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Quality Control

7.1

Periodic Quality Control Tests ..................................................... 7-1

7.2

Fluorescence ................................................................................. 7-1

7.2.1

Sensitivity Test ................................................................................ 7-1

7.2.2

Linearity Test ................................................................................... 7-2

7.2.3

Precision Test.................................................................................. 7-3

7.2.4

Cross-Talk Test ............................................................................... 7-3

7.3

Glow Type Luminescence ............................................................ 7-4

7.3.1

Sensitivity Test ................................................................................ 7-4

7.3.2

Cross-Talk Test ............................................................................... 7-5

Cleaning & Maintenance

8.1

Introduction.................................................................................... 8-1

8.2

Liquid Spills ................................................................................... 8-1

8.3

Replacing the Fuse........................................................................ 8-2

8.4

Maintenance................................................................................... 8-3

8.4.1

Daily................................................................................................. 8-3

8.4.2

Weekly............................................................................................. 8-3

8.4.3

Every Six Months............................................................................. 8-3

8.4.4

Yearly Maintenance......................................................................... 8-3

8.5

Instrument Disinfection ................................................................ 8-4

8.5.1

Disinfection Solutions ...................................................................... 8-4

8.5.2

Disinfection Procedure .................................................................... 8-5

8.6

Disinfection Certificate ................................................................. 8-6

Application Note for DNA Quantification

9.1

Introduction.................................................................................... 9-1

9.1.1

Common techniques for the quantitation of DNA ............................ 9-1

9.2

Experimental Setup ....................................................................... 9-2

9.2.1

DNA extraction ................................................................................ 9-2

9.2.2

Assay Procedure ............................................................................. 9-2

9.3

Typical Results .............................................................................. 9-3

9.3.1

Detection techniques vs detection limits.......................................... 9-3

9.3.2

Sensitivities in comparison: ............................................................. 9-4

9.3.3

Results from Plasmid and Blood DNA Sample................................ 9-4

9.3.4

DNA Purity at 260/280 nm............................................................... 9-5

9.4

Conclusion ..................................................................................... 9-5

Acknowledgements ......................................................................... 9-5

Index

June 2002

Operating Manual for GENios, GENios FL, and GENios

Plus

No: I 112 904 Rev No: 1.1

Summary of Contents for GENios

Page 1: ...Operating Manual for GENios GENios FL and GENios Plus Document Part No I 112 904 June 2002 Document revision 1 1 Software level 4 4x ...

Page 2: ... 39 02 215 21 28 Fax 39 02 215 97 441 Japan Tecan Japan Co Ltd Meiji Seimei Fuchu Building 10F 1 40 Miyamachi Fuchu City Tokyo Japan Tel 81 42 334 88 55 Fax 81 42 334 04 01 USA Tecan US P O Box 13953 Research Triangle Park NC 27709 USA Tel 1 919 361 5200 Fax 1 919 361 5201 Spain Tecan Spain Sabino de Arana 32 E 08028 Barcelona Spain Tel 34 93 490 01 74 Fax 34 93 411 24 07 Germany Tecan Deutschland...

Page 3: ...3 0 6246 8933 FAX 0043 0 6246 72770 E Mail office austria tecan com Copyright Information The contents of this manual are the property of TECAN Austria Ges m b H and are not to be copied reproduced or transferred to another person or persons without our prior written permission Copyright TECAN Austria Ges m b H All rights reserved Printed in Austria About this Manual This manual describes the GENi...

Page 4: ... situation The following notices are Note Gives helpful information Caution Indicates a possibility of instrument damage or data loss if instructions are not followed WARNING INDICATES THE POSSIBILITY OF SEVERE PERSONAL INJURY LOSS OF LIFE OR EQUIPMENT DAMAGE IF THE INSTRUCTIONS ARE NOT FOLLOWED 4 Operating Manual for GENios GENios FL and GENios Plus No I 112 904 Rev No 1 1 June 2002 ...

Page 5: ...elevant information By doing this you are qualified to receive all updates of GENios software XFLUOR free of charge as they are released We would appreciate any comments or questions you may have regarding the performance of the instrument TECAN Your partner in microplate measurement and handling Registration Form GENios Serial Number Company University Department Institute User Name Address Stree...

Page 6: ... 2 Inserting the Filter Slides 4 3 4 3 How Do I Install the Custom Filter 4 4 4 3 1 Defining the Filter 4 6 5 Instrument Features 5 1 Specifications GENios 5 1 5 1 1 Fluorescence 5 3 5 1 2 Absorbance 5 4 5 1 3 Glow Type Luminescence 5 4 5 2 Specifications GENios FL 5 5 5 2 1 Fluorescence 5 7 5 3 Specifications GENios Plus 5 8 5 3 1 Fluorescence 5 10 5 3 2 Absorbance 5 11 5 3 3 Glow Type Luminescen...

