PS12 manual
Contents
1. d Cru Norkstation Pamoen PamStation 12 User Manual LE E E PamStation12 Operator Manual Version 3 2009 PamStation 12 instrument meets the Essential Requirements of the EMC Directives 98 37 EC 73 23 EEC and 89 336 EEC for an EG statement type IIA No rights can be derived from this manual No part of this manual may be reproduced stored or transmitted by any means electronic mechanical photocopying or otherwise without the prior written permission of PamGene International B V 02009 PamGene International B V all rights reserved PamGene International B V Version 3 2009 86 Pamoen PamStation 12 User Manual LE E 6 Contents 1 Symbols used in this manual NEVER OCG UI OWN saei endi PLatuudi udin SUM nc MM xIaE Ra KIND E E I PEN FUE C 2 1 REMC SUS Cis orcs NRI RR 2 2 Description of the instrument ccccceeeeeeeeeeeeeeeeeeeesaeeeeeens 2 3 Principles of OPELAtION ccccseeceeeeeeeeeceeeeeeeseeeeeeeeeeeeeseeeeeeeas 3 PamsStation 12 System tnnntntnens 3 1 General iC NOS NE 3 2 Incubator wagon system ccceccceeseeeeeeeeeeaeeeeeeeeeaeaeeeeeeeaeaees 3 9 Dispensing and aspiration system sseessseesss 3 4 imaging SYSTEMI aces cei ases 6 iuh pass petu t buuEd od rius Ex d anaia iii 3 5 Other system parts cccccccccseseeeeeeeeeeseeeeeeeeeeeeseeeeeeeeeeeeeeaaas 3 5 1 SLE2. eeseeeeeeeeeeeereeeen Advanced protocol parameters ssssss Running man run button eeseeeeeeeeeeeeene 5 1 9 Editing an existing protocol seeesssssessss 5 1 10 Saving a PLOtOCOl cece ccecsseeeeeeeeeeeeeeeeeeeeeeeeeeeeseaeeeees 5 1 11 Loading a proloGOl ere ce erm lame Rm 5 2 Protocol UMN ETT Version 3 2009 ames PamStation 12 User Manual 6 2 aues m TT 39 ZEE ue dst 40 7 1 Status B ro a M RR E RE NRUET m 40 7 2 Error messages NR m 40 7 3 MFOUDISS MO OTIAG NIRE 41 7 3 1 EROF COGS riarena eap en ee ee eee eee eee eee 41 7 3 2 ziiiegaijelgm 42 BANSAL OIA ee 48 8 1 Safety and Electrical Compliance eeeeeeeesssssseeesseeeeeeeenenennn nennen 48 8 1 1 Safety Considerations E 48 8 1 2 Electa Compan E sssi 49 8 2 Recommended Environmental Conditions cccccccccssseeceeeceesseeeeeeeeseeeeesseeseeeessaaeees 49 8 2 1 SGT IC AN ec TANS gs E TO UE 49 8 2 2 Space SCI I II 50 8 3 LIST Ol standard ACCCSS ORC TT T TEE T 50 8 4 Installation of the PamStation 12 and PC 50 8 5 Installation of the barcode to the PC sese 51 8 6 Setting up the Evolve mail notification service ceccccccseeeseeseeee
3. Open the lid to place the disposable into the incubator by turning the big metal button at the front of the wagon AN The incubator can be hot Before placing the disposable into the incubator check that there is a fluid barrier located at the bottom of the incubator If not place a fluid barrier as described in chapter 4 4 Fluid barrier If there is a fluid barrier check its colour This should be light blue If the colour is white replace the fluid barrier Place the disposable in the incubator close the lid and turn the big button anti clockwise and press the load button The wagon will move back into the instrument and the assay will continue with the next step PamGene International B V Page 35 of 64 Version 3 2009 Evolve12 User Manual 5 2 3 Stopping a protocol A protocol can be stopped or aborted at any point during the experiment by clicking on the red stop button This should only be done when absolutely necessary The error handling screen will appear as shown below Figure 30 Error window Use the unload option to unload the disposable and or use the shutdown button to terminate the protocol The error handling screen is handled in more detail in chapter 7 Error handling PamGene International B V Page 36 of 64 Version 3 2009 Evolve12 User Manual eee Pamee 5 2 4 End of a protocol Unload disposable Before the end of an experiment the disposable must be removed from the
4. dry aspirating time in protocol too high Blockage of tubing Check tubing between aspiration head and wasted bottle for blockage Change tubing if a blockage is present Check that waste bottle is not too full If pressure was Check if tubing is connected properly between aspirating head and too low Missing waste bottle tubes Check tubing between aspiration head and waste bottle for holes Check if waste bottle is attached in the proper manner Call Support Note In most cases Evolve can be resumed and the protocol can be continued However if this error occurred during aspirating and one or more arrays were all ready aspirated there is a small chance that a broken membrane error will occur because there is no fluid left on the microarray If this happens a new protocol can be opened which comprises only of an aspiration step at the temperature of the previous protocol and can be run with the wet microarrays selected After this edit the current protocol and remove previous steps and with the aspirate step set load step at desired assay temperature and continue the assay PamGene International B V Page 42 of 64 Version 3 2009 Evolve12 User Manual E HW 2203 Message CCD get device list error Category Error Number 2203 Explanation CCD camera can t communicate with the computer solution Firewire cable Use the dx devicelist program under the kappa sdk directory to determine if all cables are
5. 80GB IDE Hard Drive 1024Mb Intel Pentium 17 inch Flat Panel screen Dell mouse two buttons 16x DVD ROM US European QWERTY keyboard Next Business Day support 3years Intel extreme graphics 2 Windows XP Intel Pro Evolve NB Alternative PCs require validation by PamGene PamGene International B V Page 55 of 64 Version 3 2009 Evolve12 User Manual a i Pamee oo 10 SUPPORT PamGene aims to provide total support to enable customers to fully realize the benefits of the PamGene technology and to maximize use of the PamStation 12 system The software components and data handling are essential interfaces to the system and are supported by a dedicated software support team through email telephone and on site visits if required Please contact support on 31 0 73 615 8900 Support amgene com PamGene International B V Nieuwstraat 30 5211NL Den Bosch The Netherlands Phone 31 73 615 80 80 General number Fax 31 73 615 80 81 PamGene International B V Page 56 of 64 Version 3 2009 Evolve12 User Manual Pa mee 11 RIGHTS AND RESTRICTIONS 11 1 Intellectual property rights The use of The PamChip microarrays is licensed among others under patent and patent applications issued to PamGene B V Any patents patent applications and other proprietary rights named in this manual are included for reference purposes only No licenses to use the processes or products covered by such patents
