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Filter-Based Fluorescence

Fluorescence Intensity

Sodium Fluorescein in phosphate buffered saline (PBS)

DL ≤10 pM, top read
Excitation 485/20 nm, Emission 528/20 nm, 510 nm mirror

Methylumbelliferone (MUB) in carbonate-bicarbonate buffer (CBB)

DL ≤0.16 ng/mL, top read
Excitation 360/40 nm, Emission 460/40 nm, 400 nm mirror

Time-Resolved Fluorescence

Europium

DL ≤ 250 fM, top read
Excitation 360/40 nm, Emission 620/40 nm, 400 nm mirror
Integration Time 20 to 16,000 µs, Delay 0 to 16,000 µs, Granularity 1-µs steps

Fluorescence Polarization

Sodium Fluorescein

5 mP standard deviation at 1 nM sodium fluorescein
Excitation 485/20 nm, Emission 528/20 nm, 510 nm mirror
Excitation range 330–700 nm (UV-transparent polarizing filter)
Emission range 400–700 nm

Luminescence Specifications

≤ 75 amol/well DL ATP in a 96-well plate (low-noise PMT), 30 amol typical

≤ 500 amol/well DL ATP in a 96-well plate (red-shifted PMT)

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Summary of Contents for Synergy H1

Page 1: ...Hybrid Multi Mode Microplate Reader Synergy H1 TM Operator s Manual ...

Page 2: ......

Page 3: ...Synergy H1 Multi Mode Reader Operator s Manual BioTek Instruments Inc Part Number 8041000 Revision K May 2016 2016 ...

Page 4: ...Tek Instruments Inc Harta is a trademark of Harta Instruments Microsoft Windows Windows 7 Windows 8 and Excel are either registered trademarks or trademarks of Microsoft Corporation in the United States and or other countries All other trademarks are the property of their respective holders Restrictions and Liabilities Information in this document is subject to change and does not represent a comm...

Page 5: ... Hazards xvi Precautions xvii CE Mark xix Directive 2014 30 EU Electromagnetic Compatibility xix Emissions Class A xix Immunity xix Directive 2014 35 EU Low Voltage Safety xix Directive 2012 19 EU Waste Electrical and Electronic Equipment xx Directive 98 79 EC In Vitro Diagnostics if labeled for this use xx Electromagnetic Interference and Susceptibility xx USA FCC CLASS A xx Canadian Department o...

Page 6: ...0 Turn on the Reader 17 11 Establish Communication 17 12 Verify Set Dispenser Calibration Values 18 13 Run a System Test 19 14 Test the Injection System 20 Operational Performance Qualification 21 Repackaging and Shipping Instructions 22 Prepare the Dispense Module for Shipment 25 Getting Started 29 Modular Design 30 External Components 31 Internal Components 31 Filter Cube 32 Injection System 33 ...

Page 7: ...r Mirror 49 Clean the Filters and Mirrors 52 Filters and Mirrors Available from BioTek 52 Preventive Maintenance 53 Overview 54 Daily Cleaning for the Dispense Module 54 Recommended Maintenance Schedule 55 Warnings and Precautions 56 Clean Exposed Surfaces 57 Inspect Clean Excitation and Emission Filters 58 Inspect Clean Mirrors 59 Materials 59 Procedure 60 Flush Purge Fluid Path 61 Run a Dispense...

Page 8: ...e Syringe 72 Instrument Qualification Process 73 Instrument System Test 74 Plate Shaker Test 74 Absorbance Testing 75 BioTek Absorbance Test Plates 75 Test Methods 75 Sample Test Report 76 Troubleshooting 76 Peak Absorbance Test 76 Alignment Test 77 Accuracy Test 77 Repeatability Test 77 Absorbance Liquid Tests 77 Test Methods 77 Gen5 Protocol Parameters 78 Results Analysis 80 Absorbance Liquid Te...

Page 9: ...njection System Testing 102 Test Method 102 Gen5 Parameters 102 Results Analysis 105 Instrument Qualification Procedures 107 Overview 108 IQ OQ PQ Description 109 Recommended Qualification Schedule 110 System Test 111 Setup 111 Test Procedure 111 Absorbance Plate Tests 112 Requirements 112 Setup 112 Test Procedure 113 Absorbance Liquid Tests 114 Absorbance Liquid Test 1 114 Materials 114 Solution ...

Page 10: ...orescence Polarization FP Test 125 Time Resolved Fluorescence TRF Test 125 Test Procedure 126 Pipette Maps 127 Corners Sensitivity and Linearity FI Tests 127 Fluorescence Polarization FP Test 128 Time Resolved Fluorescence TRF Test 129 Alternate Supplemental Tests Using Methylumbelliferone MUB 130 Materials 130 Test Solutions 131 Test Procedure 132 Pipette Map 133 Injection System Tests 134 Materi...

Page 11: ...or Based Fluorescence 142 Performance 142 Filter Based Fluorescence 143 Fluorescence Intensity 143 Time Resolved Fluorescence 143 Fluorescence Polarization 143 Luminescence Specifications 143 Dispense Read Specifications 144 Error Codes 145 Overview 146 Error Codes 147 Instrument Dimensions for Robotic Interface 151 Sample Reports 155 Synergy H1 Operator s Manual Contents ix ...

Page 12: ...l assistance refer to the information below Customer Service and Sales Internet www biotek com Phone 888 451 5171 toll free in the U S or 802 655 4740 outside the U S Fax 802 655 7941 Email customercare biotek com Service Technical Assistance Center TAC Phone 800 242 4685 toll free in the U S or 802 655 4740 outside the U S Fax 802 654 0638 E Mail tac biotek com European Coordination Center Author...

Page 13: ...Dispense Module installation instructions Chapter 7 Instrument Qualification For the Absorbance Plate Test removed the restriction on the use of the peak closest to 243 nm for the Erbium glass any peak may be used In the Fluorescence Liquid Tests section for the Corners Sensitivity Linearity tests added information on Sodium Fluorescein Kit BioTek PN 7160013 Chapter 9 Specifications Clarified the ...

Page 14: ...tating that the workarounds for kinetic assays with continuous shake apply to Synergy H1 basecode software versions lower than 2 00 the issue was addressed in v2 00 Chapter 7 Instrument Qualification Corrected the PN for the pre configured qualification TRF filter cube 8040555 Under Harta Plate Test updated instructions for checking the test plate s battery changed the location of the buffer wells...

Page 15: ...ts designed to qualify the Synergy H1 Added Gen5 protocol parameters tables for Absorbance Testing Renamed the former Chapter 7 Instrument Qualification as Chapter 8 Instrument Qualification Procedures and moved the description content to the aforementioned new chapter For the Injection System Tests corrected the volume of water that is manually pipetted on top of the green test dye solution just ...

Page 16: ...nly to specific reader models are presented in this style For example Applies only to models equipped with injectors Intended Use Statement The Synergy H1 is a hybrid multi mode microplate reader The performance characteristics of the data reduction software have not been established with any laboratory diagnostic assay The user must evaluate this instrument and PC based software in conjunction wi...

Page 17: ...k to ensure that you receive important information and updates about the product s you have purchased Register online through the Customer Resource Center at www biotek com or call 888 451 5171 or 802 655 4740 Repackaging and Shipping If you need to ship the instrument to BioTek for service or repair contact BioTek for a Service Call Notice SCN number and be sure to use the original packing materi...

Page 18: ...ower Rating The instrument s power supply or power cord must be connected to a power receptacle that provides voltage and current within the specified rating for the system Use of an incompatible power receptacle may produce electrical shock and fire hazards Warning Electrical Grounding Never use a plug adapter to connect primary power to the external power supply Use of an adapter disconnects the...

Page 19: ...ower switch and unplug the power supply before cleaning the outer surface of the instrument Warning Potential Biohazards Some assays or specimens may pose a biohazard This hazard is noted by the symbol shown here Adequate safety precautions should be taken as outlined in the assay s package insert Always wear safety glasses and appropriate protective equipment such as chemically resistant rubber g...

Page 20: ...posal Dispose of the instrument according to Directive 2012 19 EU on waste electrical and electronic equipment WEEE or local ordinances Caution Warranty Failure to follow preventive maintenance procedures may void the warranty Caution Shipping Hardware All shipping hardware e g carrier shipping screw filter reader shipping bracket must be removed before operating the instrument and reinstalled bef...

Page 21: ...1 and EN 61326 2 6 for Immunity Verification of compliance was conducted to the limits and methods of the following EN 61000 4 2 Electrostatic Discharge EN 61000 4 3 Radiated EM Fields EN 61000 4 4 Electrical Fast Transient Burst EN 61000 4 5 Surge Immunity EN 61000 4 6 Conducted Disturbances from RFI EN 61000 4 11 Voltage Dips Short Interruptions and Variations Directive 2014 35 EU Low Voltage Sa...

Page 22: ...an radiate radio frequency energy and if not installed and used in accordance with the instruction manual may cause harmful interference to radio communications Operation of this equipment in a residential area is likely to cause harmful interference in which case the user will be required to correct the interference at their own expense In order to maintain compliance with FCC regulations shielde...