Page 7: ... 3 8 5 Instrument Disinfection 8 4 8 5 1 Disinfection Solutions 8 4 8 5 2 Disinfection Procedure 8 5 8 6 Disinfection Certificate 8 6 9 Application Note for DNA Quantification 9 1 Introduction 9 1 9 1 1 Common techniques for the quantitation of DNA 9 1 9 2 Experimental Setup 9 2 9 2 1 DNA extraction 9 2 9 2 2 Assay Procedure 9 2 9 3 Typical Results 9 3 9 3 1 Detection techniques vs detection limit...

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Page 9: ...3 Observe all STOP and CAUTION statements in the manual 4 Never open the instrument while it is plugged into a power source 5 Never force a microplate or filter slide into the instrument 6 The GENios GENios FL and GENios Plus are intended for laboratory research use only Observe proper laboratory safety precautions such as wearing protective clothing and using approved laboratory safety procedures...

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Page 11: ...sorbance and glow type luminescence and can be used with 384 96 24 12 or 6 well microplates The GENios FL Reader offers fluorescence top measurement with 384 96 24 12 or 6 wells The GENios Plus Reader offers glow type luminescence absorbance and fluorescence with high sensitivity especially in the UV range and the capability of measuring fluorescence in 1536 well plates The filters are arranged in...

Page 12: ...r LED 2 emission filter 3 excitation filter 4 plate carrier Caution If the instructions given in this manual are not correctly performed the instrument may be damaged or the procedure may not be performed correctly and the safety of the instrument can not be guaranteed 2 2 Operating Manual for GENios GENios FL and GENios Plus No I 112 904 Rev No 1 1 June 2002 ...

Page 13: ... is flat level vibration free away from direct sunlight and free from dust solvents and acid vapors Allow at least 10cm distance between the back of the instrument and the wall or any other equipment Ensure that the main switch and the mains cable can be reached at all times and are in no way obstructed Place the carton in an upright position and open it Lift the instrument out of the carton and p...

Page 14: ...ng procedure 1 Ensure that the instrument is disconnected from the mains power supply 2 Open the plate compartment flap 3 Using the 2mm Allen key supplied remove the two screws holding the transport lock in place See diagram below 2 screws 4 Remove the transport lock Note The transport lock should be saved for further transportation purposes 3 2 Operating Manual for GENios GENios FL and GENios Plu...

Page 15: ...e voltage range is 100 120 220 240V If the voltage is not correct please contact your distributor Caution Do not use the instrument if the voltage setting is not correct If the instrument is switched ON with the incorrect voltage setting it will be damaged Ensure that the correct type and rating of fuse is fitted Fuse T 2A 250 slow blow WARNING RISK OF FIRE REPLACE ONLY WITH THE SAME TYPE AND RATI...

Page 16: ...ces must be proved and listed with regulations EN 60950 UL 1950 or CSA C22 2 No 950 for Data Processing Devices Switch the instrument ON using the switch in the back panel of the instrument Caution When installing or uninstalling the instrument ensure that the instrument and the computer are both switched off and disconnected from the mains before the RS 232 interface cable is connected or removed...

Page 17: ... Contact TECAN for filters other than those supplied on the standard filter slides 4 1 2 Absorbance Filters Bandpass filters which are commonly used in microplate readers for absorbance measurements usually have a bandwidth of 10 nm Therefore it is not recommended to use fluorescence filters for absorbance measurements because the bandwidth FWHM is usually larger than 10 nm This could cause a brig...

Page 18: ...EX and EM that contain standard filters are coded as A The absorbance filter slides that contain standard filters are coded as D You can order empty filter slides for ABS EX as B 126301 coded as requested EM as B 126102 coded as requested Without request it is coded as B The barcodes are labeled as follows A B C D E F G H 4 2 Operating Manual for GENios GENios FL and GENios Plus No I 112 904 Rev N...

Page 19: ...cated on the left and the emission filter on the right side of the instrument Ensure that the filter slides are only inserted into their respective compartments and that they do not get mixed up Depending on the measurements that are to be performed the appropriate filter slide containing the required wavelength filters must be inserted into the instrument The instrument can store the data for up ...

Page 20: ...art of a pencil to aid in removing the filter from the holder Do this with extreme care as the filters are easily damaged 4 For filters without aluminum housing place the filter securing ring into the filter slide on top of the filter 5 For filters with aluminum housing carefully tighten the securing screw to secure the filter 6 If there are unused openings remaining after filter insertion filter ...