6. Dispense settings in Wash step ER Soe of Crates E A desde AMNES ISP Cycle Time mE a ps m Wash Apirate oe Cycle Freq 2 l min 1 10 24 Pump settings in Wash step Wash Boo om Duration 10 sec 1 I Dispense fluid 25 pl pere ALS Aspirate settings in Wash step Figure 20 Wash window PamGene International B V Page 23 of 64 Version 3 2009 Evolve12 User Manual Pamite ma 86 Wait Step The pausing of a protocol for a specified time in seconds minimum 1 sec is accomplished with the wait step Bea Wait Duration 10 sec 1 Figure 21 Wait window Priming step Prior to a dispensing or wash step it is recommended to run a priming step for the specific dispensing needles Fluid is dispensed in small tubes at the front corners of the incubator These tubes have to be cleaned emptied regularly pE Prime Fluid Name Fluid Figure 22 Prime window PamGene International B V Page 24 of 64 Version 3 2009 Evolve12 User Manual Pa we Mail Step The mail step can be used when the user needs to do something else then standing by the instrument waiting for every step when it is going to be finished The mail step sends a mail to the user mail address telling on which step the current protocol runs Send to Subject Evolve Evolve Figure 23 Mail window Adding steps Steps can be added to the protocol list by clicking on the preferred step holding the mouse but
7. 2009 Evolve12 User Manual 4 4 Fluid barrier If an array breaks the fluid will fall onto the fluid barrier and will be absorbed A fluid barrier has to be placed in the incubator before starting an experiment The fluid barrier will protect the underlying valves tubes and pumps An error message will be displayed when the system detects a broken array This array will no longer be used by the system Placement of a fluid barrier Before running an experiment a fluid barrier has to be inserted in the incubator This is normally done during the load step of a protocol when the wagon is moved outside the PamStation 12 To insert a fluid barrier move the incubator outside the instrument during a disposable load open the lid of the incubator by turning the big metal button at the front of the wagon clockwise and place the fluid barrier in the incubator as shown on the pictures below A PamChip can be used to press the fluid barrier deeper into the incubator Quality control of fluid barrier At the end of every experiment when the PamChip has been removed the fluid barrier has to be checked for a colour change If fluid has been in contact with the fluid barrier the colour of the fluid barrier will change from blue to white If this occurs the fluid barrier must to be replaced by a new one It is advised to check the fluid barrier also prior to the start of an experiment Removal of fluid barrier This can easily be done with t
8. PamStation 12 approximately 10 20 cm of space is needed for ventilation On the front of the instrument additional space is needed for opening of the door and experimental handling In addition space needs to be available for a PC and monitor 8 3 List of standard accessories Number of Article number a be 8 Cover glass already inserted in 30023 Eee NN Fire wire cable wire cable E cable Three power cables instrument monitor and computer type can vary from country to country Syringes Needles 8 4 Installation of the PamStation 12 and PC Before installing the instrument make sure that there are no abnormalities outside the instrument damage contamination etc Make sure that the place of installation is in accordance to PamGene guidelines A computer a monitor a mouse and keyboard are normally shipped with the instrument The computer and monitor are supplied with their own power cable Installation of these can be done in the normal manner according to guidelines of the computer manufacturer A standard power cable the same as for the computer and the monitor a firewire cable and a canbus cable are supplied for the instrument AN Make sure that the instrument is switched off when the power cable is attached The power cable can be plugged in at the back of the instrument on the right side just below the power switch PamGene International B V Page 50 of 64 Version 3 2009 Evolve12 User Manual TES P
9. SS E 3 5 2 Waste bottle ccccccecccceceececeececuccecucaecueaececaeaecaneeeass 3 5 3 Fluid barrier cccccececcecececccecececcccccececececacaececsceuauuecscecass 4 Preparation before running an Experiment 4 1 Starting up instrument and PC sseeeeeeeenree 4 2 wide ERH n 4 3 WV ASS OOS m E 4 4 PNAS i apes etal one alec svete ecards sncetie se ccentboes ede edi suele aene deune 5 Evolve12 software 0 cccecececncnencnccensneneneneeeeeeeeeeneneneceneceneeeeeeeeeenenenes 5 1 Evolve12 protocol CCItOr ccccccccsssseeeeeeseeeeceeeeeaeeeeeeesaaaeeeees omooogooo a a a a a a a a o 00 tia 5 2 1 Protocol runner screen cccceececcseeeeeceesseeeseeseeesseeeeees 5 2 2 Running a protocol Poem 5 2 3 Stopping a protocol esseeeeeneeeeeeem 5 2 4 EnG Of a PO FOLOCO MRMRTMME Rr 6 PamStation 12 maintainance and shut down 6 1 Maintenance ccccececececececcecucccacacaeccecaeauauuaeauauauaeaeaeaenenenss PamGene International B V Page 1 of 64 Starting up the protocol editor ssss Protocol editor screen eesseeseseseeeeeeernrnneee Description of protocol steps eeeesssssessse Protocol NSU e Positioning of steps sessesseseseeeeeeeennnene Editing of step values