Page 23: ...irements for electrical equipment for measurement control and laboratory use Part 1 General requirements l Canadian Standards Association CAN CSA C22 2 No 61010 1 Safety requirements for electrical equipment for measurement control and laboratory use Part 1 General requirements l EN 61010 Standards see CE Mark starting on page xix Synergy H1 Operator s Manual User Safety xxi ...

Page 24: ...ground terminal Borne de terre Erde Betriebserde Borne de tierra Terra di funzionamento On Supply Marche alimentation Ein Verbindung mit dem Netz Conectado Chiuso Protective conductor terminal Borne de terre de protection Schutzleiteranschluss Borne de tierra de protección Terra di protezione Off Supply Arrêt alimentation Aus Trennung vom Netz Desconectado Aperto sconnessione dalla rete di aliment...

Page 25: ...edical device Dispositif médical de diagnostic in vitro Medizinisches In Vitro Diagnostikum Dispositivo médico de diagnóstico in vitro Dispositivo medico diagnostico in vitro Separate collection for electrical and electronic equipment Les équipements électriques et électroniques font l objet d une collecte sélective Getrennte Sammlung von Elektro und Elektronikgeräten Recogida selectiva de aparato...

Page 26: ...BioTek Instruments Inc xxiv Preface ...

Page 27: ...key features lists its package contents and provides contact information for technical assistance Product Description 2 Package Contents Accessories 3 Optional Accessories 4 Materials for Conducting Liquid Tests 5 Product Support Service 6 Technical Assistance Center TAC 6 Applications Support 6 ...

Page 28: ...Absorbance measurements are made using the reader s monochromator optics The xenon lamp allows for both UV and visible light measurements The monochromator provides wavelength selection from 230 999 nm in 1 nm increments Available read methods are endpoint area scan spectral scanning and pathlength correction For luminescence reads the Synergy H1 has a direct to PMT channel no filtering white ligh...

Page 29: ...ply bottles to syringe drives 250 µL syringes 2 Syringe thumbscrews 2 Priming plate Injector tip priming trough Dispense module communication cable Dispense module front cover Dispense module box Supply bottles 2 30 mL Supply bottle holders 2 Straps to secure bottles in the holders 6 Injector tip cleaning stylus and storage bag 8040541 7082121 7083000 19511 8042202 8042068 75107 8042197 8040534 71...

Page 30: ...ady upgrade kit contact BioTek Sales Synergy H1 Product Qualification and Maintenance IQ OQ PQ package 8040528 Additional filters and filter cubes contact BioTek Customer Care for availability and part numbers The Synergy H1 is compatible with the BioStack Microplate Stacker The BioStack rapidly and systematically transfers a stack of microplates to and from the instrument s microplate carrier Con...

Page 31: ...in and Methylumbelliferone and Time Resolved Fluorescence TRF tests using Europium BTI 7160010 contains 7160013 7160012 and 7160011 described below Kit for FI tests using Sodium Fluorescein BTI 7160013 Kit for FI tests using Methylumbelliferone BTI 7160012 Kit for TRF tests using Europium BTI 7160011 Kit for Fluorescence Polarization FP test BTI 7160014 or Invitrogen P3088 Individual Materials Sod...

Page 32: ...are version information Help About Gen5 l For troubleshooting assistance or instruments needing repair the specific steps that led to the problem and any error codes that were reported see also Error Codes starting on page 145 If you need to send an instrument to BioTek please contact the TAC for a Service Call Notice SCN number and the shipping address Package the instrument according to the inst...

Page 33: ...ct the Synergy H1 9 2 Select an Appropriate Location 9 3 Remove the Shipping Hardware 10 4 Install the Power Supply 11 5 Install the Gas Controller 11 6 Unpack and Inspect the Dispense Module 12 7 Install the Dispense Module 13 8 Connect the Host Computer 16 9 Install Gen5 Software 17 10 Turn on the Reader 17 11 Establish Communication 17 12 Verify Set Dispenser Calibration Values 18 13 Run a Syst...

Page 34: ...or a Synergy H1 model equipped with all of the available modules Your model may be different for example it may not have injection capability Perform the tasks in the order presented skipping those that do not apply to your reader s configuration Materials You will need a Phillips screwdriver supplied with the instrument to perform some of the steps in this chapter Remove the shipping hardware bef...

Page 35: ... for reuse if the instrument needs to be shipped again 2 Select an Appropriate Location Install the reader on a level stable surface in an area where ambient temperatures between 18 C 64 F and 40 C 104 F can be maintained Leave at least six inches of space between the instrument s rear panel and any other object This space ensures proper air flow in and out of the instrument The reader is sensitiv...

Page 36: ... remove the carrier shipping bracket 3 If the instrument is equipped with the filter module Open the top access door and use a screwdriver to remove the filter reader shipping bracket 4 Store the shipping hardware with the original packaging for reuse in case you need to ship the instrument Figure 2 1 Carrier shipping bracket left and filter reader shipping bracket 10 Chapter 2 Installation BioTek...

Page 37: ...e shock hazard Always connect the system power cord directly to an appropriate receptacle with a functional ground 1 Plug the power supply s cord into the power inlet on the rear of the reader 2 Connect the power cord to the power supply 3 Plug the power cord into an appropriate power receptacle 5 Install the Gas Controller Applies only to models compatible with the BioTek gas controller module If...

Page 38: ...and your BioTek representative Keep the shipping boxes and the packaging materials for the carrier s inspection BioTek will arrange for repair or replacement of your dispense module immediately Refer to Figure 2 9 and Figure 2 10 starting on page 26 1 Open the shipping box Remove the accessories box and the foam insert that contains the injector tubing and bottle holders 2 Lift out the module and ...

Page 39: ...e the clear shrouds from the tubes 3 Remove the two inlet tubes from their canisters 4 Identify the two syringe valves on the dispense module see Figure 2 4 on page 15 Each is labeled with a left pointing arrow i When installing the tubes do not use any tools Finger tighten only 5 Screw the fitting of one inlet tube into the right side of the Syringe 1 valve 6 Screw one end of one outlet tube into...

Page 40: ... shipped again 9 Thread the injector tip holder with outlet tubing connected to both ports through the hole in the top of the reader 10 Open the reader s top access door and holding the injector tip holder by the tab insert the injector tips into the appropriate holes inside the reader A magnet helps guide the tips into place and secures them in the reader Figure 2 3 Injector tip holder in its soc...

Page 41: ...into the barrel 13 Install the syringes referring to Figure 2 5 on page 16 l Hold the syringe vertically with the threaded end at the top l Screw the top of the syringe into the bottom of the syringe valve Finger tighten only l Carefully pull down the bottom of the syringe until it rests inside the hole in the bracket l Pass a thumbscrew up through this hole and thread it into the bottom of the sy...

Page 42: ...stylus packaged in a small cylinder Attach the cylinder to the back of the dispense module for storage 8 Connect the Host Computer The Synergy H1 is equipped with two communication ports USB and RS232 serial located on the back of the reader Connect one end of the supplied communication cable to the appropriate port on the reader and the other end to an appropriate port on the host computer 16 Cha...

Page 43: ...ble refer to the instructions that shipped with the USB drivers on the Gen5 software media to install the necessary drivers 1 Start Gen5 and log in if prompted 2 From the main screen select System Instrument Configuration 3 Click Add Reader and select Synergy H1 Click OK 4 Perform one of the following steps as applicable l Select Plug Play A reader must be connected to the computer and turned on t...

Page 44: ...200 80 40 with their actual measured values e g 199 3 79 7 39 9 Gen5 should display the measured calibration values 1 If you have not already done so turn on the instrument and establish communication with Gen5 2 In Gen5 go to System Instrument Configuration select the Synergy H1 and click View Modify 3 Click Setup and select the Dispenser 1 tab 4 Click Get Volumes 5 Compare the Calibration Volume...

Page 45: ...as a pending system test report view the report and then repeat steps 2 and 3 4 The results report appears and should contain the text SYSTEM TEST PASS l If required print the report and store it with your installation records Note Gen5 stores results in its database you can print a report at any time If an error code is returned refer to Error Codes starting on page 145 If the problem is somethin...

Page 46: ... the bottles 5 In Gen5 select System Instrument Control Synergy H1 and click the Prime tab 6 With Dispenser set to 1 set the Volume to 5000 µL and click Prime The syringe should move down and up repeatedly drawing fluid from the bottle and pumping it through the tubing and into the priming plate Examine the fittings no leaks should be detected If leaks are detected tighten all fittings and repeat ...

Page 47: ...r back from BioTek following service or repair or if regulatory requirements dictate that Operational Performance Qualification is necessary turn to Instrument Qualification Procedures starting on page 107 to learn about BioTek s recommended OQ PQ procedures for the Synergy H1 A Product Qualification Maintenance IQ OQ PQ package for the Synergy H1 is available for purchase BTI 8040528 Contact your...