Page 21: ...Figure A 1 Location of Securing Screw and Bar Code Label on the Filter Slide standard The EX ABS and EM filter slide can have the same coding The filter slide is now ready to be placed into the appropriate filter slide opening and be recognized by the software June 2002 Operating Manual for GENios GENios FL and GENios Plus No I 112 904 Rev No 1 1 4 5 ...

Page 22: ...en is displayed 3 Select Connect from the Instrument menu You are now connected to the GENios 4 Select Define Filter from the Setup menu 5 The following dialog box is displayed showing the filter values for the excitation and the emission filters The Define Filter Dialog Box You can choose the options from the right column of the dialog box for GENIOS 6 Enter the new wavelength for each new filter...

Page 23: ...ttes equipped with PTFE plug The cuvettes are positioned horizontally in the 4 designated areas The cuvette should be filled completely and closed tightly in order to exclude air from the measurement chamber and ensure reliablity of results Select the provided CUV pdf plate format from the plate definition file before the measurement is started The table below lists the technical specifications fo...

Page 24: ...fuse T 2A 250 slow blow Physical Outer dimensions Width 380 mm Depth 415 mm Height 170 mm Weight 18 8 kg Environmental Ambient temperature Operation 15 C 30 C 59 F 86 F Non operation 20 C 60 C 4 F 140 F Relative humidity Operation 90 non condensing Overvoltage category II Pollution degree 2 Usage Commercial Noise level 70 dBA Method of disposal Electronic waste infectious waste 5 2 Operating Manua...

Page 25: ...etting Values Measurement range Manual 1 255 0 60 000 RFU Optimal Automatic 0 60 000 RFU Extended Dynamic Range Automatic 0 6 000 000 RFU using XFLUOR c Band width for standard filters may have some tolerance 10 Needs to be adjusted for Time Resolved Measurements Lag Time is typically 40 µs No of Flashes Well Definition Characteristic Meas Time for 96 Wells 1 3 Plate carrier does not stop at the m...

Page 26: ... well plates Parameters Characteristics Sensitivity 0 4 10 6 DEA activity units well of molecular biology grade alkaline phosphatase 20 µl well using CDP Star Tropix chemiluminescent substrate mix with Emerald II enhancer 100 µl well in a Greiner 96 well white plate Use of the provide cover plate greatly reduces the cross talk 0 4 10 6 DEA activity units well correspond to 200 fg well of alkaline ...

Page 27: ...e Microplates to be Used Fluorescence see 5 2 1 Standard and time resolved top measurement 6 384 wells The instrument allows for kinetic measurements Reading may be restricted to one part of the microplate Up to four measurement parameter sets can be queued using XFLUOR June 2002 Operating Manual for GENios GENios FL and GENios Plus No I 112 904 Rev No 1 1 5 5 ...

Page 28: ...ight source High energy Xenon flash lamp Optics PMMA fibers and lenses Detector Side window low dark current photomultiplier tube Power supply Auto sensing 100 120V 220 240V 50 60 Hz Power consumption 200 VA Main fuse T 2A 250 slow blow Physical Outer dimensions Width 380 mm Depth 415 mm Height 170 mm Weight 18 8 kg Environmental Ambient temperature Operation 15 C 30 C 59 F 86 F Non operation 20 C...

Page 29: ...ement range Manual 1 255 0 60 000 RFU Optimal Automatic 0 60 000 RFU Extended Dynamic Range Automatic 0 6 000 000 RFU using XFLUOR c Band width for standard filters may have some tolerance 10 Needs to be adjusted for Time Resolved Measurements Lag Time is typically 40 µs No of Flashes Well Definition Characteristic Meas Time for 96 Wells 1 3 Plate carrier does not stop at the measurement point on ...

Page 30: ...tes equipped with PTFE plug The cuvettes are positioned horizontally in the 4 designated areas The cuvette should be filled completely and closed tightly in order to exclude air from the measurement chamber and ensure reliablity of results Select the provided CUV pdf plate format from the plate definition file before the measurement is started The table below lists the technical specifications for...

Page 31: ...e T 2A 250 slow blow Physical Outer dimensions Width 380 mm Depth 415 mm Height 170 mm Weight 18 8 kg Environmental Ambient temperature Operation 15 C 30 C 59 F 86 F Non operation 20 C 60 C 4 F 140 F Relative humidity Operation 90 non condensing Overvoltage category II Pollution degree 2 Usage Commercial Noise level 70 dBA Method of disposal Electronic waste infectious waste June 2002 Operating Ma...

