Biotek Synergy HTX Operator'S Manual Download Page 125

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Absorbance Plate Test

103

Absorbance Test Plate Results

Reader:

Synergy HTX (Serial Number: EM-04)

Basecode: P/N

1340200 (v1.00)

Date and Time: 07/08/2012 03:03:36 PM

Absorbance Plate: 7 Filter Test Plate (P/N 7260522) - S/N 161259

Last Plate Certification: December 2013

Next Plate Certification Due: December 2014

User: Administrator

Comments: Test performed during Initial OQ

Peak Absorbance Results

Well C6

Reference 586

Tolerance 3

Result PASS

Alignment Results

Wells A1 A12 H1 H12

Tolerance 0.015 0.015 0.015 0.015

Result PASS PASS PASS PASS

Wavelength = 405 nm

Accuracy Results

Wells C1 E2 G3 H6 F5 D4

Reference 0.147 0.618 1.133 1.701 2.279 2.945

Min Limit 0.124 0.586 1.090 1.647 2.168 2.807

Max Limit 0.170 0.650 1.176 1.755 2.390 3.083

Read 1 0.144 0.615 1.128 1.696 2.284 2.908

Result PASS PASS PASS PASS PASS PASS

Repeatability Results

Wells C1 E2 G3 H6 F5 D4

Read 1 0.144 0.615 1.128 1.696 2.284 2.908

Min Limit 0.138 0.604 1.112 1.674 2.210 2.816

Max Limit 0.150 0.626 1.144 1.718 2.358 3.000

Read 2 0.144 0.615 1.128 1.695 2.285 2.903

Result PASS PASS PASS PASS PASS PASS

Wavelength = 630 nm

Accuracy Results

Wells C1 E2 G3 H6 F5 D4

Reference 0.136 0.568 1.040 1.560 1.865 2.400

Min Limit 0.113 0.537 0.999 1.509 1.808 2.284

Max Limit 0.159 0.599 1.081 1.611 1.922 2.516

Read 1 0.134 0.566 1.037 1.557 1.866 2.385

Result PASS PASS PASS PASS PASS PASS

Synergy HTX Operator's Manual

Summary of Contents for Synergy HTX

Page 1: ...Operator s Manual Multi Mode Microplate Reader Synergy HTX ...

Page 2: ......

Page 3: ...Synergy HTX Multi Mode Microplate Reader Operator s Guide July 2014 2014 Part Number 1341000 Revision A BioTek Instruments Inc ...

Page 4: ... are trademarks of BioTek Instruments Inc Harta is a trademark of Harta Instruments Microsoft Windows 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 co...

Page 5: ... Directive 98 79 EC In Vitro Diagnostics if labeled for this use xiv Electromagnetic Interference and Susceptibility xiv USA FCC CLASS A xiv Canadian Department of Communications Class A xiv User Safety xv Safety Symbols xvi Introduction 1 Synergy HTX Multi Mode Microplate Reader 2 Package Contents 3 Optional Accessories 4 Product Support Service 5 Technical Assistance Center TAC 5 Returning Instr...

Page 6: ...itation and Emission Filter Wheels 34 Installing the Time Resolved Fluorescence Cartridge 37 Configuring the System for Luminescence Measurements 38 The External Dispense Module 39 Gen5 Software 41 Viewing Updating the Filter and Wavelengths Tables 42 Creating Protocols and Experiments 43 Controlling the Dispense Module 45 Recommendations for Optimum Performance 48 Incubation and Partial Plates 49...

Page 7: ...and Injector Heads 87 Clean the Tip Priming Trough and Priming Plate 88 Alternate Procedure for Models with Injectors 89 Instrument Qualification 91 Overview 92 IQ OQ PQ 92 Recommended Qualification Schedule 94 System Test 95 Absorbance Plate Test 100 Test Plate Certificates 100 Define Absorbance Test Plate Parameters 100 Run the Absorbance Plate Test 101 Results and Troubleshooting Tips 104 Lumin...

Page 8: ... Absorbance Capabilities 135 Results Analysis 136 Gen5 Test Protocols for Models with Absorbance Capabilities137 Gen5 Test Protocols for Models without Absorbance Capabilities 139 Create the Dispense Protocols 139 Specifications 143 General Specifications 144 Absorbance Specifications 145 Fluorescence Specifications 147 Luminescence Specifications 148 Models with Injectors 149 Error Codes 151 Erro...

Page 9: ...s 15 Tigan Street Winooski VT 05404 Room 304 Tower D Ocean International Center 62 Middle 4th East Ring Road Chaoyang District Beijing 100025 P R China Phone 800 242 4685 Outside US 802 655 4740 Fax 802 654 0638 Phone 86 10 85865569 Fax 86 10 85861829 Email TAC biotek com Website www biotek com Email infochina biotek com Website www biotekchina com cn BioTek Germany Service Center European Coordin...

Page 10: ...ong Gangnam gu Seoul South Korea 135 936 Zentrum Fanhöfli 8 6014 Luzern Switzerland Phone 82 0 2 562 4740 Fax 82 0 2 562 4750 Phone 41 41 2504060 Fax 41 41 2505064 Email korea biotek com Website www biotekinstruments co kr Email info biotek ch Website www biotek ch BioTek United Kingdom UK 6 Bull Street Potton Bedfordshire SG19 2NR United Kingdom Phone 44 1767 262000 Fax 44 1767 262330 Email info ...

Page 11: ...blem Caution A Caution indicates potential damage to the instrument and tells you how to avoid the problem Note Bold text is primarily used for emphasis italic Topics that apply only to specific Synergy HTX models are preceded by a notice in italics for example Applies only to Synergy HTX models with injectors This icon calls attention to important information Synergy HTX Operator s Manual ...

Page 12: ...viii Preface Revision History Rev Date Changes A 7 2014 Initial release BioTek Instruments Inc ...

Page 13: ...rposes including research and development If there is no such label the instrument may be used only for research and development or other non clinical purposes Quality Control It is considered good laboratory practice to run laboratory samples according to instructions and specific recommendations included in the assay package insert for the test to be conducted Failure to conduct Quality Control ...

Page 14: ...are no known hazards associated with the instrument However the operator should be aware of certain situations that could result in serious injury these may vary depending on the instrument model See Hazards and Precautions Hazards The following hazard warnings are provided to help avoid injury Warning Internal Voltage Always turn off the power switch and unplug the power supply before cleaning th...

Page 15: ...d safeguards specified in this manual could result in a hazardous condition Warning Software Quality Control The operator must follow the manufacturer s assay package insert when modifying software parameters and establishing reading washing or dispensing methods Failure to conduct quality control checks could result in erroneous test data Warning Reader Data Reduction Protocol No limits are appli...

Page 16: ...lorite solution bleach for more than 20 minutes Prolonged contact may damage the instrument surfaces Be certain to rinse and thoroughly wipe all surfaces Caution Power Supply Only use the power supply shipped with the instrument Operate this power supply within the range of line voltages listed on it Caution Disposal Dispose of the instrument according to Directive 2002 96 EC on waste electrical a...

Page 17: ...nd found to meet the requirements of EN 61326 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 ...

Page 18: ...ercial environment Like all similar equipment this equipment generates uses and can 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 interference in which case users will be required to correct the interference at their ow...

Page 19: ...10 1 Safety requirements for electrical equipment for measurement control and laboratory use Part 1 General requirements 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 EN 61010 Standards see CE Mark starting on page xiii Synergy HTX Operator s Manual ...

Page 20: ...g vom Netz Desconectado Aperto sconnessione dalla rete di alimentazione Caution refer to accompanying documents Attention voir documents d accompanement Achtung siehe Begleitpapiere Atención vease los documentos incluidos Attenzione consultare la doc annessa Warning risk of electric shock Attention risque de choc électrique Gefährliche elektrische schlag Precaución riesgo de sacudida eléctrica Att...

Page 21: ...tronic equipment Les équipements électriques et électroniques font l objet d une collecte sélective Getrennte Sammlung von Elektro und Elektronikgeräten Recogida selectiva de aparatos eléctricos y electrónicos Raccolta separata delle apparecchiature elettriche ed elettroniche Consult instructions for use Consulter la notice d emploi Gebrauchsanweisung beachten Consultar las instrucciones de uso Co...