Page 48: ...the reader Please contact BioTek and order part number 8040015 if you need a carrier shipping bracket and or filter reader shipping bracket When preparing to ship the Synergy H1 and or the dispense module to BioTek be sure to use the original packaging materials Other forms of commercially available packaging are not recommended and can void the warranty The shipping materials are designed to be u...

Page 49: ...hipping bracket See Figure 2 1 on page 10 7 Place the accessories in the accessories box and then seal the box with tape Figure 2 7 Repacking the Synergy H1 accessories box 8 Place the instrument in a plastic bag 9 Place the instrument in the shipping box with foam corners 10 Place the accessories box in the shipping box Seal the box with tape Synergy H1 Operator s Manual Repackaging and Shipping ...

Page 50: ...11 When finished write the SCN number on the outside of the box and ship the box to BioTek Figure 2 8 Repacking the instrument and accessories box 24 Chapter 2 Installation BioTek Instruments Inc ...

Page 51: ...ispense module from the reader Set the module aside for the moment 6 Remove the tip priming trough and store it in the dispenser accessories bag 7 Remove the two inlet tubes from the syringe valves and store them in their plastic canisters 8 Remove the two outlet tubes from the syringe valves Attach the clear plastic shrouds to the fittings of the outlet tubes Place the tubes in a plastic bag 9 Re...

Page 52: ...Figure 2 9 Packing the dispense module accessories 26 Chapter 2 Installation BioTek Instruments Inc ...

Page 53: ...Figure 2 10 Packing the dispense module Synergy H1 Operator s Manual Repackaging and Shipping Instructions 27 ...

Page 54: ...BioTek Instruments Inc 28 Chapter 2 Installation ...

Page 55: ...nts 31 Internal Components 31 Filter Cube 32 Injection System 33 Gen5 Software 35 Define the Filter Cube in Gen5 and on the Reader 35 Protocols and Experiments 36 Dispense Module Control 37 Plate Shaking Options 38 Recommendations for Optimum Performance 39 General 39 Luminescence Measurements 40 Monochromator Based Fluorescence Systems 40 Models with Injectors 40 Incubation and Partial Plates 40 ...

Page 56: ...dicates its capabilities Part Number Description H1F Filter based optics top only H1M Monochromator based optics top and bottom H1MF Monochromator and filter based optics H1FD Filter based optics and dispense module H1MD Monochromator based optics and dispense module H1MFD Monochromator and filter based optics and dispense module H1FG Filter based optics and gas controller module H1MG Monochromato...

Page 57: ...ilters mirrors and polarizing filters Preconfigured cubes are available from BioTek or you can change the filters and mirrors yourself See Filter Cube on page 32 Injection System The syringes may require replacement over time The tubing and injectors require cleaning at regular intervals Applies to models with injectors and an external dispense module See Injection System on page 31 Synergy H1 Ope...

Page 58: ...ors provide better sensitivity than 50 mirrors but they are dye specific Filters and mirrors are stored in the filter cube as described in Filter Cube Overview starting on page 44 If you run different types of fluorescence or luminescence assays you can replace the entire filter cube with a different one this is the BioTek recommended option Alternatively you can install different filters or mirro...

Page 59: ...ts on top of the reader and pumps fluid from the reagent bottles to injectors located inside the instrument Fluid is injected into one well at a time The injectors support plate types from 6 to 384 well plates Figure 3 2 Dispense module components 1 Two 250 µL syringes draw fluid from the supply bottles 2 Inlet tubes transport fluid from the supply bottles to the syringes These tubes are short pie...

Page 60: ...trolled using Gen5 see Dispense Module Control on page 37 If the injection system is not adequately primed air bubbles can get trapped in the system and affect injection volumes Air bubbles in the system can also result in fluid spraying or scattering inside the reader Both types of primes require a fluid reservoir to be present on the microplate carrier See the photo in Test the Injection System ...

Page 61: ...en5 and the reader s software exactly match the contents of the installed filter cube If you exchange or modify the filter cube you must update the Gen5 Filter Cube table and send the information to the reader 1 From the Gen5 main view select System Instrument Configuration Highlight the Synergy H1 click View Modify and then click Setup 2 If this is a new filter cube enter a unique name to identif...

Page 62: ...nstructions 1 In the Gen5 Task Manager select the Protocols icon and click Create New 2 Open the Procedure dialog double click Procedure in the menu tree 3 Select an appropriate Plate Type Gen5 stores measurements and other characteristics for individual plate types in a database It is essential that you select or define the plate type to match the assay plate Otherwise results may be invalid See ...

Page 63: ...step prime the system with the fluid to be used 1 Place the priming plate on the carrier 2 Fill the supply bottle with a sufficient volume of the fluid to be used for the prime and the assay Insert the appropriate inlet tube into the bottle 3 Select System Instrument Control Synergy H1 and click the Prime tab 4 Select the Dispenser number 1 or 2 associated with the supply bottle 5 Enter the Volume...

Page 64: ...tep to a protocol s procedure Figure 3 3 Gen5 Shake Step options Mode Speed Amplitude in 1 mm steps Frequency Linear 1 mm to 6 mm 18 Hz to 6 Hz Orbital Slow 1 mm to 6 mm 10 Hz to 3 Hz Orbital Fast 1 mm to 6 mm 14 Hz to 5 Hz Double Orbital Slow 1 mm to 6 mm 10 Hz to 3 Hz Double Orbital Fast 1 mm to 6 mm 14 Hz to 5 Hz Note Frequency is based on the Amplitude selected 38 Chapter 3 Getting Started Bio...

Page 65: ...e used For best results in most cases use at least 100 µL per well in a 96 well plate 25 µL in a 384 well plate and 5 µL in a 1536 well plate if supported Pipetting solution into 384 and greater well plates often traps air bubbles in the wells which may result in inaccurate readings A dual wavelength reading method usually eliminates these inaccuracies For best results however remove the air bubbl...

Page 66: ...en purging the lines at the beginning of each day ensures optimal performance of the dispense system See the Preventive Maintenance chapter for more information When dispensing volumes less than or equal to 20 µL well we recommend specifying a tip prime volume that is equal to the dispense volume For dispense volumes greater than 20 µL well we recommend a tip prime volume of 20 µL To avoid spillag...

Page 67: ...but shaking may stop prematurely with no error message One suggested workaround is to shorten the kinetic interval For example if your desired experiment is 25 kinetic reads with 60 minute intervals use 100 kinetic reads with 15 minute intervals Another suggestion is to perform multiple Shake steps and then a Read step with the Discontinuous Kinetic Procedure feature enabled For example if your de...

Page 68: ...BioTek Instruments Inc 42 Chapter 3 Getting Started ...

Page 69: ... more detailed information on working with these components Filter Cube Overview 44 Removing a Filter Cube 46 Installing a Filter Cube 46 Configuring the System for Luminescence Measurements 47 About the Gen5 Optics Library 47 Handling Filters and Mirrors 49 Change a Filter or Mirror 49 Clean the Filters and Mirrors 52 Filters and Mirrors Available from BioTek 52 ...

Page 70: ...n5 Optics Library to identify and manage the contents of multiple filter cubes see About the Gen5 Optics Library on page 47 The default filter cube configuration is shown below any changes are reflected in the sales order Verify that the filter cube contains the filters and mirrors that you ordered Contact BioTek or your supplier if you did not receive the expected components Position 1 Position 2...

Page 71: ...naccurate For Synergy H1 models with FP capability the cube is equipped with up to four polarizers of the following types l Excitation polarizer visible range or UV range l Emission polarizer parallel to the excitation polarizer l Emission polarizer perpendicular to the excitation polarizer Two types of excitation EX polarizers are available visible range 400 nm and above the default or UV range 3...

Page 72: ...cube and slide it to the right to remove it from its chamber Installing a Filter Cube 1 Ensure that all filters plugs and mirrors are inserted properly in the filter cube see Figure 4 2 and Figure 4 3 2 Open the front access door and locate the filter cube chamber 3 With the filter cube properly oriented the non labeled side entering the chamber gently slide the cube into the chamber You will feel...

Page 73: ...l and then update the reader s internal software to match the currently installed filter cube by selecting System Optics Library Set Reader You can also enable a Read Plate Prompt option to alert users at run time if they attempt to run an experiment that calls for filters mirrors not currently installed Brief instructions for adding a filter cube to the library are provided below refer to the Gen...

Page 74: ... 435 385 425 445 610 455 400 450 460 710 510 440 505 515 640 525 475 520 530 670 545 512 535 555 578 550 415 540 560 850 555 541 550 560 595 570 515 565 575 735 595 540 590 600 770 635 640 780 400 630 660 580 655 665 850 i If Fluorescence Polarization Cube is checked only Filter Set 1 is available for definition The filters and mirrors of Filter Set 2 must be identical to those of Filter Set 1 for...