10. imaging step prior to the read step Call Support Note Adapt the protocol and retest the sample PamGene International B V Page 47 of 64 Version 3 2009 Evolve12 User Manual Po 8 INSTALLATION The following information is supplied for guidance in selecting a location for the instrument 8 1 Safety and Electrical Compliance 8 1 1 Safety Considerations e The PamStation 12 weighs approximately 80kg and therefore must be placed on a level stable bench that is capable of supporting its weight e Protect from direct sun light e The back of the instrument should be at least 30cm from any walls to prevent overheating e There are high voltage circuits within the instrument associated with the light source The instrument case should only be opened and the light source changed by qualified engineers e here are moving parts within the instrument which may be damaged or cause injury if touched whilst in motion Do not touch them until they have stopped moving This particularly applies to the incubator which moves outside the instrument for loading e he incubator may be hot and should be handled with caution when outside the instrument e As the instrument will be used with biological materials or inflammable or toxic substances good laboratory practice for handling such materials should be observed e When the instrument is in use all casing panels and doors should be kept closed PamGene Interna
11. incubator This is normally done during the unload step Press the load button when requested and the incubator will move outside the instrument AN The wagon will move outside the instrument so make sure the area in front of the PamStation is empty The disposable can be removed by opening the lid of the incubator AN The incubator can be hot When removing the disposable check the fluid barriers for a colour change If any are white replace it as described in chapter 4 4 Fluid barrier N Close the lid before pressing the load button for the second time After pressing the load button for the second time the incubator will move inside again and red stop button will become a green run button PamGene International B V Page 37 of 64 Version 3 2009 Evolve12 User Manual a i Pamee e 5 6 PAMSTATION 12 MAINTAINANCE AND SHUT DOWN 6 1 Maintenance Syringes If syringes were used during the experiment then it is very important to clean the needles as soon as possible after usage to prevent clogging by crystallization of salts Cleaning can be done by emptying and filling the syringe first without the needle with water Thereafter attach the needle to the cylinder and empty through the needle Repeat this several times for better cleaning results If a needle is clogged an ultra sound bath can be used to facilitate cleaning Replace the syringes every 6 months Fluid barrier At the end of
12. is between 5 50 ul although 40 ul is recommended After dispensing manually or automatically the fluid is always pumped down Dispense Manual Fluid Hame fluid a Fluid Temperature ZB 45 BU Volume 25 pl 5 50 Manual Dispense Figure 15 Dispense window The manual dispense will bring the wagon outside of the instrument and allows the user to add sample to each of the arrays The manual dispense step can also be used to add reagents to the arrays or wash the arrays manually PamGene International B V Page 19 of 64 Version 3 2009 Evolve12 User Manual 686 E Pump step Using the pump step it is possible to incubate the disposable for a specified time and to control the pressure cycles There are three variables in this step e Number of cycles e Cycle time e Cycle frequency A cycle consists of pumping the liquid up through the array one time then down one time This is done by opening a valve for a very short time less then 1 sec closing the valve and then waiting for a given amount of time e g 15 cycles means pumping the fluid 15 times up then 15 times down through the array The cycle time is the time that a complete cycle will take The cycle frequency is the amount of cycles per minute The cycle time and the cycle frequency are linked If a kinetic read is required use the read step with the read by cycle option explained in the read step instead of this step cic Pump Number of C
13. is described in 5 1 11 5 1 10 Saving a protocol A protocol can be saved using the save or save as buttons located at the top of the screen in the centre im a Save button saves the protocol with the name and place already chosen mi Save as button saves the protocol and defines a new name and mn place 5 1 11 Loading a protocol Open the Evolve protocol editor as described in chapter 5 1 1 Starting up the protocol editor Double click on the New Protocol icon at the top middle of the screen QO The protocol will consist of a combination of the 9 steps shown on the left hand side of the protocol screen These pre programmed steps their functions and parameters involved are described in chapter 5 1 3 Description of protocol steps A protocol will consist of some but not necessarily all of the steps described The different steps can be integrated in the protocol by clicking holding the mouse button and dragging them to their right position If a step is not in the right position or the step has to be removed use then the position buttons that are located above the list of protocol steps as described in 5 1 5 Positioning of steps After a step is positioned correctly it can be edited to achieve the desired values oelect the step by clicking on it with the mouse An editing screen will appear on the right side of the screen After editing the step to the desired values press the up
14. on the amount of fluid and its viscosity The recommended value is around 10 seconds for 25 ul of water per array fis Manual Duration 10 sec 1 m Manual Aspirate Aspirate Figure 14 Aspiration window The instrument can be set at 20 C but cannot cool below ambient temperature The software allows the temperature to be set at 20 C however the minimum operating temperature during an experiment is ambient 3 PamGene International B V Page 18 of 64 Version 3 2009 Evolve12 User Manual Dispensing step The dispensing step adds fluids to the arrays manually or automatically The operator has several options in this step The first option is to dispense manually or automatically If the manual box is checked the wagon will move outside the instrument The operator can then manually add fluids The default option is to let the instrument dispense fluids by using up to four different syringes As described in section Dispensing and aspiration system there is room for up to four different syringes with different buffers Placement of the syringes is described in Syringes Syringe numbers 1 and 2 also called fluid 1 and 2 can only be operated at room temperature while numbers 3 and 4 can be heated If syringes 3 or 4 are selected the option to enter the preferred temperature opens up grey temperature field turns white There is no active cooling on any syringe The amount of liquid that can be added to an array