Page 22: ...xviii Preface BioTek Instruments Inc ...

Page 23: ...sts its package contents and provides contact information for technical assistance Synergy HTX Multi Mode Microplate Reader 2 Package Contents 3 Optional Accessories 4 Product Support Service 5 Technical Assistance Center TAC 5 Returning Instruments for Service Repair 5 Applications Support 5 ...

Page 24: ...ments The monochromator provides wavelength selection from 200 to 999 nm in 1 nm increments The Synergy HTX has a 4 Zone temperature control from 4 C over ambient to 50 C controlled via a software adjustable gradient Internal plate shaking with both linear and orbital modes is supported to ensure that reagents are properly mixed prior to reading Both Synergy HTX models support the reading of 6 12 ...

Page 25: ...s dummy filters or blanks 7082073 Plastic storage bag and fastener strips Time Resolved Fluorescence cartridge assembly T models only 7090523 Models with injectors an external dispense module packed separately with the following accessories Outlet tubes 2 plus 2 spare from dispense module to instrument Inlet tubes 2 from supply bottles to syringe drives 250 µl syringes 2 Syringe thumbscrews 2 Prim...

Page 26: ...ate 7272051 7270512 Additional Fluorescence Filters contact BioTek for part numbers and availability The Synergy HTX is compatible with the BioStack Microplate Stacker Contact BioTek or visit our website to learn more For Use with Liquid Tests see Chapter 5 Part Absorbance Liquid Test Solutions BioTek Wetting Agent Solution BioTek QC Check Solution 1 25 mL 125 mL 7773002 7120779 7120782 Dispense M...

Page 27: ...elp About Gen5 For troubleshooting assistance or instruments needing repair the specific steps that produce your problem and any error codes displayed in Gen5 see also Appendix C Error Codes A text file of the diagnostic history of the instrument available via Gen5 by selecting System Diagnostics History then selecting the appropriate file and clicking Export Returning Instruments for Service Repa...

Page 28: ...6 Chapter 1 Introduction BioTek Instruments Inc ...

Page 29: ...3 Remove the Microplate Carrier Shipping Screw 11 4 Install the Fluorescence Lamp Assembly 12 5 Select an Appropriate Location 13 6 Connect the Power Supply 14 7 Unpack and Inspect the Dispense Module 15 8 Install the Dispense Module 18 9 Connect the Host Computer 20 10 Install Gen5 Software 20 11 Turn on the Reader 20 12 Establish Communication 21 13 Set the Dispenser Calibration Values 22 14 Run...

Page 30: ...he reader is damaged notify the carrier 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 reader immediately 1 Open the outer shipping box Remove the foam blocks to access the inner box 2 Carefully open the inner shipping box Remove the accessories box and set it aside Remove the ver...

Page 31: ...1 Unpack and Inspect the Reader 9 Figure 1 Unpacking the reader accessories box Synergy HTX Operator s Manual ...

Page 32: ...refully set the reader upright 4 Locate the supplied plastic tool storage pocket Place the screws and washers inside the bag Use the supplied fastener strips to attach the pocket to the back of the reader for storage Do not block any air vents See Figure 2 on the next page 5 Place the panel back into the inner shipping box for storage Important Reattach the shipping panel before repackaging the Sy...

Page 33: ...plate Carrier Shipping Bolt Important Remove the microplate carrier shipping bolt before turning on the Synergy HTX 1 Pull down the microplate loading door on the front of the reader 2 Using the supplied wrench remove the carrier shipping bolt with its o ring and warning tag Synergy HTX Operator s Manual ...

Page 34: ...olt before repackaging the Synergy HTX for shipment Please contact BioTek if you have misplaced the bolt PN 1342008 and or its o ring PN 49259 4 Install the Fluorescence Lamp Assembly Applies only to Synergy HTX models with fluorescence capability Important Do not touch the glass lenses Fingerprints on the condenser lens or heat absorber may negatively affect performance shipping bolt shipping bol...

Page 35: ...n its lower left and right corners The lamp compartment is on the far left 3 Orient the lamp assembly as shown below Slide the assembly all the way into the compartment 4 Plug the lamp cables into the power source located to the right of the lamp Either cable can be plugged into either socket 5 Close the hinged door Figure 4 Installing the fluorescence lamp assembly replacement lamp PN 7080500 5 S...

Page 36: ...ader in their aligning plates at this time Refer to the BioStack Operator s Manual for more information 6 Connect the Power Supply Warning Power Rating The power supply 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...

Page 37: ...llow the repackaging instructions may void your warranty During the unpacking process inspect the packaging module and accessories for shipping damage If the reader is damaged notify the carrier 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 reader immediately 1 Open the outer shi...

Page 38: ...accessories box Remove and identify its contents see Figure 7 on the next page 2 inlet tubes packaged in plastic cylinders 4 outlet tubes packaged in plastic bags 2 syringes packaged in boxes 1 priming plate 2 reagent bottles 1 injector tip priming trough small plastic cup 1 plastic tool storage bag with fastener strips 2 metal thumbscrews 1 stylus wire packaged in a small plastic cylinder 1 dispe...

Page 39: ...ule 17 Figure 7 Unpacking the dispense module s accessories Accessories box Bottom foam end cap with cutouts Syringes 2 Dispense module cover Outlet tubes 4 Inlet tubes 2 Top foam end cap Dispense module cable Synergy HTX Operator s Manual ...

Page 40: ... clear plastic shrouds from the tubes Put the other two bags in a safe place they are spares 4 Remove the two inlet tubes from their plastic canisters 5 Identify the two syringe valves on the dispense module Each is labeled with a left pointing arrow When installing the inlet and outlet tubes do not use any tools Finger tighten only 6 Screw the fitting of one inlet tube into the right side of the ...

Page 41: ... syringe until it rests inside the hole in the bracket d Pass a thumbscrew up through this hole and thread it into the bottom of the syringe Hold the syringe to prevent it from rotating while tightening the thumbscrew Finger tighten only 12 Locate the dispenser cable Plug one end into the port on the left side of the dispenser Plug the other end into the dispenser port on the rear of the reader 13...

Page 42: ...the host computer Figure 8 RS 232 serial and USB ports on the rear panel injector model shown 10 Install Gen5 Software The Synergy HTX is controlled by BioTek s Gen5 software running on a host computer There is a certain sequence of events that must be followed to ensure that the software is properly installed and configured Please follow the instructions provided in the Gen5 Getting Started Guide...

Page 43: ... From the Task Manager select Setup Go to System Menu 3 Select System Instrument Configuration and click Add 4 Set the Reader Type to Synergy HTX 5 Set the Com Port to the computer s COM port to which the reader is connected If using the USB cable the information can be found via the Windows Control Panel under Ports in the Hardware Device Manager area of System Properties e g USB Serial Port COM5...

Page 44: ...Applies only to Synergy HTX models with injectors Before you use the dispenser with the Synergy HTX you must set the calibration values in Gen5 1 Power on the instrument and establish communication 2 In Gen5 go to System Instrument Configuration select the Synergy HTX and click View Modify 3 Click Setup and then select the Dispenser 1 tab 4 On the keyboard press CTRL SHIFT M to enter maintenance m...

Page 45: ...ation Enter the information if required and click OK 3 The results report will appear with text that reads SYSTEM TEST PASS You may wish to print the report and store it with your Installation records The software stores system test information in its database you can retrieve it at any time If an error code is returned turn to Appendix C Error Codes and look up the code If the problem is somethin...

Page 46: ...h injectors 1 If necessary press the button above the power switch to eject the microplate carrier 2 Place the tip priming trough in the left rear pocket of the carrier 3 Place the priming plate on the carrier Figure 10 Installing the tip priming trough and priming plate on the microplate carrier 4 Fill the two reagent bottles with distilled or deionized water Place the bottles in their holders an...

Page 47: ... and up repeatedly drawing fluid from the bottle The fluid should pump through the tubing and dispense into the priming plate Examine the fittings no leaks should be detected If leaks are detected tighten all fittings and repeat the prime If leaks are still detected contact BioTek s Technical Assistance Center 8 When the prime finishes set Volume to 2000 µL and click Purge to clear the fluid lines...