Page 75: ...the information to the reader It is critical that the Gen5 Filter Cube table reflect the actual location and characteristics of the filters and mirrors in the installed filter cube See instructions on page 35 Gather the following tools l 7 64 hex key l Lens paper l Cotton swab l Linen or cloth gloves To remove a filter plug or mirror i Handle with care The mirrors are seated on a shelf in the bott...

Page 76: ...filters If you accidentally touch a mirror or polarizing filter with your bare fingers see Preventive Maintenance starting on page 53 for cleaning instructions Caution When removing or replacing a filter or C clip filter retainer do not use a sharp tool Use several layers of lens paper and your finger or a cotton swab to remove and replace filters and clips Using a sharp instrument such as a flat ...

Page 77: ...plug into the desired location b Make note of the filter position number EX1 EX2 or EM1 EM2 c Using your fingers squeeze the sides of the C clip retainer and then insert it into the top of the hole containing the new filter Cover your finger with several layers of lens paper and then push down on all sides of the retainer until it sits flush against the filter d Gently wipe both sides of the filte...

Page 78: ...ilable from BioTek Bandpass filters plugs retainer clips mirrors empty filter cubes and other supplies are available for purchase from BioTek Visit www biotek com and go to the Accessories page to see the most up to date list Contact BioTek Customer Care with any questions 52 Chapter 4 Filters and Mirrors BioTek Instruments Inc ...

Page 79: ...tinue to perform to specification Overview 54 Recommended Maintenance Schedule 55 Warnings and Precautions 56 Clean Exposed Surfaces 57 Inspect Clean Excitation and Emission Filters 58 Inspect Clean Mirrors 59 Flush Purge Fluid Path 61 Run a Dispense Protocol Optional 62 Empty Clean the Tip Priming Trough 63 Clean the Priming Plate 63 Clean the Dispense Tubes and Injectors 63 ...

Page 80: ...ssageways Take special care when using molecules that are active at very low concentrations e g enzymes inhibitors Remove any residual reagent in the dispense lines using a suitable cleaning solution review the reagent s package insert for specific recommendations Flushing the tubing at the end of each day letting the DI water soak overnight and then purging the lines at the beginning of each day ...

Page 81: ...frequently than shown here Task Daily Quarterly As Needed All models Clean exposed surfaces ü Inspect clean excitation and emission filters if equipped ü Inspect clean mirrors if equipped annually Decontaminate the instrument before shipment or storage Models with injectors and an external dispense module Flush purge the fluid path ü Optional Run a Dispense protocol ü Empty clean tip prime trough ...

Page 82: ...inated instruments Caution The buildup of deposits left by the evaporation of spilled fluids within the read chamber can impact measurements Be sure to keep System Test records before and after maintenance so that changes can be noted Warning The instrument with all available modules weighs up to 55 pounds 24 95 kg depending on the model Use two people when lifting and carrying the instrument Impo...

Page 83: ...plate carrier and all exposed surfaces of the instrument 4 Wipe all exposed surfaces of the dispense module if used 5 Wipe all exposed surfaces of the gas controller module if used 6 If detergent was used wipe all surfaces with a cloth moistened not soaked with water 7 Use a clean dry cloth to dry all wet surfaces Models with injectors If the tip priming trough overflows or other spills occur insi...

Page 84: ... for identifying the filters and their unique characteristics It also contains instructions for replacing filters if necessary 3 Inspect the glass filters for speckled surfaces or a halo effect This may indicate deterioration due to moisture exposure over a long period of time If you have any concerns about the quality of the filters contact your BioTek representative 4 Using cotton balls or lens ...

Page 85: ...for removing excessive debris from an optical surface If the contamination is not dislodged by the flow of gas please follow the cleaning instructions below l The purpose of the cleaning solvent is only to dissolve any adhesive contamination that is holding debris on the surface The towel needs to absorb both the excessive solvent and entrap the debris so that it can be removed from the surface Su...

Page 86: ... linen or cloth gloves grasp the mirror by its edges and lift it out of the cube 6 Wet an absorbent towel such as a Kimwipe not lens paper with anhydrous reagent grade ethanol Wear gloves and use enough toweling so that solvents do not dissolve oils from your hands that can seep through the toweling onto the coated surface 7 Drag the trailing edge of the ethanol soaked Kimwipe across the surface o...

Page 87: ...4 Click the Prime tab and select Dispenser 1 5 Set the Volume to 5000 µL Keep the default prime rate 6 Click Prime to start the process When the process is complete carefully remove the priming plate from the carrier and empty it 7 Repeat the process for Dispenser 2 Leave the water in the system overnight or until the instrument will be used again Purge the fluid from the system see below and then...

Page 88: ...just the Rate to support the dispensing volume 3 Add another Dispense step with the same parameters selecting Dispenser 2 4 Add a quick Read step with parameters relevant to your reader model this is necessary because Gen5 requires that a Read step follow the Dispense step 5 Save the protocol with an identifying name such as Dispense Observation 6 Fill the supply bottles with the DI H 2 O Tween so...

Page 89: ...ires dispensing Gen5 prompts the user to empty the tip prime trough Clean the Priming Plate Applies only to models equipped with injectors Clean the priming plate regularly to prevent bacteria growth and residue buildup Wash the plate in hot soapy water using a small brush to clean in the corners Rinse thoroughly and allow it to dry completely Clean the Dispense Tubes and Injectors Applies only to...

Page 90: ...om the syringe drive Caution Do not bend the injector tips A bent tip may not dispense accurately Clean the Dispense Tubes and Injectors As discussed on page 54 some reagents can crystallize and clog the tubing and injectors Daily flushing and purging can help to prevent this but more rigorous cleaning may be necessary if reagent has dried in the tubing or injectors To clean the dispense tubes soa...

Page 91: ...t procedures that need to be performed only occasionally Decontamination 66 Required Materials 66 Procedure for Models without the Dispense Module 67 Procedure for Models with the Dispense Module 68 Dispense Module Syringe Replacement 71 Syringe Maintenance Position 71 Replace the Syringe 72 ...

Page 92: ...ach laboratory must ensure that decontamination procedures are adequate for the biohazards they handle Wear prophylactic gloves when handling contaminated instruments Gloved hands should be considered contaminated at all times keep gloved hands away from eyes mouth and nose Eating and drinking while decontaminating instruments is not advised Mucous membranes are considered prime entry routes for i...

Page 93: ...us solution of 0 50 sodium hypochlorite bleach If the effects of bleach are a concern 70 isopropyl alcohol may be used Check the percent NaClO of the bleach you are using Commercial bleach is typically 10 0 NaClO prepare a 1 20 dilution Household bleach is typically 5 0 NaClO prepare a 1 10 dilution 3 Wet a cloth or paper towel with the bleach solution or alcohol and then thoroughly wring it out s...

Page 94: ...isopropyl alcohol may be used Check the percent NaClO of the bleach you are using Commercial bleach is typically 10 0 NaClO prepare a 1 20 dilution Household bleach is typically 5 0 NaClO prepare a 1 10 dilution 3 Open the plate carrier door and slide out the plate carrier 4 Wet a cloth or paper towel with the bleach solution or alcohol and then thoroughly wring it out so that liquid does not drip...

Page 95: ...the fluid lines 12 Empty the beaker containing the outlet tubes Put the tubes back in the empty beaker 13 If sodium hypochlorite bleach was used perform the next procedure Rinse the Fluid Lines Otherwise or after performing the Rinse procedure repeat steps 1 13 for SYRINGE 2 Dispenser 2 Rinse the Fluid Lines Perform this procedure only if decontamination was performed using sodium hypochlorite 1 P...

Page 96: ...cedure If you are unable to prime the system due to an equipment failure decontaminate the instrument and the dispense module as follows 1 Perform the procedures under Clean the Dispense Tubes and Injectors on page 63 2 Prepare an aqueous solution of 0 50 sodium hypochlorite bleach If the effects of bleach are a concern 70 isopropyl alcohol may be used Check the percent NaClO of the bleach you are...

Page 97: ...en the Dispense Accuracy and Precision tests fail If cleaning the injection system does not eliminate performance problems or if a syringe is leaking perform these instructions to replace a faulty syringe Contact BioTek TAC to order replacement syringes To change a syringe first use Gen5 to put the syringe in its maintenance position Syringe Maintenance Position Do not change the syringe position ...

Page 98: ... box 4 Hold the syringe vertically with the threaded end at the top Screw the top of the syringe into the bottom of the syringe valve Finger tighten only 5 Carefully pull down the bottom of the syringe until it rests inside the hole in the bracket 6 Pass the thumbscrew used to hold the old syringe up through this hole and thread it into the bottom of the syringe Hold the syringe from rotating whil...

Page 99: ... describes the materials and relevant Gen5 protocols used to execute the tests explains how to analyze test results and provides troubleshooting tips in the event of a failure Instrument Qualification Procedures starting on page 107 contains the actual step by step test procedures Instrument System Test 74 Plate Shaker Test 74 Absorbance Testing 75 Luminescence Testing 83 Fluorescence Testing 87 I...