15. patent applications or proprietary rights are granted by or implied by PamGene International B V It is the responsibility of the user to determine whether a license is required and obtained 11 2 Trademarks PamStation 12 and PamChip are trademarks of PamGene BV registered in one or more countries 11 3 Disclaimer PamGene International B V reserves the rights to change its products and services at any time to incorporate technological developments This manual is subject to change without notice Although this manual has been carefully prepared with every precaution to ensure accuracy PamGene International B V can assume no liability for any errors or omissions or for any direct of indirect damages resulting from application of this information PamGene International B V Page 57 of 64 Version 3 2009 Evolve12 User Manual LE E Pamee LEE E Note pages PamGene International B V Page 58 of 64 Version 3 2009 Evolve12 User Manual LE E Pamee LEE E Note pages PamGene International B V Page 59 of 64 Version 3 2009 Evolve12 User Manual LE E Pamee LEE E Note pages PamGene International B V Page 60 of 64 Version 3 2009 Evolve12 User Manual LE E Pamee LEE E Note pages PamGene International B V Page 61 of 64 Version 3 2009 Customer Support Pamgene International B V Nieuwstraat 30 5211 NL s Hertogenbosch The Netherlands 31 73 615 80 80 general 31 3 615 89
16. 00 customer support 31 73 615 80 81 support pamgene com u 2009 PamGene International B V all rights reserved
17. Don t apply vertical pressure on the opened door Behind the door at the front four slots are visible labelled 1 to 4 Slots 3 and 4 can be heated between 45 and 80 degrees There is no active cooling Turn the switch above the slot 90 degrees clockwise to open it Make sure that the flat side of the syringe is at the instrument panel and between the two metal holders of the slot Turn the switch to it original position the slot will close and the syringe will be held in place PamGene International B V Page 11 of 64 Version 3 2009 Evolve12 User Manual AN Position of waste fluid tubing The waste tubing should not be blocked by a syringe which is positioned in position 3 or 4 but should move in front of the white block as indicated in the figure below Figure 9 Inside door of the PamStation 12 and placement of a syringe 4 3 Waste bottle Before starting an assay check that the waste bottle is attached in the proper way to the instrument screwed to the red cap and check that the bottle is empty The bottle needs to be positioned straight in the dedicated holder at the right hand side of the instrument If needed empty the waste bottle by unscrewing the glass part of the bottle Dispose of the contents in the appropriate way There is no sensor to warn of a full waste bottle If a bottle is full aspiration AN errors could occur and it can damage the aspiration pump PamGene International B V Page 12 of 64 Version 3
18. LILI BERR RRR RRR RRR RRR Re MELCTI EARAMETER MICROARRAY ANALYZER Step List Logs Ii Load Dispasable 30 C 7 Selected Image Contrast i Brightness em PERE EERE ERE REESE REREEHEEEREREEREEREERERHEREHREEREERHEREEERENE r Show last BURR Auto power off amp L oogde P 4 Temperature and cycle COOP Initialize 3 Load initialize and run Figure 28 Protocol runner window Click on the initialize button The computer will now connect itself to the instrument Make sure that the instrument is connected and turned on A large green run button will pop up Click on the green RUN button to activate and start the protocol This button will now change into a red stop button PamGene International B V Page 33 of 64 Version 3 2009 Evolve12 User Manual Pam While this red button can be used to stop a protocol at any time it should only be used when absolutely necessary The next screen which appears is Array 1 Aray 2 Array 3 Array 4 1 41 lt 1 4 41 I 4 4 I I 4 Article number Array layout pe tine E m Sample annotation Data protocol Figure 29 Run entry window A dialog window appear where the following requires to be filled in Barcode This will automatically be filed in if you use the barcode reader and point to the barcode on the PamChip otherwise you can manually enter the 9 di
19. Runner C instrument protocols Example PS12Protocol HERE Evolve 1 MULTI PARAMETER MICROARRAR ANALYZER Step List Logs M VR ecent Documents Desktop My Documents My Computer My Network File name Example PS1 2Protocal Places Files of type Protocols P S12Protocal Cancel me Figure 27 Open protocol runner window Select your protocol file Click on open You have now successfully loaded your protocol into the protocol runner Protocol list Located on the left side of the screen in the complete list of the protocol steps in this protocol To view a step more completely leave the mouse pointer for a few seconds and the information will automatically pop up PamGene International B V Page 31 of 64 Version 3 2009 Evolve12 User Manual Load initialize and run buttons At the bottom left of the screen under the protocol list are two buttons the load button that has to be pressed to move the wagon outside the instrument will ask the operator when this is needed and the initialize button to make contact between the software and the instrument After pressing the initialize button the green run button will appear This button has to be pressed to start running the protocol chapter 5 2 2 figure 28 point 3 If the green run button is pressed it will change to a red stop button This red button should be used if a run suddenly needs to be aborted Shoul