Page 48: ... OQ PQ procedures for the Synergy HTX An Installation Operational Performance Qualification IQ OQ PQ package for the Synergy HTX is available for purchase PN 1340508 Contact your local BioTek dealer for more information Repackaging and Shipping Instructions Warning If the reader and or dispense module has been exposed to potentially hazardous material decontaminate it to minimize the risk to all w...

Page 49: ...contact information Perform these steps to prepare the reader for shipment 1 Contact BioTek s Technical Assistance Center for a service authorization number before returning equipment for service See page 6 for contact information 2 Decontaminate the reader and if attached the dispense module according to the instructions provided in Chapter 5 3 If you will also be shipping the dispense module per...

Page 50: ...er box with tape 14 Write the service authorization number in large clear numbers on the outside of the box Ship the box to BioTek Perform these steps to prepare the dispense module for shipment 1 If you have not already done so Contact BioTek s Technical Assistance Center for a service authorization number and shipping address before returning equipment for service See page 6 for contact informat...

Page 51: ...e bottom foam end cap in the accessories box Place the dispense module cover on top of the accessories 7 Cover the accessories with the top foam end cap place the dispense module cable inside the top of the end cap and seal the box with tape 8 Locate the original outer shipping box and foam end caps Insert the bottom foam end cap Lower the dispense module box into the end cap 9 Insert the accessor...

Page 52: ...30 Chapter 2 Installation BioTek Instruments Inc ...

Page 53: ...Assembly and Filter Wheel Access 33 Excitation and Emission Filter Wheels 34 Installing the Time Resolved Fluorescence Cartridge 37 Configuring the System for Luminescence Measurements 38 The External Dispense Module 39 Gen5 Software 41 Viewing Updating the Filter and Wavelengths Tables 42 Creating Protocols and Experiments 43 Controlling the Dispense Module 45 Recommendations for Optimum Performa...

Page 54: ... as described in Appendix A Specifications The plate is positioned so that well A1 is in the left rear corner of the carrier A spring clip holds the plate securely in place The microplate loading door helps to ensure a light impermeable measurement chamber When a plate read is initiated the carrier slides into the measurement chamber and then moves on the X and Y axes to align each microwell with ...

Page 55: ...fluorescence lamp assembly and the excitation and emission filter wheels are accessible via a hinged door on the front of the instrument To open the door slip your finger into the notch on the right side and pull the door downward A diagram showing the location of the lamp assembly and the orientation of the excitation and emission filter wheels is printed on the inside of the hinged door For mode...

Page 56: ...ission Filter Wheels Synergy HTX models with fluorescence capability are equipped with one excitation filter wheel and one emission filter wheel readers with luminescence capability use an emission filter wheel only A monochromator is used for absorbance measurements A filter in the excitation wheel selects the narrow band of light to which the sample will be exposed A filter in the emission wheel...

Page 57: ...r table and then download the information to the reader The Synergy HTX does not automatically detect which filters are installed See page 42 for information on updating Gen5 s filter table Removing the Filter Wheels The filter wheels can be removed if different filter wheels need to be installed It is important to note that 485 20 Supporting metal bracket Thumbscrew EXCITATION Filter wheel Direct...

Page 58: ...umbscrew and slide the filter wheel s supporting metal bracket straight out of the compartment Note The emission filter wheel will spring out when removed This is because a shutter behind the wheel closes quickly to protect the PMT Important When removing or replacing a filter or C clip filter retainer do not use a sharp instrument Use several layers of lens paper and your finger to remove and rep...

Page 59: ...RF assay can be run The TR cartridge allows light from the xenon flash bulb to be input to the fluorescence optical system within the Synergy HTX Excitation wavelengths are selected by adjusting the monochromator from 200 to 999 nm in 1 nm increments with a fixed bandwidth of 10 nm The Synergy HTX automatically detects the presence of the TR cartridge At the start of a time resolved fluorescence a...

Page 60: ... contents ensure that there are no empty locations and there is at least one plug installed If your tests require that the light emitted from the samples remain unfiltered the emission filter wheel should have an empty location in it Remove the emission filter wheel and examine its contents ensure that there is an empty location If you made any changes to either filter wheel you must update Gen5 s...

Page 61: ...ctors The dispense module pumps fluid from the supply bottles to injector heads located inside the instrument Fluid is injected into one well at a time Figure 16 Dispense module components Two 250 µL syringes draw fluid from the supply bottles Inlet tubes transport fluid from the supply vessels to the syringes These tubes are short pieces of opaque PTFE Teflon tubing connected to stainless steel p...

Page 62: ...ed to deliver fluid from the syringes to the instrument Inside the Synergy HTX two Teflon tubes transport fluid from the tubing ports on the rear of the instrument to the two injectors As shown below both injectors are positioned directly above the bottom fluorescence optical probe Syringe 1 Syringe 2 Bottom probe Figure 17 Close up view of the injectors inside the instrument The tubing and inject...

Page 63: ... on the microplate carrier The priming plate is about the same size as a standard microplate and is placed on the microplate carrier for a Prime operation to prime the dispense system with fluid The tip priming trough is a small removable priming cup located in the left rear of the carrier and is used for performing the Tip Prime before dispensing The trough holds up to 1 5 mL of liquid and must b...

Page 64: ...ight the Synergy HTX reader and click View Modify Click Setup and then click the Fluorescence Luminescence tab Regarding the Absorbance Wavelengths table The Synergy HTX performs absorbance reads in the range of 200 to 999 nm Click the Absorbance tab to specify and calibrate 6 wavelengths to be made available as default selections within a protocol s Reading Parameters dialog To change the setting...

Page 65: ...r analyzing the data retrieved from the reader At a minimum a protocol must specify the procedure for the assay you wish to run After creating a protocol create an experiment that references the protocol You ll run the experiment to read plates and analyze the data Figure 19 Defining the procedure within a Gen5 protocol Synergy HTX Operator s Manual ...

Page 66: ... select Protocols Create New 2 Select Protocol Procedure If prompted to select a reader select the Synergy HTX and click OK 3 Select a plate type The assay plate must match the plate type selected in Gen5 Otherwise the results of the read may be invalid 4 Add steps to the procedure for shaking or heating the plate dispensing fluid reading the plate and more Click Validate to verify that the reader...

Page 67: ...lick OK 3 Select Plate Read or click the Read Plate icon 4 Click OK when the Load Plate dialog appears The plate will be read 5 When the read is complete measurement values will appear in Gen5 To view them select the desired data set e g 528 20 645 40 from the Data drop down list 6 If you have not already done so save the file with an identifying name Controlling the Dispense Module Applies only t...

Page 68: ...nergy HTX and click the Prime tab 4 Select the Dispenser number 1 or 2 associated with the supply bottle 5 Enter the Volume to be used for the prime from 5 to 5000 µL The minimum recommended prime volume is 1100 µL 6 Select a prime Rate in µL second 7 Click Prime to start the process 8 When the process is complete carefully remove the priming plate from the carrier and empty its contents If the pr...

Page 69: ...ct the appropriate Dispenser number 1 or 2 associated with the syringe 3 Click Maintenance The syringe plunger will move to its furthest from home position The syringe can then be disconnected from the drive bracket and unscrewed from the valve 4 See Install Dispense Module Components in Chapter 2 Installation for information on installing removing the syringes Important Do not change the syringe ...

Page 70: ... microplates Check for this by reading an empty microplate Dual wavelength readings can eliminate this problem or bring the variation in density readings to within acceptable limits for most measurements Inaccuracy in pipetting has a large effect on measurements especially if smaller volumes of liquid are used For best results use at least 100 µL per well in a 96 well plate and 25 µL in a 384 well...

Page 71: ...on For models with injectors 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 Incubation and Partial Plates When performing a partial plate read that includes an incubation step the following recommendations can reduce the eff...

Page 72: ...50 Chapter 3 Getting Started BioTek Instruments Inc ...