Page 100: ...e LED on the power switch will flash If this occurs press the carrier eject button to stop the beeping If necessary initiate another system test using Gen5 to try to retrieve an error code from the reader Refer to Error Codes starting on page 145 for information on error codes and troubleshooting tips Refer to Sample Reports on page 155 to see a sample System Test Report for Synergy H1 Plate Shake...

Page 101: ...te Calibration Certificate containing a table with Absorbance OD Standards for each filter at each wavelength supported by the plate The certificate for test plate PN 7260522 also contains Wavelength Accuracy Standards tables with Expected Peak nm values with Test Ranges for the C6 glass filter Before the Absorbance Plate Test can be performed the OD Standard values and Expected Peak Test Range co...

Page 102: ...meets its repeatability specification by conducting repeated reads of each neutral density filter on the test plate and comparing the results Sample Test Report Refer to Sample Reports on page 155 to see a sample Absorbance Plate Test Report for Synergy H1 Troubleshooting If a test fails try the troubleshooting tips below If the test continues to fail contact BioTek TAC Important Do not remove fil...

Page 103: ...eadings and caused changes l Check the microplate carrier to ensure it is clear of debris Absorbance Liquid Tests BioTek Instruments Inc has developed a series of liquid test procedures for testing your reader s absorbance system Test Methods Absorbance Liquid Test 1 confirms repeatability and alignment of the reader when a solution is used in the microplate If these tests pass then the lens place...

Page 104: ...5 of that of the stock high level solution and that the second dilution low level solution will have an absorbance value near 50 of that of the stock solution Gen5 Protocol Parameters The information in this section represents the recommended reading parameters for the referenced Gen5 protocol s It is possible that your tests will require modifications to some of these parameters such as the Plate...

Page 105: ... plate Wavelengths 2 450 nm 630 nm Read Speed Normal Delay after plate movement 100 msec Data Reduction Define two Delta OD transformations 450 630 nm one per Read data set Synergy H1 Abs Test 3 prt Parameter Setting Plate Type 96 WELL PLATE Shake Step Linear 30 seconds default frequency Kinetic loop Set a Run Time Interval combination to read the plate five times with minimal delay Detection Meth...

Page 106: ...n 0 013 the well meets the test criteria 3 The plate is read five times in the Turnaround position at 405 nm Calculate the Mean OD of those five reads for each well in columns 11 and 12 4 Perform a mathematical comparison of the Mean values for each well in its Normal and Turnaround positions that is compare A1 to H12 A2 to H11 B1 to G12 H2 to A11 To pass the test the differences in the compared M...

Page 107: ... the Normal plate position data from Linearity Test and in the Turnaround position o Compare the mean reading for well A1 to its mean reading when in the H12 position Next compare the mean values for the H1 well to the same well in the A12 position The difference in the values for any two corresponding wells should be within the Accuracy specification for 96 well plates If the four corner wells ar...

Page 108: ...chieve high pipetting accuracy when conducting linear dilutions an R2 value of at least 0 9900 is considered adequate Troubleshooting If an absorbance liquid test fails try the following If a test continues to fail contact BioTek TAC l Check the microwells and plate carrier for debris that may have shifted and caused changes l Ensure the microplate is properly seated in the carrier l As applicable...

Page 109: ... 02884 attomole photon is applied to determine an ATP concentration and subsequent limit of detection for the instrument under test Gen5 Protocol Parameters The information in this section represents the recommended reading parameters for the referenced Gen5 protocol s Synergy H1 F LumTest_Harta prt Parameter Setting Plate Type If present 8030015 Harta w o 8032028 adapter otherwise Costar 96 black...

Page 110: ... After Plate Movement 350 msec Dynamic Range Standard Read Height 7 00 mm READ STEP 3 Detection Method Luminescence Read Type Endpoint Optics Type Filters Step Label Battery check Read wells A7 A8 Filter Set 1 filter cube Excitation Plug Emission Hole Gain 60 Integration Time 0 01 00 MM SS ss Delay After Plate Movement 350 msec Dynamic Range Extended Read Height 10 00 mm 84 Chapter 7 Instrument Qu...

Page 111: ...escribed on page 119 1 Determine if the plate s battery is functioning properly If A8 0 2 A7 the battery is good Otherwise it requires replacement A replacement battery is included with each new and recalibrated Harta Luminometer Reference Microplate 2 On the Harta plate s calibration certificate locate the NIST measurement for the A2 position Convert it to attomoles A2 NIST measurement 0 02884 3 ...

Page 112: ...ted PMT please contact BioTek TAC Troubleshooting If a test fails try the suggestions below If a test continues to fail contact BioTek s Technical Assistance Center TAC l Ensure that the reading is performed through a hole in the filter cube not through a glass filter l Verify that the filter cube definition in Gen5 matches the physical item l The optical probe s may need to be cleaned contact Bio...

Page 113: ...rmine pass fail The protocols and their spreadsheets were fully validated in accordance with BioTek Instruments Product Validation policies and procedures Gen5 version 2 06 or higher is required The package also contains a User Guide that describes the test methods helps you get started with using the plate and provides important information for cleaning and maintaining the test plate The guide al...

Page 114: ...olarization The optional FP Test measures high and low polarized samples to verify the instrument s ability to measure polarization of a liquid fluorophore and confirm that the excitation and emission polarizers are properly oriented in the instrument This test is conducted using only the top optics l Time Resolved Fluorescence The optional TRF Test measures a fluorescent compound Europium and buf...

Page 115: ...7 50000 Read Speed Normal Delay after plate movement 350 msec Measurements per data point 40 Lamp Energy Low faster Read Height 7 00 mm Read Step 2 Kinetic loop Run time 0 01 30 Interval 0 00 06 16 reads Detection Method Fluorescence intensity Read Type Endpoint Kinetic Optics Type Filters Step Label Sensitivity Read Buffer Read well C9 D9 E9 Filter Set 1 Green Excitation 485 20 nm Emission 528 20...

Page 116: ...ormal Delay after plate movement 350 msec Measurements per data point 40 Lamp Energy Low faster Read Height 7 00 mm Read Step 4 Detection Method Fluorescence intensity Read Type Endpoint Kinetic Optics Type Filters Step Label Linearity Read Read well C1 F5 Filter Set 1 Green Excitation 485 20 nm Emission 528 20 nm Optics Position Top 510 nm Gain Auto Scale to High Wells C1 50000 Read Speed Normal ...

Page 117: ... Auto Scale to High Wells D7 50000 Read Speed Normal Delay after plate movement 350 msec Measurements per data point 100 Lamp Energy Low faster Read Height 7 00 mm Top only Read Step 2 Kinetic loop Run time 0 01 30 Interval 0 00 06 16 reads Detection Method Fluorescence Read Type Endpoint Kinetic Optics Type Monochromators Step Label Sensitivity Read Buffer Read Well C9 D9 E9 Wavelengths 1 Excitat...

Page 118: ... 50000 Read Speed Normal Delay after plate movement 350 msec Measurements per data point 100 Lamp Energy Low faster Read Height 7 00 mm Top only Read Step 4 Detection Method Fluorescence intensity Read Type Endpoint Kinetic Optics Type Filters Step Label Linearity Read Read well C1 F5 Filter Set 1 Green Excitation 485 nm Emission 528 nm Optics Position Top or Bottom Gain Auto Scale to High Wells C...

Page 119: ... Scale to High Wells A8 10000 Read Speed Normal Delay after plate movement 350 msec Measurements per data point 60 Lamp Energy Low faster Read Height 7 00 mm Synergy H1 TRF prt Parameter Setting Plate Type Costar 96 white opaque 3912 Delay Step 3 minutes Shake Step Linear 30 seconds default frequency Read Step 1 Kinetic loop Run time 0 00 30 Interval 0 00 02 16 reads Detection Method Time resolved...

Page 120: ... 00 03 16 reads Detection Method Time resolved fluorescence Read Type Endpoint Kinetic Optics Type Filters Step Label Sensitivity Read Buffer Read well A6 B6 C6 Filter Set 1 TRF Excitation 360 40 nm Emission 620 40 nm Optics Position Top 400 nm Gain Auto Use first filter set sensitivity from FIRST Read Step Read Speed Normal Delay after plate movement 350 msec Measurements per data point 20 Lamp E...

Page 121: ...D7 80000 Read Speed Normal Delay after plate movement 350 msec Measurements per data point 40 Lamp Energy Low faster Read Height 7 00 mm Read Step 2 Kinetic loop Run time 0 01 30 Interval 0 00 06 16 reads Detection Method Fluorescence intensity Read Type Endpoint Kinetic Optics Type Filters Step Label Sensitivity Read Buffer Read well C9 D9 E9 Filter Set 1 Blue Excitation 360 40 nm Emission 460 40...

Page 122: ...0000 Read Speed Normal Delay after plate movement 350 msec Measurements per data point 40 Lamp Energy Low faster Read Height 7 00 mm Synergy H1 M_FI_T_MUB prt Parameter Setting Plate Type Costar 96 black opaque 3915 Read Step 1 Kinetic loop Run time 0 01 00 Interval 0 00 04 16 reads Detection Method Fluorescence intensity Read Type Endpoint Kinetic Optics Type Monochromators Step Label Sensitivity...