20. The excitation light required for detection is provided by three LEDs The life of the light source is in excess of 60 000 hours The light is guided to a position above one array of the PamChip through an angled mirror which also allows reading by a 12 bit CCD camera focused on the array The light of each LED is guided through a different filter The LED s can detect fluorescein Cy3 and Cy5 dyes Table 1 Overview of installed filter characteristics Designation Dye Excitation Emission wavelength wavelength Filter 1 FITC 460 490 915 550 Filter 2 530 560 575 645 Filter 3 590 650 665 735 3 5 Other system parts 3 5 1 Syringes If an automated dispensing step or an automated wash step is part of the protocol a syringe filled with the appropriate buffer has to be placed inside the instrument at the position mentioned in the Evolve protocol Four syringes are supplied with the instrument and more syringes and needles can be acquired at PamGene 3 5 2 Waste bottle The waste bottle can be found outside the instrument at the right hand side The waste bottle is connected to the instrument with two tubes a small one going to the aspiration needle and a bigger one to the aspiration pump The waste bottle must be emptied at the end of the day PamGene International B V Page 8 of 64 Version 3 2009 Evolve12 User Manual oon 4 PN P amier 3 5 3 Fluid barrier The fluid barrier comprises of three different parts a ru
21. a syringe filled with the appropriate buffer has to be placed at the position described in the Evolve protocol inside the instrument Preparation of the syringes A syringe comprise of three parts a needle a glass casing and a metal plunger Assembly is done by screwing the needle on top on the glass casing and inserting the plunger into the casing AN Caution is needed when handling the needle Fluids can be placed into the syringe by unscrewing the needle from the glass casing Insert the casing into a container with the desired fluid and pull on the plunger Wipe the outside of the casing and screw back the needle Make sure no air bubbles are present in the syringe otherwise this will cause problems with dispensing Cleaning of Syringes If syringes were used during the experiment then it is very important to clean the needles as soon as possible after usage to prevent clogging by crystallization of salts Cleaning can be done by emptying the syringe without the needle and filling it with distilled water Assemble the needle to the cylinder and empty through the needle Repeat this several times for better cleaning results If a needle is clogged an ultra sound bath can be used to facilitate cleaning of the needle It is recommended to replace the needles every 6 months Placement of syringes in the PamStation 12 The PamStation 12 can hold 4 different syringes To place a syringe in the PamStation 12 open the door AN
22. ameer N Make sure that the PC and the instrument are switched off when the firewire cable and the canbus cable are attached The firewire cable and the canbus cable can be attached at the appropriate places at the left hand side at the back of the instrument and at the back of the computer The locations of these cables are labelled with their names at the back of the instrument and on the back of the computer Figure 33 left Placement of canbus and firewire cable at back of instrument right Placement of canbus and firewire cable at back of computer 8 5 Installation of the barcode to the PC e Download the vy USBCOMDriver zip e Uncompress the folder e Connect barcode device to a free USB slot e Barcode device should beep PamGene International B V Page 51 of 64 Version 3 2009 Evolve12 User Manual BM e Place Barcode into the USB COM Mode by scanning the following barcode found on page 26 of the barcode user manual E URA e New hardware will be detected e Wait for the Found New Hardware Wizard to appear e Ahardware wizard window will appear Select No not this time e Second dialog windows appears Select Install from a list or specific location and click next e Click on Browse e Navigate to the driver location the folder and click Open e Click OK e Click Next e You will be notified that this driver is certified by micr
23. ations Filter1 2 Fluorescein Filter 2 Cy3 Filter 3 Cy5 C mount Kappa Camera CCD size 7 inch Incubator Properties Temperature range Ambient 3 C 80 C minimum 20 C Temperature accuracy lt 0 5 C from set value Temperature uniformity across lt 0 5 C SD across the PamChip disposable disposable Heating rate Maximum 5 C min from 30 C to 80 C Cooling rate Maximum 5 C min from 80 C to 30 C Washing Module lt 5 deviation at 10 ul array 596 at 50 ul array Dispense reproducibility 596 CV over 10 ul 50 ul range Thermal regulation Up to 80 C accuracy 5 C no active cooling possible Wash boitle 100 ml glass Waste Liquid Disposal As according to local rules PamGene International B V Page 54 of 64 Version 3 2009 TT Pamee seas 9 2 Power Supply Requirements Voltage Frequency Maximum Power Average Power requirement Evolve12 User Manual 110V 115V 50 60 Hz As is standard a surge protected constant power supply should be used Up to three outlets may be required for the instrument and associated computer equipment PC Information Hardware Dimensions Weight Manufacturer and Model Operating System Hard Drive Memory Capacity Processor Type Also Included Support Graphics Card Software Networking Software Network Card Instrument Control Software 414mm Height Width 185mm Depth 439mm 12 34kg Dell Optiplex GX520 Windows XP
24. bber casing an absorption powder and a lid that is coloured with a colouring agent The fluid barrier is placed in the incubator and protects the underlying valves tubes and pumps If an array breaks the fluid will fall onto the fluid barrier and will be absorbed and an error message will be displayed If this happens the top of the fluid barrier will change colour from light blue to white If this happens the fluid barrier should be replaced There are five fluid barriers supplied with the instrument More can be ordered from PamGene Figure 6 Fluid barrier Figure 7 Fluid barrier in incubator PamGene International B V Page 9 of 64 Version 3 2009 Evolve12 User Manual Pon 4 PREPARATION BEFORE RUNNING AN EXPERIMENT Make sure the instrument and the PC are connected properly as described in chapter 8 4 Installation of the PamStation 12 and PC 4 1 Starting up instrument and PC Turn on the instrument by switching on the power supply using the switch at the back left hand side of the instrument above the power cord Figure 8 Power switch at left side of the instrument Turn on the PC and the Monitor Log into the system using the standard Microsoft log in prompt Log in as pamstation Password Is pamgene PamGene International B V Page 10 of 64 Version 3 2009 Evolve12 User Manual eee Pamee 86 4 2 Syringes If an automated dispense step or an automated wash step is part of the protocol