Page 73: ...Warnings and Precautions 54 Cleaning Exposed Surfaces 55 Inspect Clean Excitation and Emission Filters 56 Flush Purge the Fluid Path 57 Running a Dispense Protocol Optional 58 Empty Clean the Tip Priming Trough 59 Clean the Priming Plate 59 Clean the Internal Components 60 Required Materials 61 Removing the Reader s Shroud 62 Removing the Internal Tubes and Injector Heads 64 Cleaning the Internal ...

Page 74: ...sageways Flushing the tubing at the end of each day letting the DI water soak them overnight and then purging the lines at the beginning of each day ensures optimal performance of the dispense system Perform a visual inspection of the dispensing accuracy before conducting an assay that requires a dispense step to verify instrument performance It is important to keep the dispensing lines scrupulous...

Page 75: ...performed more frequently than presented in the schedule Task Page Daily Quarterly As Needed All models Clean exposed surfaces 55 Inspect clean excitation and emission filters if equipped 56 Decontamination see Chapter 5 before shipment or storage Models with injectors only Flush purge the fluid path 57 Optional Run Dispense protocol 58 Empty clean tip prime trough 59 Clean priming plate 59 Clean ...

Page 76: ...low water or other cleaning solution to run into the interior of the instrument If this happens contact BioTek s Technical Assistance Center Important Do not apply lubricants to the microplate carrier or carrier track Lubrication on the carrier mechanism or components in the carrier compartment will attract dust and other particles which may obstruct the carrier path and cause the reader to produc...

Page 77: ...instrument 2 Moisten a clean cotton cloth with water or with water and mild detergent Do not soak the cloth 3 Wipe the plate carrier and all exposed surfaces of the instrument 4 Wipe all exposed surfaces of the dispense module if used 5 If detergent was used wipe all surfaces with a cloth moistened with water 6 Use a clean dry cloth to dry all wet surfaces 7 Reassemble the instrument as necessary ...

Page 78: ...ace the right secures the emission EM filter wheel Remove each thumbscrew and slide the filter wheel s supporting metal bracket straight out of the compartment Chapter 3 Getting Started contains illustrations for identifying the filter wheels and their unique characteristics This chapter also contains instructions for replacing filters if necessary 3 Inspect the glass filters for speckled surfaces...

Page 79: ...ader if prompted 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 s...

Page 80: ... Set the Dispense Volume to 100 µL or an amount to match your assay protocol Select a Rate adjust the rate to support the dispensing volume Click OK to close the dialog and add the Dispense step to the list 3 Add another Dispense step with the same parameters selecting Dispenser 2 4 Add a quick Read step with the following parameters Gen5 requires that a Read step follow the Dispense step Define a...

Page 81: ...er To empty clean the tip prime trough 1 Extend the microplate carrier and carefully remove the tip priming trough from its pocket in the left rear of the carrier 2 Wash the trough in hot soapy water Use a small brush to clean in the corners 3 Rinse the trough thoroughly and allow it to dry completely 4 Replace the trough in the microplate carrier When starting a Gen5 experiment that includes disp...

Page 82: ...te carrier should be cleaned The procedures in this section should be performed in succession Start with Removing the Reader s Shroud and execute the procedures that meet your needs in the order in which they are presented Finish with Reassembling the Components We recommend running a System Test in Gen5 before and after performing these cleaning procedures This will verify that all systems are fu...

Page 83: ...mponents Screwdriver 1 8 hex key 3 32 hex key For cleaning the internal dispense tubes and injector heads as well as for wiping the surface under the plate carrier Mild detergent Clean lint free cotton cloths Deionized or distilled water Stylus stored in a plastic cylinder affixed to the rear of the dispense module or reader PN 2872304 For cleaning the optical probes Clean cotton swabs Isopropyl a...

Page 84: ...lready done so purge the dispense module of fluid 2 Clear the work surface around the reader so you can easily access all sides of the instrument 3 Disconnect power and all cables Set the dispense module aside 4 Remove four black mounting screws one at the bottom rear corner on each side and two at the top center of the rear panel When reinstalling the shroud press down firmly on the top to mainta...

Page 85: ...e Internal Components 63 5 Stand facing the front of the instrument Grasp both sides of the shroud slide it toward you and pull it straight off the instrument Set the shroud aside Synergy HTX Operator s Manual ...

Page 86: ... sure to align the tubing ports with the injector heads as shown in this diagram Look for the SYRINGE 1 and SYRINGE 2 labels on the instrument s rear panel SYRINGE 1 SYRINGE 2 Internal tubes 2 black for delivering fluid to the injector heads Injector heads 2 white for dispensing fluid into microwells Cable clamp for holding the tubes in place Tubing ports 2 for connecting the two internal dispense...

Page 87: ... cable clamp to release the tubes 2 Locate the tubing ports on the reader s rear wall Turn each tube s thumbscrew counterclockwise and gently pull the tube from the port 3 Locate the injector heads Turn each tube s thumbscrew counterclockwise to disconnect the tube from the injector head Cable clamp Synergy HTX Operator s Manual ...

Page 88: ...reventive Maintenance 4 Turn the injector heads counterclockwise and gently pull them out of their sockets Be sure to seat the injector tips securely when reinstalling See the photo on page 77 BioTek Instruments Inc ...

Page 89: ... dissolve any hardened particles 2 Flush each tube by holding it vertically under a stream of water from a faucet To clean the injector heads Do not remove the o ring from the injector head see photos below 1 Gently insert the stylus into each injector head pipe to clear any blockages The stylus should be stored in a plastic cylinder affixed to the rear of the dispense module or reader 2 Stream wa...

Page 90: ...her indicator is the CV in the Corners liquid test it may increase due to the Noise in the chamber from any spilled fluorescing compounds To access the optical probes the first step is to unplug the reader and remove its shroud cover If you haven t already done this turn to page 62 now for instructions We recommend cleaning the internal tubes and injector heads along with the optical probes Instru...

Page 91: ...e removed adjusted for cleaning the optic probes Thumbscrew to access ground wire Heater thermistor connectors 2 Thumbscrews 2 black for holding the incubator housing in place Screw for lowering and raising the top optical probe Top probe hanger with two shoulder screws Top optical probe Incubator housing Synergy HTX Operator s Manual ...

Page 92: ...tructions to remove a few more components and then clean the optical probes 1 Disconnect the heater and thermistor wires To do this depress the small tab pictured below and separate the connectors 2 Remove the thumbscrew located in the left rear of the instrument and set it aside This exposes the ground wire Depress tab to separate the connectors BioTek Instruments Inc ...

Page 93: ...ernal Components 71 3 Lift the ground wire and move it off to the side 4 Locate the two black thumbscrews that hold the incubator housing in place Remove both of them and set them aside Synergy HTX Operator s Manual ...

Page 94: ...Rotate the screw not the ring around it 6 Gently lift the left side of the incubator housing and carefully slide it out Rotate counterclockwise Note When replacing the incubator housing the two forks on its right side should wrap around the holding screws The forks should not slide under the fixed foam housing BioTek Instruments Inc ...

Page 95: ...move the top optical probe s holding screw 8 Gently pull the optical probe up and out of its socket to expose it for cleaning Soak the end of the probe in alcohol for one minute maximum Wipe with lens cleaning tissue and set aside Synergy HTX Operator s Manual ...

Page 96: ... the top probe hanger down and slide to the left to remove it Turn the hanger upside down to clean the absorbance lens see instructions on the next page Do not touch the lens with your fingers Inspect the block for spills or other contamination Carefully clean with mild detergent if necessary Clean with alcohol and lens tissue Shoulder screws BioTek Instruments Inc ...

Page 97: ...nstallation must be performed by BioTek service personnel If the lens does fall out contact BioTek TAC 11 Use a cotton swab moistened with alcohol to gently clean the lens on the top probe hanger 12 Slide the microplate carrier out of the way Use a cotton swab moistened with alcohol to clean the lens on the instrument surface Absorbance Lens Probe hanger bottom view Synergy HTX Operator s Manual ...

Page 98: ...Follow the instructions under Cleaning the Optical Probes to at a minimum disconnect the incubator wires detach the ground wire lower the top optic probe hanger and remove the incubator housing steps 1 through 6 2 Manually slide the microplate carrier to the left to engage the support pin and then away from the center surface Microplate carrier fully extended BioTek Instruments Inc ...

