument power cord into the surge protector or line conditioner 8 Plug the surge protector or line conditioner into an electrical outlet IMPORTANT The voltage of the electrical outlet must match the v
116. s Quality assessment by Then Qubit 2 0 Fluorometer Transfer a 2 0 uL aliquot of the unenriched to a 0 2 mL H q PCR Tube then see Quality control using the Qubit 2 0 Fluorometer on page 60 Optional Guava Transfer a 1 0 uL aliquot of the unenriched ISPs to a easyCyte 5 Flow 1 5 mL Eppendorf LoBind Tube Refer to the on Cytometer Sphere Particles ISPs Quality Assessment Using the Guava easyCyte 5 Flow Cytometer User Bulletin Pub no 4470082 available on the lon Community website http ioncommunity lifetechnologies com Demonstrated protocol Transfer a 1 0 uL aliquot of the unenriched ISPs to a Quality assessment by 1 5 mL microcentrifuge tube Put the sample on ice the Applied Biosystems then refer to Demonstrated Protocol lon Sphere Attune Acoustic Particles ISPs Quality Assessment using the Applied Focusing Cytometer Biosystems Attune Acoustic Focusing Cytometer User Bulletin Pub no 4477181 available on the lon Community website http ioncommunity lifetechnologies com 11 Life Technologies Demonstrated Protocols have been successfully demonstrated by Life Technologies research and development but not formally validated lon PGM Template OT2 200 Kit User Guide 47 Chapter 4 Enrich the template positive lon PGM Template OT2 200 lon Sphere Particles Prepare reagents then fill the 8 well strip 3 Fill the remaining wells in the
117. ssing steps Do not introduce amplified DNA into library preparation laboratory or work area Instrument clearances Ion OneTouch 2 Instrument and Ion OneTouch ES Space Requirements and Clearances Position the instrument so that the front is a minimum of 12 in 30 5 cm from the front of the laboratory bench Place the instrument at least 40 in 1 meter away from major sources of electronic noise such as refrigerators or microwaves For more information refer to the Ion Personal Genome Machine System Site Preparation Guide for use with Ion Personal Genome Machine Sequencer Ion Torrent Server and Ion OneTouch 2 System Pub no MAN0007516 lon PGM Template OT2 200 Kit User Guide 13 i Getting started Workflow Unpack and install the lon OneTouch 2 Instrument and lon OneTouch ES on page 14 h Check the firmware on page 15 h Initialize the lon OneTouch 2 Instrument on page 15 h Perform a verification run on page 15 Procedural guidelines e Use good laboratory practices to minimize cross contamination of products When designing the laboratory layout consider the need for space separation of pre and post PCR activities Separate the amplicon source and post PCR activities from pre PCR activities Dedicate laboratory supplies and or equipment to the appropriate space to significantly reduce the potential for contamination e Unless otherwise specified thaw reagents
118. t potential hazards or convey important safety information In this document the hazard symbol is used along with one of the following user attention words e CAUTION Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices e WARNING Indicates a potentially hazardous situation that if not avoided could result in death or serious injury e DANGER Indicates an imminently hazardous situation that if not avoided will result in death or serious injury Symbol English Francais A Caution risk of danger Attention risque de danger Consult the manual for further safety Consulter le manuel pour d autres information renseignements de s curit A Moving parts Parties mobiles A Caution hot surface Attention surface chaude D On Off On Off marche arr t lon PGM Template OT2 200 Kit User Guide Appendix E Safety Safety information for instruments not manufactured by Thermo Fisher Scientif IC Symbol English Francais Protective conductor terminal main ground Borne de conducteur de protection mise a la terre principale Do not dispose of this product in unsorted municipal waste CAUTION To minimize negative environmental impact from disposal of electronic waste do not dispose of electronic waste in unsorted municipal waste Follow local municipal waste or
119. therwise expressly stated in writing by Life Technologies ANY PRODUCT NOT COVERED BY AN EXPRESS WRITTEN WARRANTY IS SOLD AND PROVIDED AS IS WITHOUT WARRANTY OF ANY KIND STATUTORY EXPRESS OR IMPLIED Any description of Ion Torrent Product recited in Life Technologies quotation is for the sole purpose of identifying Ion Torrent Product and any such description is not part of any contract between Life Technologies and buyer and does not constitute a warranty that Ion Torrent Product shall conform to that description Any sample or model used in connection with Life Technologies quotation is for illustrative purposes only and is not part of any contract between Life Technologies and buyer and does not constitute a warranty that Ion Torrent Product will conform to the sample or model No affirmation of fact or promise made by Life Technologies whether or not in Life Technologies quotation shall constitute a warranty that Ion Torrent Product will conform to the affirmation or promise Unless otherwise specified in writing in documentation shipped with Ion Torrent Product or otherwise agreed by Life Technologies in writing Life Technologies does not provide service or support for custom products or other products made to buyer s specifications THE WARRANTIES IDENTIFIED IN THIS CLAUSE ARE LIFE TECHNOLOGIES SOLE AND EXCLUSIVE WARRANTIES WITH RESPECT TO Ion Torrent PRODUCT AND ARE IN LIEU OF ALL OTHER WARRANTIES STATUT