25. ciple The PamStation 12 is an integrated processor for up to 3 PamChip 4 arrays It consists of several modules linked by a transport system which delivers the chip to each module as required This is achieved by the use of a fast stepper motor Typically wagon movement from one position to the next is carried out in less than 10 seconds The instrument is capable of processing 1 2 or 3 chips 4 8 or 12 microarrays respectively at a time Figure 2 PamStation 12 The modules within the instrument are Incubator Wagon Dispensing syringe and aspiration needle s Imager 3 2 Incubator wagon system The incubator also referred to as the wagon is the heart of the instrument It carries the chips between the loading incubation washing and reading positions It also incubates the chip and facilitates the pressure pumping action during hybridization and washing The incubator comprises a_ heating m block a lid three cover glasses and Figure 3 Incubator in load position three fluid barriers The incubator is capable of operating at a temperature range of 20 85 C The instrument can be set at 20 C but cannot cool below ambient temperature The software allows the temperature to be set at 20 C however the minimum operating temperature during an experiment is ambient 3 PamGene International B V Page 6 of 64 Version 3 2009 Evolve12 User Manual AN The incubator may be hot when it is outsid
26. connected Check that the firewire cable is connected to the computer in the proper socket and to the instrument Check the can card cable Restart computer and instrument Call Support Note If this error is given during a protocol and the problem was a loose firewire cable there is a small possibility that the protocol can be resumed Otherwise the computer and instrument will have to be restarted and the protocol run again E HW 2204 Message CCD get device count error Category Error Number 2204 Explanation CCD camera can t communicate with the computer oolution Firewire cable Use the dx devicelist program under the kappa sdk directory to determine if all cables are connected Check that the firewire cable is connected to the computer in the proper socket and to the instrument Check the can card cable Restart computer and instrument Call Support Note If this error is given during a protocol and the problem was a loose firewire cable there is a small possibility that the protocol can be resumed Otherwise the computer and instrument will have to be restarted and the protocol run again PamGene International B V Page 43 of 64 Version 3 2009 Evolve12 User Manual E HW 2400 Message Sound error in combination with a wait time error Category Error Number 2400 Explanation The syringe used for automated dispensing is completely empty oolution Syringe empty Fill syringe with the appropria
27. d it be pressed an error handling screen will pop up See chapter 7 Error handling Assay information At the bottom of the screen in the centre the actual incubator temperature is shown This does not always reflect the temperature of your sample which remains at the one programmed into your temperature or load step The amount of pumping cycles completed is also registered here Pumping cycles carried out during a wash step are not counted chapter 5 2 2 figure 28 point 4 Images Images taken during a protocol are grouped according to the number of cycles being pumped This image list is shown in the middle of the protocol screen When clicking on a cycle number all the images with that number of pumping cycles will be shown as thumbnails It is possible to select one thumbnail to get a bigger picture on the screen chapter 5 2 2 figure 28 point 6 and 7 PamGene International B V Page 32 of 64 Version 3 2009 Evolve12 User Manual k Pame 5 2 2 Running a protocol After opening the protocol runner and loading the desired protocol see chapter 5 2 1 Protocol runner screen the following screen will be shown The name of the protocol will appear at the top of the screen The steps of your protocol will appear at the left hand side of the screen 2 Protocol steps 5 Cycle 1 Opening Saving a protocol 6 Image from selected cycle Protocol Runner PRISES See ew Le TT LILII
28. date button located at the bottom right of the screen If the protocol is finished it can be saved by using the save or the save as buttons that are located at the top middle of the screen PamGene International B V Page 29 of 64 Version 3 2009 Pamee 5 2 Evolve12 User Manual Protocol runner Starting up the protocol runner Open Evolve12 protocol runner by double clicking on the Evolve12 symbol In ZIIEEZEII Runnera It is also possible to open the protocol runner from the evolve protocol runner screen by pressing the running man run button located next to the save buttons at the top middle of the screen See chapter 5 1 8 Running man run button 5 2 1 Protocol runner screen 2 Protocol steps 5 Image list 1 Opening Saving a protocol 6 Image from selected cycle Protocol Runner Seles Seer eee LEELEE CELLET TP PP Be NN 1173 S O OS zd hh CJ ane BEHBRERN oe 7 Selected Image UL b COCO PEEP ron TE x 5 L Show lest Brightness E 4 z s 3 Load initialize and run Figure 26 Protocol runner window PamGene International B V Page 30 of 64 Version 3 2009 Evolve12 User Manual P amier Open protocol You can now select a protocol to run Click on the Open icon found at the top of the screen The following screen will appear lt Protocol