Page 99: ...mbling the Components Perform these steps in the order listed to reassemble the components Refer to the page numbers shown for further instructions and photos demonstrating the steps 1 Slide the microplate carrier back into the instrument page 76 2 Insert the two injector heads into their sockets in the top probe hanger Do not touch the absorbance lens with your fingers Ensure that the injector he...

Page 100: ...he injector heads as shown below Do not overtighten the thumbscrews Here is the top probe hanger ready for reinstallation with injector heads and internal dispense tubes attached 4 Replace the top probe hanger and shoulder screws using the 3 32 hex key page 74 BioTek Instruments Inc ...

Page 101: ...ports taking care to align the correct port with the correct injector head page 65 10 Slide the two internal dispense tubes into the cable clamp and close the clamp page 65 11 Review the steps you just performed to make sure the components have been properly reassembled 12 Slide the shroud onto the instrument page 63 13 Replace the four screws to securely attach the shroud to the base page 62 Perf...

Page 102: ...80 Chapter 4 Preventive Maintenance BioTek Instruments Inc ...

Page 103: ...ed Materials 83 Procedure for Models without Injectors 84 Routine Procedure for Models with Injectors 85 Clean Exposed Surfaces 85 Decontaminate the Fluid Lines 86 Rinse the Fluid Lines 87 Clean the Internal Tubing and Injector Heads 87 Clean the Tip Priming Trough and Priming Plate 88 Alternate Procedure for Models with Injectors 89 ...

Page 104: ...rs for Disease Control and Prevention CDC Neither BioTek nor the CDC assumes any liability for the adequacy of these solutions and methods Each laboratory must ensure that decontamination procedures are adequate for the Biohazard s they handle Wear prophylactic gloves when handling contaminated instruments Gloved hands should be considered contaminated at all times keep gloved hands away from eyes...

Page 105: ...ve to bleach Deionized or distilled water Safety glasses Surgical mask Protective gloves Lab coat Biohazard trash bags 125 mL beakers Clean lint free cotton cloths Additional materials for models with injectors Screwdriver Small brush for cleaning the tip priming trough and priming plate Optional Mild detergent Synergy HTX Operator s Manual ...

Page 106: ...ure to check the percent NaClO of the bleach you are using this information is printed on the side of the bottle Commercial bleach is typically 10 0 NaClO if this is the case prepare a 1 20 dilution Household bleach is typically 5 0 NaClO if this is the case prepare a 1 10 dilution 3 Moisten a cloth with the bleach solution or alcohol Do not soak the cloth 4 Manually open the plate carrier door sl...

Page 107: ...aces 1 Turn off and unplug the instrument 2 Prepare an aqueous solution of 0 50 sodium hypochlorite bleach As an alternative 70 isopropyl alcohol may be used if the effects of bleach are a concern Be sure to check the percent NaClO of the bleach you are using this information is printed on the side of the bottle Commercial bleach is typically 10 0 NaClO if this is the case prepare a 1 20 dilution ...

Page 108: ... tubes from the instrument s rear panel Place the ends of the outlet tubes in an empty beaker and set the beaker on the work surface 4 Launch Gen5 select System Instrument Control and click the Prime tab 5 Select Dispenser 1 enter a Volume of 5000 µL and keep the default dispense Rate 6 Place the priming plate on the carrier it is not used but the reader requires its presence 7 Run two prime cycle...

Page 109: ... one prime cycle with 5000 µL This delay will allow any residual sodium hypochlorite to diffuse into the solution and be flushed out with the next prime 7 Empty the beaker containing the outlet tubes 8 Wipe all surfaces with deionized water 9 Discard the used gloves and cloths using a biohazard trash bag and an approved biohazard container Clean the Internal Tubing and Injector Heads Turn to Chapt...

Page 110: ...nd plate 3 To decontaminate soak the trough and plate in a container of 0 5 sodium hypochlorite or 70 isopropyl alcohol for 20 to 30 minutes 4 If decontaminating in bleach solution remove the trough and plate and thoroughly rinse with DI water If decontaminating with alcohol remove the trough and plate and let them air dry 5 Discard the used gloves and cloths using a biohazard trash bag and an app...

Page 111: ... 0 NaClO if this is the case prepare a 1 20 dilution Household bleach is typically 5 0 NaClO if this is the case prepare a 1 10 dilution 3 Slide the microplate carrier out of the instrument 4 Moisten a cloth with the bleach solution or alcohol Do not soak the cloth 5 Use the cloth to wipe All surfaces of the shroud All surfaces of the plate carrier The instrument s rear panel The exposed surfaces ...

Page 112: ...ater 9 Use a clean dry cloth to dry all wet surfaces on the instrument and the Dispense module 10 Reassemble the instrument and dispense module as necessary 11 Discard the used gloves and cloths using a biohazard trash bag and an approved biohazard container BioTek Instruments Inc ...

Page 113: ...tial and ongoing performance of the Synergy HTX and the external dispense module if used Overview 92 IQ OQ PQ 92 Recommended Qualification Schedule 94 System Test 95 Absorbance Plate Test 100 Luminescence Tests 106 Absorbance Liquid Tests 109 Fluorescence Tests 116 Dispense Module Tests 132 ...

Page 114: ...ualification Performance Qualification and Preventive Maintenance procedures Microsoft Excel spreadsheets are provided for performing the calculations and checklists data sheets and logbooks are provided for recording results Contact your local BioTek dealer for more information IQ OQ PQ Installation Qualification confirms that the reader and its components have been supplied as ordered and ensure...

Page 115: ...hat are comparable to previous PQ and OQ tests confirms that the equipment is operating according to specification initially and over time Performance Qualification confirms that the reader consistently meets the requirements of the tests performed at your laboratory The recommended PQ procedure consists of performing the System Test Absorbance Plate Test a series of Liquid Tests and if the extern...

Page 116: ...t 1 or Liquid Test 2 Liquid Test 3 Fluorescence Tests Corners Test Sensitivity Linearity Tests Tests for Injector Models Injector System Test Dispense Accuracy and Precision Tests If applicable to your reader model Regarding Liquid Tests 1 and 2 If you have an Absorbance Test Plate run Liquid Test 1 If you do not have an Absorbance Test Plate run Liquid Test 2 Liquid Test 3 is optional it is provi...

Page 117: ...rbance Wavelength table to values that will confirm operation of the reader at its limits We recommend 200 and 999 nm the lower and upper limits of the monochromator and any four wavelengths in between that best represent your assays and or the lowest and highest wavelength values used in your lab From Gen5 s main screen select System Instrument Configuration Highlight Synergy HTX and click View M...

Page 118: ...ny time We recommend that you print and save the reports to document that the test was performed 7 If the test failed look up the error code in Appendix B Error Codes to determine its cause If the cause is something you can fix turn off the reader fix the problem and then turn the reader back on and retry the test If the test continues to fail or if the cause is not something you can fix contact B...

Page 119: ...4 SW1 S1 1100 1000 AF T Voltage Reference Test Min Low High Max 24V Power 1949 Xenon Flash 1431 1741 2164 2473 Motor Power 2054 Tungsten Lamp 12 1724 ABSORBANCE Optics Test Ref Meas Gain Resets 1 200 1 73 2 Light 13454 38437 Dark 9874 9884 Delta 3580 28553 2 352 1 46 4 Light 12848 39920 Dark 9878 9885 Delta 2970 30035 3 620 2 75 2 Light 12829 39979 Dark 9870 9884 Delta 2959 30095 4 790 2 10 2 Ligh...

Page 120: ...Delta 2 1048 1044 4 Delta 3 1792 1784 8 Delta 4 7312 7312 0 Carrier Top Fluorescence Probe Upper Left x 9744 y 232 Lower Left x 9732 y 5756 Lower Right x 1044 y 5748 Upper Right x 1056 y 224 Delta 1 9744 9732 12 Delta 2 1056 1044 12 Delta 3 224 232 8 Delta 4 5748 5756 8 Carrier Absorbance Probe Upper Left x 11292 y 1796 Lower Left x 11284 y 7316 Lower Right x 2592 y 7316 Upper Right x 2604 y 1796 ...