120. trument into one of the PGM ports on the Torrent Server Turn the power switch ON Initial start up takes 3 minutes Note You may hear sounds from the instrument This is normal Log in to the Torrent Browser with your ionadmin account username and password Click the gear icon at top right of the screen then select Configure from the dropdown menu Click Admin Interface at the bottom of the page Click Update OneTouch Device Click Update You see the progress bar during the update displayed on the Ion OneTouch 2 Instrument Wait for the update to complete and the instrument to reset Touch Options on the Ion OneTouch 2 Instrument home page to ensure that the new version of the firmware update is installed lon PGM Template OT2 200 Kit User Guide 99 OAT Appendix C Supplemental procedures Initialize the lon OneTouch 2 Instrument Initialize the lon OneTouch 2 Instrument Set up the lon OneTouch 2 Instrument 100 Before operating the Ion OneTouch 2 Instrument for the first time you must perform the one time initialization on the instrument Initialization primes the pumps and tubing lines for reliable operation Perform initialization at any time before the first run lon PGM OneTouch Plus Reaction Filter Assembly Instrument display Clamp handle to access the Amplification Plate in the heat block lon OneTouch DL Injector Hub Built in centrifuge to install the Recover
121. tubing and disposable injector are attached to the Amplification Plate An Amplification Plate supplied in the lon PGM Template OT2 200 Kit has ion on the tab Note Before use inspect the plate port of the Amplification Plate to ensure that it is straight and perpendicular to the plate If you have questions about the plate contact Technical Support 4 Insert the Amplification Plate a Hold the disposable injector connected to the disposable tubing in one hand and the Amplification Plate in the other hand aN CAUTION PHYSICAL INJURY HAZARD The pointed end of the disposable injector can puncture your skin Keep your hand away from the point of the disposable injector b Insert the Amplification Plate into the heat block so that the single plate port aligns with the left hole of the lon OneTouch 2 Instrument 5 Pull the handle of the heat block to close the block The disposable tubing is under the handle lon PGM Template OT2 200 Kit User Guide 103 rE Appendix C Supplemental procedures Initialize the lon OneTouch 2 Instrument 6 Place the lid over the heat block so that the notch of the lid is over the inlet and outlet holes of the heat block and the lid rests on the disposable tubing IMPORTANT Do not restrict the flow of liquid in the disposable tubing 7 Thread the disposable tubing through the Ion OneTouch DL Tubing Catch 8 Install the disposable tubing in the p
122. vert lon OneTouch Oil bottle 450 mL size 3 times to mix 3 Fill the Reagent Tube Aalf full with Oil Minimize bubbles Note It is not necessary to re mix the Oil 4 Insert the filled Reagent Tube into the left front port and screw the Reagent Tube firmly into place one quarter turn on the instrument lon PGM Template OT2 200 Kit User Guide 23 TM 5 Chapter 3 Prepare template positive lon PGM Template OT2 200 lon 4 Sphere Particles Set up the lon OneTouch 2 Instrument Install the lon IMPORTANT Use only the lon PGM OT2 Recovery Solution provided as part of the PGM OT2 Ion PGM Template OT2 200 Kit for the Ion OneTouch 2 Instrument Do not use a Recovery Solution recovery solution from a kit that is used with the Ion OneTouch DL configuration 1 Ensure that the Recovery Solution is clear If it is clear proceed to the next step If the Recovery Solution is not clear heat the bottle of Recovery Solution in a 30 C bath until the Recovery Solution is clear 2 Fill the appropriate lon OneTouch Reagent Tube with Ion OneTouch Recovery Solution on the right front port If you are Then Using a NEw 1 Use a new Reagent Tube from the kit Template Kit 2 Discard the used Reagent Tube and Sipper Tube Appropriately discard the Recovery Solution 3 Use fresh gloves to attach the Luer Lok end of a new lon OneTouch Sipper Tube to the right front port Do not let th
123. y Tubes and Recovery Router Waste Container Oil waste tray Pinch valve to hold lon OneTouch Oil A disposable tubing lon PGM OT2 Recovery Solution Figure 1 lon OneTouch 2 Instrument layout lon PGM Template OT2 200 Kit User Guide Appendix C Supplemental procedures Initialize the lon OneTouch 2 Instrument Materials required for this procedure Provided in the lon PGM Template OT2 Supplies 200 Kit Part no 4480981 e 2 lon OneTouch Reagent Tubes e lon OneTouch Recovery Router e 2 lon OneTouch Recovery Tubes e lon OneTouch 2 Amplification Plate e 21lon OneTouch Sipper Tubes Provided in the lon PGM Template OT2 Solutions 200 Kit Part no 4481105 e lon OneTouch Oil e lon PGM OT2 Recovery Solution Note This protocol has been validated using only the material specified Substitution may adversely affect performance and safety IMPORTANT Use only the lon PGM Template OT2 200 Kit Cat no 4480974 with this user guide and with the Ion OneTouch 2 Instrument Do not use the kit with the Ion OneTouch System Do not mix reactions or disposables including plates solutions and kit reagents from other template preparation kits Install the lon OneTouch Recovery Tubes and lon OneTouch Recovery Router 1 On the instrument display touch Open Lid wait until the lid clicks open then lift and hold the side of the centrifuge lid IMPORTANT

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