29. e has to be taken every number cycles With the read step it is not necessary to program in a pump step as one is already automatically included PamGene International B V Page 21 of 64 Version 3 2009 Evolve12 User Manual Ba Read Kinetic f Unce Image will be take after MEM Filter Name Exposure Ti given cycles vis ES niter 200 Pumping E nw Number of Cycles 1 Cycle Time 20 sec T Cycle Free 3 l min 1 10 5 Figure 18 Read kinetic window The imaging system is optimised for reading when the arrays contain fluid Do not AN perform a read step after an aspiration or washing step since the array will not contain sufficient fluid for optimal imaging Temperature step This step sets the temperature of the assay between 20 80 C B Temperature 30 1C 20 80 Temperature Figure 19 Temperature window PamGene International B V Page 22 of 64 Version 3 2009 Evolve12 User Manual 686 Wash step The wash step consists of a dispensing step a pumping step and an aspiration step Information about these steps is given in the appropriate chapters earlier in this manual The cycle counter in the protocol runner does not count pumping being done in the wash step aj Wash M Dispense fluidi ae Manual mtr i mM fi Wash Aspirate l sec Vid Name Fluid Temperature 45 60 Volume 5 H 5 54 Wash Fluid 1 z Temperature control
30. e is a maximum temperature for unloading the wagon 68 degrees Solution No hardware adjustments Note Edit the protocol to prevent unloading of the wagon Then run that protocol open new protocol E HW 3301 Message Wagon cover not closed Category Error Number 3301 Explanation The cover of the wagon was not closed properly Solution Wagon cover Close the wagon cover Call Support Note Use the resume option The protocol should resume normally E HW 4000 4100 Message Can errors Category Error Numbers E HW 4000 4100 Explanation There is a problem with the can card in the computer or with the communication between the can card and the instrument Solution Cable problem Check that all cables between computer and instrument are connected properly Turn off computer and instrument and turn on again Call Support Note This error will normally only occur before running a protocol Run the protocol again PamGene International B V Page 46 of 64 Version 3 2009 Evolve12 User Manual P ameer No error number Message Pictures are out of focus Category Error Number No error number Explanation The cover of the wagon was not closed properly Solution Liquid was The imaging system is optimized for reading when the arrays contain removed from fluid Do not perform a read step after an aspiration or washing step array in the since the array will not contain sufficient fluid for optimal
31. e the instrument A cover glass covers each PamChip array during hybridization or incubation to prevent evaporation of fluid The lid of the incubator heats these cover glasses to prevent condensation The cover glasses can be taken out easily to facilitate cleaning The sample is made to flow back and forth through the array by changing the pressure Pressure sensors inside the incubator detect broken arrays A consumable fluid barrier in the incubator protects the valves tubing and pumps from liquids These fluid barriers comprise an absorbent material with a blue colouring agent A colour change of blue to white will be noticeable if the fluid barrier comes into contact with fluids 3 3 Dispensing and aspiration system The dispensing unit is located behind the door at the front of the instrument It consists of four motor driven syringes The needles of two of these syringes numbers 3 and 4 may be heated The four needles are positioned over the active array All four syringes are capable of dispensing into all four arrays of the PamChip Liquid level is detected by a sensor which detects the top flange of the syringe as it descends Each syringe can hold up to 5 ml At the rear of the compartment is an aspirating needle that removes waste fluid into a 100 ml glass bottle Figure 4 The wash head unit PamGene International B V Page 7 of 64 Version 3 2009 Evolve12 User Manual eee Pamee eens 3 4 Imaging system
32. eeeeeeaaeeeeeeeeeeessneeeeees 52 9 PamStation 12 Specifications sscsesesesssesescsssesesssssssescesesesesssesseceseveverseseeseesevevereesnenens 54 9 1 Instrument specifications NR Rc n 54 9 2 xi S ace da are Ue g emcee eee TE 55 10 SUPPO aaa san sete eee 56 11 allein S and TO SUA ONS eem 57 11 1 Intellectual property rights ccc ccccceecccccsseeeceeseeeceeececseucesseaeeecsaueeeessseeessseeessageees 57 e WAC Sy aan ET qtu nemi uM Ses Mice uode qq inc Een DE eT eUED CC NmUEDE 57 13 DRGAIME e 57 PamGene International B V Page 2 of 64 Version 3 2009 ames PamStation 12 User Manual 1 SYMBOLS USED IN THIS MANUAL oymbols are ANSI IEC approved American National Standard Institute and International Electro technical Commission International Standardization Organization Read this carefully Caution Risk of personal injury to the user or a safety hazard Caution Lift carefully Heavy item could cause injury Caution Hot surface Caution High Voltage Pap P Protect from direct sun light PamGene International B V Page 3 of 64 Version 3 2009 bet Pame PamStation 12 User Manual 2 INTRODUCTION 2 1 Intended use The PamStation 12 instrument is solely intended for the processing of up to 3 x PamChip 4 microarrays and is intended for research purposes only 2 2 Description of the instrument T
33. every experiment when the disposable is removed the fluid barriers have to be checked for a colour change If any fluid has come into contact with a fluid barrier the colour of the fluid barrier will change from blue to white If this occurs the fluid barrier has to be replaced by a new one After every experiment the fluid barrier has to be inspected for a change in colour Replacing the fluid barrier can easily be done by using a set of pliers and gently lifting the old fluid barrier out of the incubator The inside of the fluid barrier consists of an absorbent powder which can be AN spilled if the fluid barrier is not gently removed The new fluid barrier can be placed into the incubator as described in chapter 4 4 Fluid barrier The fluid barrier should be changed once a month for optimal effect Cover glass After every experiment the cover glass has to be removed from its position in the wagon and inspected for dirt When the wagon is in the unload position pull the cover glass gently from the wagon to remove it Clean it with water and a piece of optical quality cloth Replace the cover glass with the right side up This side contains the word top PamGene International B V Page 38 of 64 Version 3 2009 Evolve12 User Manual Pamen 6 2 Shutdown After an experiment has finished and no additional experiments are planned press the shutdown button as indicated with the black arrow in the picture below Switc