Page 121: ...r PASS Zone 4 25 3 Min 25 3 Max 25 3 Range PASS Thermistor PASS 0000 Dispenser 1 005 0 010 0 020 0 040 0 080 0 200 0 Dispenser 2 005 0 010 0 020 0 040 0 080 0 200 0 Reviewed Approved By _______________________________ Date ________________ Technical Support In the U S BioTek Instruments Inc Tel 800 242 4685 Fax 802 654 0638 In Europe BioTek Instruments GmbH Tel 49 0 7136 9680 Fax 49 0 7136 968 111...

Page 122: ...hs used in your assays The Wavelength Accuracy Standards section contains Expected Peak wavelength values for the filter in position C6 on the plate Each value has a valid test range associated with it For example an Expected Peak value may be 586 nm with tolerance values of 6 4 or a test range of 580 to 590 nm The instructions provided below and on the following page are guidelines Refer to the G...

Page 123: ...enter the Expected Peak wavelength value and adjust the Test Range values so the range displayed in parentheses is 580 to 590 as demonstrated above 9 Review all of the values you entered and then click OK to save the data The information you just entered will be available in Gen5 each time the Absorbance Plate Test is performed It may need to be modified after the annual recertification of your te...

Page 124: ...t should show PASS See page 104 for information on results and troubleshooting tips in the event of failures A sample test report is shown on page 104 Gen5 stores the results in a database they can be retrieved and printed at any time We recommend you print and save the report to document that the test was performed BioTek Instruments Inc ...

Page 125: ...lls C1 E2 G3 H6 F5 D4 Reference 0 147 0 618 1 133 1 701 2 279 2 945 Min Limit 0 124 0 586 1 090 1 647 2 168 2 807 Max Limit 0 170 0 650 1 176 1 755 2 390 3 083 Read 1 0 144 0 615 1 128 1 696 2 284 2 908 Result PASS PASS PASS PASS PASS PASS Repeatability Results Wells C1 E2 G3 H6 F5 D4 Read 1 0 144 0 615 1 128 1 696 2 284 2 908 Min Limit 0 138 0 604 1 112 1 674 2 210 2 816 Max Limit 0 150 0 626 1 1...

Page 126: ...wing Peak Absorbance When the test is performed the C6 filter is scanned at the test range s defined by the user in the Absorbance Test Plate dialog To verify wavelength accuracy the wavelength of the maximum absorbance is compared with the peak wavelength value entered in the software which comes from the data sheet supplied with the Test Plate The accuracy of the wavelength should be 3 nm 2 nm i...

Page 127: ...d linear To further verify this you can perform a regression analysis on the Test Plate OD values in a program such as Microsoft Excel An R Square value of at least 0 990 is expected If the test fails Check the filters in the Test Plate to ensure they are clean If necessary clean them with lens paper Do not remove the filters from the test plate and do not use alcohol or other cleaning agents Veri...

Page 128: ...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 you cannot see it 3 Place the Harta plate adapter on the reader s carrier and then place the test plate on top of the adapter 4 Create an experiment based on Synergy HTX LumTest_Harta prt protocol and read the ...

Page 129: ...certified 3 Calculate the signal to noise ratio A2 Mean of the buffer cells 3 Standard deviation of buffer cells 4 Calculate the detection limit A2 NIST measurement in attomoles signal to noise ratio If the reader is equipped with the low noise PMT the detection limit must be 60 amol to pass If the reader is equipped with the red shifted PMT the detection limit must be 500 amol to pass To determin...

Page 130: ...tection Method Luminescence Read Type Endpoint Integration Time 0 10 00 MM SS ss Delay After Plate Movement 100 msec Dynamic Range Standard Filter Sets 1 Excitation Plug Emission Hole Optics Position Top Gain Sensitivity 150 Read Height 1 00 mm Read Step 3 Read Wells A7 A8 Label Battery Check Detection Method Luminescence Read Type Endpoint Integration Time 0 01 00 MM SS ss Delay After Plate Movem...

Page 131: ...nt types of problems 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 placement and optical system cleanliness are proven Liquid Test 2 can be used to test the alignment repeatability and linearity of the reader if an Absorbance Test Plate is not available Liquid Test 3 is provided for sites requiring proof...

Page 132: ...which may be formulated by diluting a dye solution available from BioTek A or from the ingredients listed below B Solution A BioTek QC Check Solution No 1 PN 7120779 25 mL PN 7120782 125 mL Deionized water 5 mL Class A volumetric pipette 100 mL volumetric flask 1 Pipette a 5 mL aliquot of BioTek QC Check Solution No 1 into a 100 mL volumetric flask 2 Add 95 mL of DI water cap and shake well The so...

Page 133: ...ommend shaking the plate for four minutes This will allow any air bubbles in the solution to settle and the meniscus to stabilize Alternatively wait 20 minutes after pipetting the test solution before reading the plate 39BRead the Plate 1 Using Gen5 read the microplate five times at 405 nm using the Normal read mode single wavelength no blanking Save the data after each read Normal plate position ...

Page 134: ...ommended Stock solution A or B see page 110 A 0 05 solution of deionized water and Tween 20 Prepare the Dilutions Create a percentage dilution series beginning with 100 of the original concentrated stock solution A or B in the first tube 90 of the original solution in the second tube 80 in the third tube all the way to 10 in the tenth tube Dilute using the 0 05 solution of deionized water and Twee...

Page 135: ...mewhat difficult to achieve high pipetting accuracy when conducting linear dilutions an R Square value of at least 0 99 is considered adequate 4 Calculate the results for Repeatability Calculate the mean and standard deviation for the five readings taken in Step 1 at each concentration Only one row of data needs to be analyzed For each mean below 2 000 OD calculate the allowed deviation using the ...

Page 136: ...strument Example If the mean of well A1 in the normal position is 1 902 where the specified accuracy is 1 0 0 010 OD then the expected range for the mean of the same well in the H12 position is 1 873 to 1 931 OD 1 902 x 1 0 0 019 0 010 0 029 which is added and subtracted from 1 902 for the range If the four corner wells are within the accuracy range the reader is in alignment Absorbance Liquid Tes...

Page 137: ...S solution 3 Pipette the three solutions into a new 96 well microplate 150 µL of the 100 Test Solution into all wells of columns 1 and 2 150 µL of the 75 Test Solution into all wells of columns 3 and 4 150 µL of the 50 Test Solution into all wells of column 5 and 6 Read the Plate 1 Using Gen5 read the microplate five times using Normal mode single wavelength at 340 nm no blanking Save the data aft...

Page 138: ...s an R Square value of at least 0 99 is considered adequate Fluorescence Tests The Corners Test uses fluorescent compounds to verify that the plate carrier is properly aligned in relation to the fluorescence probe s We recommend running the test for both the top and bottom probes The Sensitivity Test uses a fluorescent compound and buffer solution to test the fluorescence reading capability of the...

Page 139: ...he microplates and solutions used in this procedure Microplates should be perfectly clean and free of dust or bottom scratches Use new microplates from sealed packages Manufacturer part numbers are subject to change Methylumbelliferone can be used as an alternative or supplemental method for performing these tests for the top probe See the instructions starting on page 123 Buffer NIST traceable So...

Page 140: ...ly dissolve Allow the solution to dissolve for five minutes with intermittent vortexing before preparing the titration dyes Wrap the vial containing the SF stock solution in foil to prevent exposure to light Discard any open unused solutions after seven days 1 The Sodium Borate solution does not require further preparation proceed to step 2 If you are using PBS prepare the solution now Optional bu...

Page 141: ...glass bottom microplate see the map on page 121 Create an experiment based on the Synergy HTX FI_B prt protocol and read the plate When complete save the experiment using a unique name 4 Perform the tests using the Top optics Pipette the test solutions into a new 96 well solid black or quartz microplate see the map on page 121 Create an experiment based on the Synergy HTX FI_T prt protocol and rea...

Page 142: ...han or equal to Bottom 5 mm 30 pM 10 pg mL Top 3 mm 53 pM 20 pg mL Linearity Test 1 Calculate the Mean of the four wells for each concentration in columns 1 5 2 Perform linear regression using these values as inputs x y 1000 Mean of the 1000 pM wells 500 Mean of the 500 pM wells 250 Mean of the 250 pM wells 125 Mean of the 125 pM wells 62 5 Mean of the 62 5 pM wells 3 Calculate the R Square value ...