Page 123: ...l Sensitivity Read Buffer Read well C9 D9 E9 Filter Set 1 Excitation 360 nm Emission 460 nm Optics Position Top Gain Auto Use first filter set gain from FIRST Read Step Read Speed Normal Delay after plate movement 350 msec Measurements per data point 100 Lamp Energy Low faster Read Height 7 00 mm Read Step 3 Detection Method Fluorescence intensity Read Type Endpoint Kinetic Optics Type Filters Ste...

Page 124: ...g the three buffer wells find the Median Standard Deviation and corresponding Mean 3 Calculate the Mean for the 16 reads of the SF or MUB Concentration well D7 4 Calculate the Signal to Noise Ratio SNR using the Mean SF or MUB Concentration Buffer Median STD with its corresponding Buffer Mean SF or MUB Mean BufferMean 3 BufferSTD 5 Calculate the Detection Limit Sodium Fluorescein Using the known c...

Page 125: ...00 Mean of the 100 nM wells 50 Mean of the 50 nM wells 25 Mean of the 25 nM wells 12 5 Mean of the 12 5 nM wells 6 25 Mean of the 6 25 nM wells 3 Calculate the R2 value it must be 0 9500 to pass Fluorescence Polarization FP Test 1 Using the raw data from the Parallel read o Calculate the Mean Blank wells A6 H6 o Calculate the Signal for each HPR well Subtract the Mean Blank from its measurement va...

Page 126: ...Signal Parallel LPR Signal Mean G Factor Perpendicular LPR Signal 1000 8 Calculate the Standard Deviation of the PLPR in mP The Standard Deviation of the PLPR must be 5 mP to pass Time Resolved Fluorescence TRF Test 1 Calculate the Mean and Standard Deviation of the 16 reads for each of the buffer wells A6 B6 C6 2 Among the three buffer wells find the Median Standard Deviation and corresponding Me...

Page 127: ...act BioTek TAC for instructions l Review the pipetting instructions to verify the plate was correctly prepared l Does the Plate Type setting in the Gen5 protocol match the plate you used l For injector models spilled fluid inside the reader may be fluorescing which can corrupt your test results If you suspect this is a problem contact BioTek TAC for assistance l When testing Fluorescence Polarizat...

Page 128: ...ree place precision balance is used to weigh the plate The Precision Test is a measure of the variation among volumes dispensed to multiple wells and uses the green test dye solution For each volume dispensed 80 μL 20 μL and 5 μL to four columns the CV of 32 absorbance readings is calculated Pass Fail criteria depends on the per well volume dispensed 2 0 for 80 μL 7 0 for 20 μL and 10 0 for 5 μL C...

Page 129: ... test RECORD the weight TARE the balance PIPETTE 150 µL well of DI water into all 12 columns Place the plate back on the carrier Click OK to perform the Read steps Shake Step Linear 15 seconds default frequency Read Step Detection Method Absorbance Read Type Endpoint Optics Type Monochromator Step label 80 ul Read_Disp 1 or 2 Wells A1 H4 Wavelengths 2 405 nm 750 nm Speed Normal Read Step Detection...

Page 130: ... Dispense Step Dispenser 1 or 2 Wells A9 H12 Tip prime before this dispense step 5 µL Dispense 5 µL at 225 µL sec Plate Out In Comment Weigh the plate 5 uL test RECORD the weight TARE the balance PIPETTE 150 µL well of DI water into all 12 columns Set the plate aside and click OK Read Step Define a brief Read step for a single well The measurement value will not be used The step is only necessary ...

Page 131: ...lculated coefficient of variation CV and Accuracy Error For each volume dispensed 80 μL 20 μL 5 μL for each injector 1 2 1 Calculate the Standard Deviation of the 32 wells 2 Calculate the Mean of the 32 wells 3 Calculate the CV Standard Deviation Mean x 100 4 Calculate the Accuracy Error ActualWeight ExpectedWeight ExpectedWeight 100 Expected Weights for 32 wells 80 μL 2 560 g 20 μL 0 640 g 5 μL 0...

Page 132: ...BioTek Instruments Inc 106 Chapter 7 Instrument Qualification Process ...

Page 133: ...the various test methods describes the materials and relevant Gen5 protocols used to execute the tests explains how to analyze test results and provides troubleshooting tips in the event of a failure Overview 108 IQ OQ PQ Description 109 Recommended Qualification Schedule 110 System Test 111 Absorbance Plate Tests 112 Absorbance Liquid Tests 114 Luminescence Test 119 Fluorescence Plate Tests 120 F...

Page 134: ... page 110 to determine which qualification tests shall be conducted for your Synergy H1 model and to meet your site s regulatory requirements A Product Qualification Package BTI 8040528 for the Synergy H1 is available for purchase The package contains test procedures Gen5 protocols checklists and logbooks for performing Installation Qualification Operational Qualification Performance Qualification...

Page 135: ... major repair or upgrade to the hardware or software l Although out of tolerance failures will be detected by the OQ tests results should be compared with those from the routine Performance Qualification tests and previous OQ tests to monitor for trends l The successful completion of the OQ procedure in combination with results that are comparable to previous PQ and OQ tests confirms that the equi...

Page 136: ...dels with absorbance capability Absorbance Plate Test ü ü Absorbance Liquid Test 1 or Liquid Test 2 ü ü Optional Absorbance Liquid Test 3 or 340 nm Absorbance Plate Test using BTI 7260551 ü ü Models with fluorescence capability Corners Sensitivity Linearity FI Tests ü ü Fluorescence Polarization FP Test ü ü Time Resolved Fluorescence TRF Test ü ü Models with luminescence capability Luminescence Te...

Page 137: ...g execution a message box will appear in Gen5 Close the box the System Test Report will contain the error code that was generated by the failure 2 When the test is complete a dialog will appear requesting additional information Enter any required information and then click OK 3 The test report will appear it will show either SYSTEM TEST PASS or SYSTEM TEST FAIL ERROR error code DETECTED If the tes...

Page 138: ...ioTek 1 Obtain the current Test Plate Calibration Certificate 2 Start Gen5 and select System Diagnostics Test Plates Add Modify Plates 3 Click Add The Absorbance Test Plate dialog appears 4 Select the appropriate Plate Type and then enter the plate s serial number 5 Enter the Last Certification and Next Certification dates from the calibration label on the Test Plate 6 If the wavelength values in ...

Page 139: ...e displayed in parenthesis is 580 to 590 8 Review all of the values that you entered When finished click OK to save the information Test Procedure 1 In Gen5 select System Diagnostics Test Plates Run If prompted select the desired Test Plate and click OK 2 When the Absorbance Test Plate Options dialog appears enter any required information 3 If applicable check the Perform Peak Wavelength Test box ...

Page 140: ...re comparable then the results from these tests will be your baseline for future tests Document your new test procedure and save all test results Materials Manufacturer part numbers are subject to change l New 96 well clear flat bottom microplate Corning Costar 3590 recommended l Stock Solution A or B which may be formulated by diluting a dye solution available from BioTek A or from the materials ...

Page 141: ...edure i Be sure to use a new microplate Debris fingerprints or scratches may cause variations in readings 1 Using freshly prepared stock solution Solution A or B prepare a 1 2 dilution using deionized water one part stock one part deionized water the resulting solution is a 1 2 dilution 2 Pipette 200 μL well of the stock solution into column 1 3 Pipette 200 μL well of the diluted solution into col...

Page 142: ...n the tenth tube Dilute using the 0 05 solution of deionized water and Tween 20 This solution can also be made by diluting the BioTek wetting agent 200 1 Tube Number 1 2 3 4 5 6 7 8 9 10 Volume of original concentrated solution mL 20 18 16 14 12 10 8 6 4 2 Volume of 0 05 Tween solution mL 0 2 4 6 8 10 12 14 16 18 Absorbance expected if original solution is 2 000 OD at 200 µL 2 0 1 8 1 6 1 4 1 2 1 ...

Page 143: ... pipette s l Beakers and graduated cylinder l Precision balance with readability to 0 010 g l Buffer solution described below l Gen5 protocol Synergy H1 Abs Test 3 prt described on page 79 Buffer Solution l Deionized water l Phosphate Buffered Saline PBS pH 7 2 7 6 Sigma tablets P4417 or equivalent l β NADH Powder β Nicotinamide Adenine Dinucleotide Reduced Form Sigma bulk catalog number N 8129 or...

Page 144: ...nto all wells of columns 1 and 2 l 150 μL of the 75 Test Solution into all wells of columns 3 and 4 l 150 μL of the 50 Test Solution into all wells of column 5 and 6 4 Create a Gen5 experiment based on the Synergy H1 Abs Test 3 protocol and read the plate l Save the experiment Refer to the instructions on page 81 to perform calculations and determine pass fail l Troubleshooting tips are provided o...