Page 32: ...s Lag Time 0 2000 µs Gain setting Values Measurement range Manual 1 255 0 60 000 RFU Optimal Automatic 0 60 000 RFU Extended Dynamic Range Automatic 0 6 000 000 RFU using XFLUOR c Band width for standard filters may have some tolerance 10 Needs to be adjusted for Time Resolved Measurements Lag Time is typically 40 µs No of Flashes Well Definition Characteristic Meas Time for 96 Wells 1 3 Plate car...

Page 33: ...y to 96 well plates Luminescence can only be measured in 96 well plates Parameters Characteristics Sensitivity 0 4 10 6 DEA activity units well of molecular biology grade alkaline phosphatase 20 µl well using CDP Star Tropix chemiluminescent substrate mix with Emerald II enhancer 100 µl well in a Greiner 96 well white plate Use of the cover plate greatly reduces the cross talk 0 4 10 6 DEA activit...

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Page 35: ... either to the CCITT interface standard or to the EIA RS 232 C interface standard 6 2 Pin Designation The illustration below shows the pin assignment of the DB 9 connector fitted to the instrument Pin 5 Pin 1 Pin 6 Pin 9 All connected devices must be approved and listed as per EN 60950 UL 1950 or CSA C22 2 No 950 for Data Processing Devices June 2002 Operating Manual for GENios GENios FL and GENio...

Page 36: ...on 7 RTS Request To Send 8 CTS Clear To Send 9 No connection The connecting cable used to connect the instrument to the computer should be wired as given below INSTRUMENT COMPUTER TD connected to RD RD connected to TD GND connected to GND RTS connected to CTS DSR connected to DCD and DTR Use the computer handbook to find the correct pin connections 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7...

Page 37: ...puter 1 Start stop synchronous system asynchron 2 Start bit length 1 3 Stop bit length 1 4 Data format 8 databits 5 Parity bit none 6 Baud Rate 9600 6 5 Signal Levels The table below lists the voltage levels and their definitions VOLTAGE LEVEL DATA SIGNALS CONTROL SIGNALS 3 to 12 SPACE Logic 0 ON 3 to 12 MARK Logic 1 OFF 3 to 3 UNDEFINED RANGE UNDEFINED RANGE June 2002 Operating Manual for GENios ...

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Page 39: ...cceptance criteria to the laboratory s primary application Ideally these tests must be performed with the laboratory s own plates fluorophore buffers volumes and all the appropriate settings filters flashes delays top bottom reading and so on 7 2 Fluorescence 7 2 1 Sensitivity Test Use a black 96 well plate for example Greiner flat bottom black microplate Apply 200 µl of 1 nM Fluorescein in 0 01 M...

Page 40: ... µl and the molecular weight 376 g Mol The sensitivity is expected to be 1 5pg fluorescein per well 7 2 2 Linearity Test 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H B B B B B B B B Use a 96 well plate for example a Greiner flat bottom black microplate Prepare a 1 1 dilution series of fluorescein in 0 01 M NaOH solution each column will take a different concentration compare figure Highest concentra...

Page 41: ...th 1µM Fluorescein Fill the wells with buffer that are indicated with the letters N and B 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B N N N N N N N N N N N N N N N N Select extended dynamic range and perform a measurement with 10 flashes Compute the MVB Mean Value of Blanks using the wells indicated with the letter B and the SD...

Page 42: ...00 µl of chemiluminescent substrate CDP Star with Emerald II enhancer source Tropix per well and mix thoroughly Incubate at room temperature for 20 minutes Integrate 2 seconds per well using gain 100 Chemiluminescent signal will change very slowly over the following hour when using the above maximum enzyme concentration The procedure to achieve the sensitivity is as follows Plot the blanked values...

Page 43: ... B B Select maximum dynamic range and adjust gain to get a RLU Relative Luminescence Units reading greater than 106 Measurement should be performed 10 minutes after preparation but not later than 30 minutes Calculation method Compute MVB B from all wells except the nearest neighbor wells N and the well filled with alkaline phosphatase for example E5 Calculate the SD Standard Deviation of MVB Use f...

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Page 45: ... 8 2 Liquid Spills See 8 3 Replacing the Fuse See 8 4 Instrument Disinfection 8 2 Liquid Spills 1 Wipe up the spill immediately with absorbent material 2 Dispose of contaminated material appropriately 3 Clean the instrument surfaces with mild detergent 4 For biohazard spills clean with a 5 10 solution of bleach in deionized water 5 Wipe cleaned areas dry WARNING ENSURE THAT THE MICROPLATE IS REMOV...