Page 143: ... placing a plate in the instrument blow the bottom of the plate with an aerosol duster Perform these steps carefully and refer to the grid on the next page For the Corners test light gray wells Pipette 200 µL of the 3 3 nM SF solution into wells A1 A3 A10 A12 H1 H3 and H10 H12 If using a Hellma plate Pipette 200 µL of buffer into the wells surrounding the 3 3 nM wells CBUF in the grid For the Sens...

Page 144: ...µL 1 0 nM BUF E 1 0 nM 0 5 nM 0 25 nM 0 125 nM 0 0625 nM BUF F 1 0 nM 0 5 nM 0 25 nM 0 125 nM 0 0625 nM G CBUF CBUF CBUF CBUF CBUF CBUF CBUF CBUF H 3 3 nM 3 3 nM 3 3 nM CBUF CBUF 3 3 nM 3 3 nM 3 3 nM CBUF applies only to the Hellma Quartz plate Gen5 Protocol Reading Parameters The following tables contain the recommended settings for the Gen5 protocols Your tests may require modifications to some ...

Page 145: ...ue 30000 Delay after Plate Movement 100 msec Measurements per Data Point 50 Dynamic Range Standard Light Source Tungsten Read Step 2 Kinetic Loop Run time 1 minute 35 seconds Interval 6 seconds Reads 16 Read Step Label Sensitivity Read Buffer Read Wells C9 E9 Filter Set EX 485 20 nm EM 528 20 nm Optics Position Bottom Gain Auto Use first filter set gain from FIRST Read Step Delay after Plate Movem...

Page 146: ...d Light Source Tungsten Read Step 4 Read Step Label Linearity Read Read Wells C1 F5 Filter Set EX 485 20 nm EM 528 20 nm Optics Position Bottom Gain Auto Scale to High Wells Scale Well C1 Scale Value 30000 Delay after Plate Movement 100 msec Measurements per Data Point 50 Dynamic Range Standard Light Source Tungsten Protocol Name Synergy HTX FI_T prt Parameter Default Setting Plate Type Costar 96 ...

Page 147: ... 1 00 mm Read Step 2 Kinetic Loop Run time 1 minute 35 seconds Interval 6 seconds Reads 16 Read Step Label Sensitivity Read Buffer Read Wells C9 E9 Filter Set EX 485 20 nm EM 528 20 nm Optics Position Top Gain Auto Use first filter set gain from FIRST Read Step Delay after Plate Movement 100 msec Measurements per Data Point 50 Dynamic Range Standard Light Source Tungsten Read Height 1 00 mm Read S...

Page 148: ... to High Wells Scale Well C1 Scale Value 30000 Delay after Plate Movement 100 msec Measurements per Data Point 50 Dynamic Range Standard Light Source Tungsten Fluorescence Tests Using Methylumbelliferone As an alternative to using Sodium Fluorescein Methylumbelliferone MUB can be used to test the top optics for the fluorescence system Required Materials BioTek offers a liquid test kit PN 7160012 c...

Page 149: ...nd dissolve the contents of two CBB capsules do not dissolve the outer gelatin capsule into 200 mL of the water Stir the solution preferably using a stir table until the CBB is completely dissolved 2 Prepare the MUB stock solution Add 1 mL of 100 methanol to the 10 mg vial of MUB Make sure all of the dye has completely dissolved and is well mixed This yields a 10 mg mL stock solution Wrap the solu...

Page 150: ...ponding Mean 3 Calculate the Mean for the 16 reads of the MUB Concentration well D7 4 Calculate the Signal to Noise Ratio SNR using the Mean MUB Concentration and the Buffer Media STD with its corresponding Buffer Mean MUB Mean Buffer Mean 3 Buffer STD 5 Calculate the Detection Limit using the known concentration value of MUB and the calculated SNR 17 6 SNR Optic Probe Detection Limit must be less...

Page 151: ... buffer solution into wells C2 F5 Discard the tips Pipette 150 µL of the 17 6 ng mL 100 nM solution into wells C1 F1 Pipette 150 µL of the 17 6 ng mL 100 nM solution into wells C2 F2 Mix the wells using the pipette Do not discard the tips Aspirate 150 µL from wells C2 F2 and dispense into wells C3 F3 Mix the wells using the pipette Do not discard the tips Aspirate 150 µL from wells C3 F3 and dispe...

Page 152: ... parameters below such as Plate Type or Gain see Troubleshooting on page 120 The Plate Type setting should match the plate you are actually using The following table contains the recommended settings for the Synergy HTX FI_MUB prt protocol Protocol Name Synergy HTX FI_ MUB prt Parameter Default Setting Plate Type Costar 96 black opaque Mfr 3915 Read Step 1 Kinetic Loop Run time 45 seconds Interval...

Page 153: ...el Sensitivity Read Buffer Read Wells C9 E9 Filter Set EX 360 40 nm EM 460 40 nm Optics Position Top Gain Auto Use first filter set gain from FIRST Read Step Delay after Plate Movement 100 msec Measurements per Data Point 50 Dynamic Range Standard Light Source Tungsten Read Height 1 00 mm Read Step 3 Read Step Label Linearity Read Read Wells C1 F5 Filter Set EX 360 40 nm EM 460 40 nm Optics Positi...

Page 154: ...t volumes The balance is tared with the empty plate and then the 80 µL dispense is performed for columns 1 4 The fluid is weighed and the balance is tared again with the plate on the balance This process is repeated for the 20 µL and 5 µL dispenses It is assumed that the solutions used are at room temperature A precision balance three place is used to weigh the plate The Precision Test is a measur...

Page 155: ...croplates BioTek s Green Test Dye Solution PN 7773003 undiluted or one of the alternate test solutions provided in the next section 100 mL graduated cylinder and 10 mL pipettes if not using BioTek s Green Test Dye Solution Gen5 software installed on the host PC Gen5 protocols as defined by the procedure on page 138 Alternate Test Solutions 80 µL of test solution with 150 µL of deionized water shou...

Page 156: ...µL of the solution When finished remove the priming plate from the carrier 2 In Gen5 create an experiment based on Synergy HTX Disp 1 Test prt 3 Place a new 96 well microplate on the balance and tare the balance 4 Place the plate on the microplate carrier Running a dispense procedure without placing a plate in the reader will result in contamination of the reader from spilled liquid When each disp...

Page 157: ...he green dye solution Procedure for Models without Absorbance Capabilities If you will not be using a BioTek absorbance reader for this procedure prepare your reader to perform two reads with the following characteristics 80 µL Read 20 5 µL Read Primary Wavelength 405 nm 630 nm Reference Wavelength 750 nm 750 nm Plate Columns 1 4 5 12 1 If you have not already done so create the necessary Gen5 pro...

Page 158: ...n test dye solution Carefully set the plate aside 10 Close the experiment without saving it If you are not using a BioTek reader for taking the absorbance measurements read the plate using the wavelengths shown in the table on the previous page and then perform the Results Analysis as described on page 137 11 Configure Gen5 to communicate with the reader 12 Create an experiment based on the Synerg...

Page 159: ...ht Expected Weight Expected Weight 100 Expected Weights for 32 wells 80 μL 2 560 g 20 μL 0 640 g 5 μL 0 160 g It is assumed that one gram is equal to one milliliter Dispense Volume To pass CV must be To pass Accuracy Error must be 80 μL 2 0 2 0 20 μL 7 0 5 0 5 μL 10 0 20 0 Failures If any tests fail prime the fluid lines and rerun the test s If the test s fail again the injector heads may require ...

Page 160: ...ested comment Weigh the plate 20 uL test RECORD the weight TARE the balance Place the plate back on the carrier Click OK to continue 5 Dispense Dispenser select 1 or 2 depending on the protocol Dispense to wells A9 H12 Tip prime before this dispense step 5 µL Dispense 5 µL at rate 225 µL sec 6 Plate Out In Suggested comment Weigh the plate 5 uL test RECORD the weight PIPETTE 150 ul well of DI wate...