Page 145: ... switch on the back of the plate 2 Check the battery by pressing the test button on the back of the plate and ensuring that the test light turns on The test light may be difficult to see in bright light change your angle of view or move to a darker environment if necessary If the light does not turn on replace the battery 3 Place the adapter on the reader s microplate carrier and then place the Ha...

Page 146: ...he Fluorescence Test Plate User Guide contains a procedure for cleaning the plate and then creating and running experiments based on supplied Gen5 protocols As described in the User Guide when each experiment is finished Gen5 exports the measurement data to a prepared Microsoft Excel xls file The worksheet s within that file calculate results and determine pass or fail i For use with the Synergy H...

Page 147: ...using your particular plates solutions and so on If results are comparable then the results from these tests will be your baseline for future tests Document your new test procedure and save all test results Materials Kits containing the microplates and solutions required by the Liquid Tests are available for purchase see Materials for Conducting Liquid Tests on page 5 i Microplates should be clean...

Page 148: ...ergy H1_M_FI_T_SF prt Corners Sensitivity Linearity tests Top optics Sodium Fluorescein SF Synergy H1_M_FI_T_MUB prt Sensitivity and Linearity tests Top optics Methylumbelliferone alternate supplemental test for Top optics l Filter set definitions as applicable Green SF Blue MUB Filter Set Name Green Excitation Band Pass 485 20 Mirror Dichroic Top 510 nm 440 505 515 640 Emission Band Pass 528 20 F...

Page 149: ...lma Quartz 96 well titration plate Mfr 730 009 QG or equivalent l If testing only the Top optics o A new clean 96 well solid black microplate such as Corning Costar 3915 or equivalent l Excitation filter 485 20 nm installed l Emission filter 528 20 nm installed l 510 nm dichroic mirror installed Fluorescence Polarization FP Test The FP Test may be conducted using the same plate as for the Top Corn...

Page 150: ...ity FI Tests If using BioTek s sodium fluorescein powder BTI 98155 be sure to hold the vial upright and open it carefully the material may be concentrated at the top If a centrifuge is available spin down the tube before opening When diluting the sodium fluorescein powder in buffer it takes time for the powder to completely dissolve Allow the solution to dissolve for five minutes with intermittent...

Page 151: ...al preparation Time Resolved Fluorescence TRF Test As described in the Materials section the recommended test solutions are available from Invitrogen Corporation or BioTek l Shake the FluoSpheres container vigorously for 30 seconds prior to pipetting Alternatively sonicate or vortex the container l Mix 10 μL of FluoSpheres with 10 mL of deionized water in a 15 mL conical bottom polypropylene sampl...

Page 152: ...d on Synergy H1_FP prt and read the plate 4 If your reader is equipped with the monochromator based fluorescence system perform the Sensitivity Linearity tests for that system l Create an experiment based on Synergy H1_M_FI_B_SF prt bottom optics and read the plate l Create an experiment based on Synergy H1_M_FI_T_SF prt top optics and read the plate 5 If your reader is equipped with Time Resolved...

Page 153: ...the 1 nM SF solution into well D7 l Pipette 200 µL of the buffer solution into wells C9 D9 and E9 Using a multi channel pipette with just four tips installed l Pipette 150 μL of the buffer into wells C2 F5 Discard the tips l Pipette 150 μL of the 1 nM SF solution into column 1 l Pipette 150 μL of the 1 nm SF solution into column 2 Mix the wells using the pipette Do not discard the tips l Aspirate ...

Page 154: ...nM 3 3 nM BUF BUF 3 3 nM 3 3 nM 3 3 nM Fluorescence Polarization FP Test The plate used for testing Corners Sensitivity Linearity of the Top optics can also be used for this test l Pipette 200 μL of the green polarization buffer BUF into wells A6 H6 l Pipette 200 μL of the green high polarization reference HPR into wells A7 B7 l Pipette 200 μL of the green low polarization reference LPR into wells...

Page 155: ...y done so shake the vial of 20 pM europium suspension vigorously for 30 seconds prior to pipetting Alternatively sonicate or vortex the vial l Pipette 200 μL of the 20 pM europium suspension Eu into well A8 1 2 3 4 5 6 7 8 9 10 11 12 A DI Eu B DI C DI D E F G H Synergy H1 Operator s Manual Fluorescence Liquid Tests 129 ...

Page 156: ...les Sigma 3041 l 100 methanol BTI 98161 l A new clean 96 well solid black plate such as Corning Costar 3915 or equivalent A Greiner SensoPlate Mfr 655892 may also be used l Excitation filter 360 40 installed l Emission filter 460 40 nm installed l 400 nm dichroic mirror installed l Deionized or distilled water l Various beakers graduated cylinders and pipettes l 95 ethanol for cleaning clear botto...

Page 157: ...ater l Stir the solution preferably using a stir table until the CBB is completely dissolved 2 Prepare the MUB stock solution l Add 1 mL of 100 methanol to the 10 mg vial of MUB l Make sure all of the dye has completely dissolved and is well mixed This yields a 10 mg mL stock solution l Wrap the solution in aluminum foil to prevent exposure to light 3 Prepare the dilutions Label each with MUB and ...

Page 158: ... an experiment based on Synergy H1_FI_T_MUB prt and read the plate 3 If your reader is equipped with the monochromator based fluorescence system perform the Sensitivity Linearity tests for that system l Create an experiment based on Synergy H1_M_FI_T_MUB prt and read the plate 4 Save the experiments Refer to the instructions starting on page 98 to perform calculations and determine pass fail l Tro...

Page 159: ... the tips l Aspirate 150 μL from column 2 and dispense into column 3 Mix the wells using the pipette Do not discard the tips l Aspirate 150 μL from column 3 and dispense into column 4 Mix the wells using the pipette Do not discard the tips l Aspirate 150 μL from column 4 and dispense into column 5 Mix the wells using the pipette Do not discard the tips l Aspirate 150 μL from column 5 Discard the s...

Page 160: ...alance with capacity of 100 g minimum and readability of 0 001 g 50 200 μL hand pipette and disposable tips Deionized water Supply bottles 250 mL beaker New 96 well clear flat bottom microplates Green Test Dye Solution BTI 7773003 undiluted or one of the alternate test solutions provided on the next page 100 mL graduated cylinder and 10 mL pipettes if not using BioTek s Green Test Dye Solution Gen...

Page 161: ...e N 3 Na 0 100 gram Deionized water make to 1 liter Test Procedure for Models with Absorbance Capability 1 Prime both dispensers with 4000 μL of deionized or distilled water 2 Remove the inlet tubes from the supply bottles Prime both dispensers with the Volume set to 2000 μL This prevents the water from diluting the dye 3 Fill a beaker with at least 20 mL of the green dye solution Prime both dispe...

Page 162: ...rrier l 5 μL well is dispensed to columns 9 12 l When prompted remove the plate and weigh it Record the weight l Manually pipette 150 μL of deionized or distilled water into all 12 columns on top of the green test dye solution l Place the plate on the carrier for the shake and read steps 9 When the experiment is complete save the file with an identifying name 10 Remove the plate from the carrier a...

Page 163: ...ance 6 Place the plate on the microplate carrier Running a dispense protocol with no plate on the carrier will contaminate the reading chamber with spilled fluid When each dispense step is finished you will weigh the plate record the weight tare the balance with the plate on it and then place the plate back on the carrier for the next step 7 Initiate a plate read Gen5 will prompt you to empty the ...

Page 164: ...xperiment based on the Other Reader protocol and read the plate 12 When the experiment is complete save the file with an identifying name 13 Remove the plate from the carrier and set it aside 14 Repeat the procedure using the Synergy H1 Disp 2 Test No Read protocol and a new microplate 15 When the tests are complete l Prime both dispensers with at least 5000 µL of deionized water to flush out the ...

Page 165: ...t g 20 µL weight g 5 µL weight g Expected weight 2 5600 g Expected weight 0 6400 g Expected weight 0 1600 g Accuracy Error Accuracy Error Accuracy Error Must be 2 0 P F Must be 5 0 P F Must be 20 0 P F Standard Deviation Standard Deviation Standard Deviation Mean Mean Mean CV CV CV Must be 2 0 P F Must be 7 0 P F Must be 10 0 P F Reader Model Tested By Reviewed Approved By Reader S N Reading Date ...

Page 166: ...t g 20 µL weight g 5 µL weight g Expected weight 2 5600 g Expected weight 0 6400 g Expected weight 0 1600 g Accuracy Error Accuracy Error Accuracy Error Must be 2 0 P F Must be 5 0 P F Must be 20 0 P F Standard Deviation Standard Deviation Standard Deviation Mean Mean Mean CV CV CV Must be 2 0 P F Must be 7 0 P F Must be 10 0 P F Reader Model Tested By Reviewed Approved By Reader S N Reading Date ...