Page 46: ...holder out On Off Switch 0 ON 1 OFF Fuse compartment Mains power socket Pull the fuse holder out and replace the defective fuse s with the spare fuse s Ensure that the fuse s has have the correct rating T 2 250V slow blow Caution Risk of Fire For continued protection replace only with the same type and rating of fuse Replace the fuse holder Reconnect the mains cable Turn the instrument on If the f...

Page 47: ...ary 8 4 2 Weekly Clean the cover and the transport with a mild detergent 8 4 3 Every Six Months Clean the filters using an optical cleaning solution Lens tissue recommended 8 4 4 Yearly Maintenance Check the belts tension between 50 an 60 Hz tension meter necessary Perform a FlouCheck test June 2002 Operating Manual for GENios GENios FL and GENios Plus No I 112 904 Rev No 1 1 8 3 ...

Page 48: ...y be held by the customs authorities 8 5 1 Disinfection Solutions If the laboratory has no specific disinfection procedure the following procedure should be used to disinfect the instrument The instrument should be disinfected using a solution such as Lysetol Manufacturer Schülke Mayr Ges m b H Aseptisol Manufacturer Bode Chemie Hamburg If neither of these solutions is available 70 ethanol should ...

Page 49: ... cotton wool that has been soaked in the disinfecting solution 4 Ensure that the same disinfection procedure is performed with the plate carrier 5 Repeat the disinfection procedure on any accessories which are also being moved or returned 6 After the disinfection procedure has been performed ensure that the disinfection certificate is completed See 8 5 for an example of the disinfection certificat...

Page 50: ... or disinfected to remove or inactivate any biological material that could be dangerous to the service personnel or that it has never been exposed to any hazardous biological material Name Firm Address Country Signature I declare that the instrument in this package has been decontaminated or disinfected to remove or inactivate any biological material that could be dangerous to the service personne...

Page 51: ...the absorbance method are the relative contribution of nucleotides and single stranded nucleic acids to the signal the interference caused by contaminants commonly found in nucleic acid preparations the inability to distinguish between DNA and RNA and the relative insensitivity of the assay an A260 of 0 1 corresponds to a 5 µg ml dsDNA solution referring to 1 cm pathlength for measurements in a cu...

Page 52: ...using the QIAprepR 96 Turbo BioRobot Kit from QIAGENR and the QIA_96 Turbo T Gemini script on a GENESIS RSP 150 with a Te VacS B from TECAN Genomic DNA Extraction Genomic DNA was extracted from human blood samples using a Genomic DNA Extraction Kit on a GENESIS RSP 150 with a Te MagS from TECAN 9 2 2 Assay Procedure The extracted DNA was quantified using UV absorbance at 260 nm and fluorescence me...

Page 53: ...ilution series of λ DNA from two different DNA samples Roche and Molecular Probes measured with absorbance at 260 nm Figure 2 Dilution series of λ DNA measured with PicoGreen fluorescence at 485 nm excitation and 535 nm emission June 2002 Operating Manual for GENios GENios FL and GENios Plus No I 112 904 Rev No 1 1 9 3 ...

Page 54: ...ot allow to quantify with absorbance 1 10 µl blood fresh EDTA 5 10 µl blood old EDTA 2 20 µl blood fresh EDTA 6 20 µl blood old EDTA 3 10 µl blood fresh Citrate 7 10 µl blood old Citrate 4 20 µl blood fresh Critrate 8 20 µl blood old Critrate Treatment 1 2 3 4 5 6 7 8 485 535 1 02 1 06 2 99 4 15 3 88 3 92 1 23 2 27 Table 1 Calculated DNA concentrations in µg ml stock solution of the same samples w...

Page 55: ...quantitation with absorbance This application example clearly demonstrates the advantage of having 2 detection techniques in one instrument Both techniques offer a variety of advantages depending on the method of extraction and amount of available DNA DNA quantitation by UV absorbance measurement is easy to perform inexpensive and allows a rapid check of DNA purity 260 280nm ratio although the ext...

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Page 57: ... 7 1 G GENIOS 2 2 Glow Type Luminescence 5 5 5 17 7 4 H Hardware Specifications 6 1 I Instrument Features 5 1 Installation 3 1 Power Requirements 3 3 Safety 1 1 Transport Lock 3 2 M Maintenance 8 1 Q Quality Control 7 1 T Test Cross Talk 7 3 7 5 Linearity 7 2 Precision 7 2 Sensitivity 7 1 7 4 June 2002 Operating Manual for GENios GENios FL and GENios Plus No I 112 904 Rev No 1 1 Index ...

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