Page 161: ...ata Set Name resembling Delta OD 20 and 5 uL_Disp Formula DS1 DS2 applied to wells A5 to H12 4 This step is optional The results analysis worksheet at the end of this chapter requires the calculation of the Standard Deviation Mean and CV of the ODs read for each dispense volume in each plate six sets of calculations By identifying the wells by their dispense volumes in the Plate Layout Gen5 will c...

Page 162: ...t RECORD the weight TARE the balance Place the plate back on the carrier Click OK to continue 3 Dispense Dispenser select 1 or 2 depending on the protocol Dispense to wells A5 H8 Tip prime before this dispense step 20 µL Dispense 20 µL at rate 250 µL sec 4 Plate Out In Suggested comment Weigh the plate 20 uL test RECORD the weight TARE the balance Place the plate back on the carrier Click OK to co...

Page 163: ... Normal Two Wavelengths 630 and 750 nm 3 Create Data Reduction steps to calculate Delta OD values Select Protocol Data Reduction and select Custom Within this dialog click the Select Multiple Data Sets button and then click the DS2 radio button Set the Data In for DS1 to the 80 µL Read step at 405 nm Set the Data In for DS2 to the 80 µL Read step at 750 nm Click OK to return to the dialog In the N...

Page 164: ...nd CV of the ODs read for each dispense volume in each plate six sets of calculations By identifying the wells by their dispense volumes in the Plate Layout Gen5 will calculate these values for you Open the Plate Layout dialog Define three Assay Control names as Disp_80 Disp_20 and Disp_5 Assign Disp_80 to wells A1 to H4 Assign Disp_20 to wells A5 to H8 Assign Disp_5 to wells A9 to H12 After runni...

Page 165: ... weight 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...

Page 166: ......

Page 167: ...s This appendix contains BioTek s published specifications for the Synergy HTX General Specifications 144 Absorbance Specifications 145 Fluorescence Specifications 147 Luminescence Specifications 148 Models with Injectors 149 ...

Page 168: ...idity 10 to 85 relative humidity noncondensing Power Supply 24 volt 90W external power supply compatible with 100 240 V 10 50 60 Hz Power Consumption 100 VA max 130 VA max with injectors Incubation Temperature control range from 4º over ambient to 50ºC Temperature variation 0 5 C at 37 C tested with Innovative Instruments Inc temperature test plate Top and bottom incubation controlled via software...

Page 169: ...plate sweep read speed 0 000 to 1 000 OD 1 0 0 010 OD Linearity by liquid dilution 96 well plate normal read speed 0 000 to 2 000 OD 1 0 0 010 OD Delay after plate movement 100 ms 2 000 to 3 000 OD 3 0 0 010 OD Delay after plate movement 100 ms 384 well plate normal read speed 0 000 to 2 000 OD 2 0 0 010 OD Delay after plate movement 100 ms 2 000 to 2 500 OD 3 0 0 010 OD Delay after plate movement...

Page 170: ... 2 nm λ precision 0 2 nm standard deviation λ bandpass 2 4 nm Resolution 0 0001 OD Increment 1 nm Minimum kinetic interval 450 nm Sweep mode 20 seconds 96 well plate Time elapse from plate in to plate out 450 nm Sweep mode 35 seconds 96 well plate BioTek Instruments Inc ...

Page 171: ...robes can be installed only in the top position and the 5 mm probe can be installed only in the bottom position Sensitivity Specifications apply to the Normal mode of reading 5 mm optical probe Bottom reading DL Sodium Fluorescein in PBS 30 pM Excitation 485 20 Emission 528 20 DL Propidium Iodide in PBS 62 5 ng mL Excitation 485 20 Emission 645 40 3 mm optical probe Top reading DL Methylumbellifer...

Page 172: ...inescence Specifications Luminescence 60 amol well DL ATP in a 96 well plate low noise PMT 20 amol typical 500 amol well in a 96 well plate red shifted PMT 10 second integration PMT sensitivity 150 16 blank wells BioTek Instruments Inc ...

Page 173: ... 1 µl or 2 0 whichever is greater Precision Dispensing a 200 µL solution of deionized water 0 1 Tween 20 and dye at room temperature 2 0 for volumes of 50 200 mL 4 0 for volumes of 25 49 mL 7 0 for volumes of 10 24 mL 10 0 for volumes of 5 9 mL Injection Speeds 225 250 275 and 300 µL sec Maximum Delay between End of Dispense Process and Start of Read Process 96 384 well plates default probe height...

Page 174: ...150 Appendix A Specifications BioTek Instruments Inc ...

Page 175: ...Appendix B Error Codes This appendix lists and describes Synergy HTX error codes that may appear in Gen5 Error Codes Overview 152 Contact Info BioTek Service TAC 152 Error Codes 153 ...

Page 176: ...d run a system test for diagnostic purposes In Gen5 select System Diagnostics Run System Test Having the system test report before calling the BioTek s Technical Assistance Center can speed the resolution of the error If an error message appears while an experiment is in process and after having received measurement data it is your responsibility to determine if the data is valid Contact Info BioT...

Page 177: ... power supply has failed Verify connection to AC mains Power supply connection to instrument is loose or broken Contact BioTek TAC 150x Temperature is out of range External 24 volt power supply is low x 1 Zone 1 x 2 Zone 2 x 3 Zones 1 and 2 x 4 Zone 3 x 5 Zones 1 and 3 x 6 Zones 2 and 3 x 7 Zones 1 2 and 3 x 8 Zone 4 x 9 Zones 1 and 4 x A Zones 2 and 4 x B Zones 1 2 and 4 x C Zones 3 and 4 x D Zon...

Page 178: ...n a filter wheel but it was not found Verify that the plug is physically installed and that this is accurately reflected in the filter table 2313 Empty hole not found in emission filter wheel Protocol contains an empty hole in the emission filter wheel but it was not found Verify that an empty hole is in the emission filter wheel and that this is accurately reflected in the filter table 2326 TRF c...

Page 179: ...A Plate not in carrier for system prime operation Place priming plate in carrier 2C01 2C05 2C07 Dispenser configuration incorrect Volume calibration data is invalid Volume calibration is needed to override defaults Verify that the dispenser calibration values have been loaded into Gen5 See the Installation chapter in the Synergy HTX Operator s Manual Contact BioTek TAC 2D09 Tip prime volumes speci...

Page 180: ...a with lower humidity Too much light in read chamber Ensure the instrument enclosure is completely installed and secured 390Y 391Y Absorbance reference channel dark range outside of limits measurement 100 counts where Y readset Absorbance measurement channel dark range outside of limits where Y readset Humidity is outside the environmental specification of instrument Note the specification in the ...

Page 181: ...e Read Process y readset Absorbance Optics Test Too much light in read chamber Ensure the instrument enclosure is completely installed and secured Check for spill in chamber or dirty absorbance optics 3F1y Absorbance measurement out of range Absorbance Read Process y readset Absorbance Optics Test Too much light in read chamber Ensure the instrument enclosure is completely installed and secured Ch...

Page 182: ...PMT test failed at 500 volts Too much light in chamber Door not closed completely Ensure that the Gen5 Filter Table accurately reflects the physical configuration of the excitation or emission filter wheels Ensure that instrument case is completely installed and secured Sensitivity set too high try adjusting sensitivity setting 4E18 PMT saturation detected Too much light in chamber Door not closed...

Page 183: ...xis rails are dusty or rusty Dirt in the roller bearings is causing them to jam An object may be obstructing the path 5002 5202 Excitation filter wheel axis failed to home Filter wheel not inserted correctly Filter wheel obstructed Filter not clipped in Gear teeth of filter wheel binding with gear teeth of the motor Remove filter wheel spin wheel by hand and reinsert 5003 5203 Emission filter whee...

Page 184: ...ing the syringe drive 5800 5801 Carrier x axis obstructed Carrier y axis obstructed Shipping screw still installed An object may be obstructing the carrier s path 5A00 5A01 Carrier x axis obstructed Carrier y axis obstructed Plate has hit something Plate cover not accounted for when creating plate dimension file Tip prime trough dislodged 5B00 Plate height violation Plate is inside chamber when it...

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