Page 167: ...his appendix contains BioTek s published specifications for the Synergy H1 General Specifications 140 Absorbance Specifications 141 Fluorescence Specifications 142 Luminescence Specifications 143 Dispense Read Specifications 144 ...

Page 168: ...lter based Fluorescence FI FP Dimensions Approximately 18 25 D x 14 75 W x 13 H 46 4 cm D x 37 5 cm W x 33 cm H Note For dimensions that include installation with a BioStack refer to the BioStack Operator s Manual Weight For a model equipped with all available modules excluding the power supply and dispense module 55 lbs 24 95 kg Environment Operational temperature 64 to 104 F 18 to 40 C Humidity ...

Page 169: ...er plate movement 100 ms 2 2 5 OD 3 0 010 OD delay after plate movement 100 ms 384 well plate normal read speed 0 2 OD 2 0 010 OD delay after plate movement 100 ms 2 2 5 OD 5 0 010 OD delay after plate movement 100 ms 96 well and 384 well plate sweep read speed 0 1 OD 1 0 010 OD Linearity By liquid dilution 96 well plate normal read speed 0 2 OD 1 0 010 OD delay after plate movement 100 ms 2 2 5 O...

Page 170: ...p with filters The following sets of requirements apply to 96 well plates Monochromator Based Fluorescence Excitation Range 250 700 nm with low noise PMT 250 900 nm with red shifted PMT Emission Range 250 700 nm with low noise PMT 300 700 nm for emission scans up to 900 nm with red shifted PMT Bandpass 18 nm Excitation and Emission Performance Sodium Fluorescein in phosphate buffered saline PBS DL...

Page 171: ...ion 360 40 nm Emission 620 40 nm 400 nm mirror Integration Time 20 to 16 000 µs Delay 0 to 16 000 µs Granularity 1 µs steps Fluorescence Polarization Sodium Fluorescein 5 mP standard deviation at 1 nM sodium fluorescein Excitation 485 20 nm Emission 528 20 nm 510 nm mirror Excitation range 330 700 nm UV transparent polarizing filter Emission range 400 700 nm Luminescence Specifications 75 amol wel...

Page 172: ... 384 well microplates Detection Method Absorbance Fluorescence FI FP TRF Luminescence Volume Range 5 1000 µL with a 5 20 µL tip prime Accuracy 1 µL or 2 0 whichever is greater Precision 2 0 for volumes of 50 200 µL 4 0 for volumes of 25 49 µL 7 0 for volumes of 10 24 µL 10 0 for volumes of 5 9 µL 144 Chapter A Specifications BioTek Instruments Inc ...

Page 173: ...Appendix B Error Codes This appendix lists and describes Synergy H1 error codes that may appear in Gen5 Overview 146 Error Codes 147 ...

Page 174: ...g A100 indicate conditions that require immediate attention If this type of code appears turn the instrument off and on If the System Test does not conclude successfully record the error code and contact BioTek s Technical Assistance Center If an error code appears in Gen5 you may want to run a System Test for diagnostic purposes In Gen5 select System Diagnostics Run System Test Contact Info BioTe...

Page 175: ...rly threaded Refer to Install the Dispense Module starting on page 13 for instructions Restart the reader 2B0A Priming plate not detected Place the priming plate on the carrier 2B04 Dispenser syringe 1 or 2 respectively failed position verify Generally this error indicates the syringe was not properly installed Make sure the syringe s thumbscrews are properly threaded Refer to Install the Dispense...

Page 176: ...e row 1 columns in plate Ex 87 8 1 12 column 3 NOTE If this code is returned during an area scan it indicates the scan point corresponding to the row column equivalent in the currently defined scan map NOT the actual well where the error occurred 4Exy Detector saturated too much light Relative Fluorescing Units RFU reached 99999 x 0 1 y 0 6 This error can indicate one of several scenarios It is po...

Page 177: ...me Generally this error indicates the filter cube is not seated properly in the reader Remove it ensure each filter or plug is properly positioned and reinstall it securely Restart the reader 5403 Filter cube failed positional verify Generally this error indicates the filter cube is not seated properly in the reader Remove it ensure each filter or plug is properly positioned and reinstall it secur...

Page 178: ... microplate is properly and securely seated in the carrier and nothing is obstructing carrier movement inside the reading chamber Verify that the Plate Type defined in the Gen5 Protocol matches the plate you are using This error can also occur if the plate type is correct but the lid was left on the plate If you wish to read the plate with a lid on it create a new plate type in Gen5 and add the he...

Page 179: ...ws the location of the microplate carrier in reference to the exterior surfaces of the Synergy H1 and the mounting holes on the bottom Use the illustrations to facilitate system setup with a robotic instrument such as the BioStack Microplate Stacker Dimensions are in inches ...

Page 180: ...Figure C 1 Top view 152 Appendix C Instrument Dimensions for Robotic Interface BioTek Instruments Inc ...

Page 181: ...Figure C 2 Bottom view The arrows point to special mounting holes for alignment caps for operation with the BioStack note that the model shown is not gas ready Synergy H1 Operator s Manual 153 ...

Page 182: ...ardware is included in the BioStack s alignment kit for correct positioning with the Synergy H1 Refer to the Installation chapter in the BioStack Operator s Manual for instructions 154 Appendix C Instrument Dimensions for Robotic Interface BioTek Instruments Inc ...

Page 183: ...Appendix D Sample Reports This appendix contains sample System Test and Absorbance Plate Test reports for the Synergy H1 ...

Page 184: ..._______________________________ SYSTEM SELF TEST 8040200 Version 2 00 1506126 S1 S2 S3 1110 0100 DMF Voltage Reference Test Min Low High Max Mono System Flash 1412 1750 2318 3098 Filter System Flash 2338 2922 3507 Switched 24V Power 1886 ABSORBANCE Optics Test Ref Meas Gain Resets 1 230 1 91 2 Light 13389 39922 Dark 10613 10655 Delta 2776 29267 2 285 2 91 8 Light 13064 39639 Dark 10647 10679 Delta...

Page 185: ...ASS Filter PCB Bias current offset 0 4 counts PASS Offset voltage 1701 counts PASS 750V measurement 53 8 counts PASS 750V noise 7 counts 750V offset 1704 counts Reset offset 1732 counts Reference bias 3 6 counts PASS Reference offset 10614 counts PASS Reference noise 0 2 counts PASS Filter Fluorescence Top Probe Reference 400V 500V 600V Gain 2 03 1 18 1 00 Light 11737 11716 12020 Dark 10641 10619 ...

Page 186: ... x 9652 y 8612 Delta 1 116 116 0 Delta 2 9652 9652 0 Delta 3 8612 8616 4 Delta 4 2396 2404 8 Carrier Bottom Mono Fluorescence Upper Left x 1856 y 10460 Lower Left x 1856 y 4252 Lower Right x 11624 y 4248 Upper Right x 11624 y 10464 Delta 1 1856 1856 0 Delta 2 11624 11624 0 Delta 3 10464 10460 4 Delta 4 4248 4252 4 Carrier Absorbance Upper Left x 1888 y 8580 Lower Left x 1888 y 2372 Lower Right x 1...

Page 187: ... 6612 6616 4 Delta 4 396 404 8 Carrier Test Sensors Middle Sensor x 20628 Tested 20636 Delta 8 Probe Height 25 99 mm Filter Mirror Slider 4424 Mono Probe Changer 3072 Backlash 44 Excitation Monochromator Absorbance B 0 00241697 C 0 50917959 305LP Edge 779 17 Tested 780 18 Emission Monochromator Top Fluorescence B 0 00231930 C 1 28592360 Bottom Fluorescence B 0 00029174 C 0 21822651 INCUBATION Temp...

Page 188: ...enser 2 005 2 010 2 020 2 040 1 079 9 199 8 Filter Cube Default Filter Set 1 Blue Ex 360 40 Mirror Top 400 nm Em 460 40 Filter Set 2 Green Ex 485 20 Mirror Top 510 nm Em 528 20 Reviewed Approved By _______________________________________ Date ________________ ...

Page 189: ...ults Wells C1 E2 G3 H6 F5 D4 Reference 0 127 0 577 1 133 1 663 1 915 2 457 Min Limit 0 104 0 545 1 090 1 610 1 857 2 339 Max Limit 0 150 0 609 1 176 1 716 1 973 2 575 Read 1 0 138 0 584 1 140 1 672 1 922 2 468 Result PASS PASS PASS PASS PASS PASS Repeatability Results Wells C1 E2 G3 H6 F5 D4 Read 1 0 138 0 584 1 140 1 672 1 922 2 468 Min Limit 0 131 0 573 1 123 1 651 1 898 2 389 Max Limit 0 144 0 ...

Page 190: ...1 271 1 238 1 586 Min Limit 0 128 0 433 0 855 1 253 1 221 1 565 Max Limit 0 140 0 452 0 882 1 289 1 256 1 607 Read 2 0 134 0 443 0 869 1 271 1 238 1 586 Result PASS PASS PASS PASS PASS PASS Reviewed Approved By _______________________________________ Date ________________ ...

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