34. git lot number of the PamChip Arrays The user can select which arrays will be employed in the run Article number The user requires to select which article number associated with the PamChip Array layout The array layout will be automatically selected based on the article number selected However the user may override this article array layout Sample Annotation The user needs to create a sample annotation file will be used in the run It will be automatically filled in otherwise can be overridden by the user PamGene International B V Page 34 of 64 Version 3 2009 Evolve12 User Manual Ba Run data analysis after the run The user can select whether to automatically run data analysis after a run Copy data to the network The user can select whether to copy the results to a network drive The OK button will only activate if you have filled in Barcode Article number and clicked on the Please annotate samples Now the protocol has begun and will move to the first step The protocol will always start with a load step During the load step the instrument will ask the operator to press the load button Please press the Load button The load button is situated at the bottom left of your screen After pressing the load button the wagon will move outside the instrument 1 The wagon will move outside the instrument so make sure the area in front of the PamStation is empty
35. h off the power button at the back right hand side of the instrument The computer and monitor can be turned off in the normal way aD Protocol Runner MULI Pea Shutdown button Figure 31 Shutdown window PamGene International B V Page 39 of 64 Version 3 2009 Evolve12 User Manual 7 ERROR HANDLING 7 1 Status Lights On the PamStation 12 there two lights A red light and a green light These lights indicate the status of related to the instrument control software called Evolve Green Red Off Off instrument control software not active Green Red On Off instrument control software is ready to run a protocol Green Red Off On instrument control software is currently running a protocol Green Red On On Not possible 7 2 Error messages When an error occurs the error screen will appear as shown below Figure 32 Error message window PamGene International B V Page 40 of 64 Version 3 2009 LEE 7 3 7 3 1 Evolve12 User Manual There are five different options available zi Quit the current protocol The disposable will stay inside the B instrument Resumes the current protocol Caution the step will start completely ey afresh So if the protocol is aborted during aspiration of the fourth array the other arrays will be aspirated again pen Open an existing protocol You can also create a new protocol or edit ii an existing one in the protocol editor save it and open it with this option Unl
36. he PamStation 12 is an integrated microfluidic workstation consisting of an incubator dispensing and aspiration needle s and an imaging module It is designed and optimized for PamGene s flow through microarray technology The PamStation 12 is specifically intended for the processing of up to 3 x PamChip 4 microarrays Both the instrument and the PamChip microarrays are products of PamGene International BV Figure 1 PamChip 4 microarray PamGene International B V Page 4 of 64 Version 3 2009 686 Pamoen PamStation 12 User Manual LE E 6 2 3 Principles of operation In the PamStation 12 experiments are carried out in PamChip 4 microarrays The surface of these microarrays is composed of a substrate consisting of aluminium oxide This material is approximately 60 um thick and has a porous structure with long branched interconnected capillaries with a diameter of about 200 nm The sample such as mRNA DNA or proteins are labelled with fluorescent dyes In case of measuring kinases a fluorescently labelled antibody to detect the phosphorylated peptides on the arrays is added to the sample The sample is then dispensed onto the PamChip and the chip placed in the PamStation 12 Once inside the instrument the sample is hybridized incubated at a pre determined temperature During hybridization incubation the sample is pumped back and forth through the substrate to maximize binding kinetics and minimize analysis time On tradi
37. he aid of tweezers Figure 10 Fluid barrier in incubator PamGene International B V Page 13 of 64 Version 3 2009 Evolve12 User Manual 686 Deere 5 EVOLVE12 SOFTWARE The PamStation 12 uses Evolve12 software for the creation and running of protocols Evolve comprises of two different parts Evolve12 editor for creating a protocol and Evolve12 runner for the running of a protocol Table 2 Evolve software parts Creating a protocol Evolve12 Protocol Editor Running of Experiment Evolve12 Protocol Runner Chapter 5 1 will describe the usage of the editor and the making of a protocol while chapter 5 2 will describe the protocol runner 5 1 Evolve12 protocol editor This section of the manual is to help scientists and engineers in designing and making protocols 5 1 1 Starting up the protocol editor Open evolve by double clicking on the Evolve12 Protocol Editor icon Prakacal Editar PamGene International B V Page 14 of 64 Version 3 2009 Evolve12 User Manual Pame 5 1 2 Protocol editor screen The protocol editor screen comprises of 6 different areas as shown in figure 11 Protocol Editor CEE E EEE EEEE EEE a M ML TTT amp ammumnamuuEHERRARRRRARRARRERRARARRRRRARRRRREEEEZEEEERERRRREER Ta i Load Disposable 30 3C i Aspirate Dispense fy Unload Disposable 3 4 ra Mail Prime 3 Adding a step B Un 4 Protocol list 3 7 E 5 Positioning deleting steps i 35 3 1 Opening Sav
38. ing a protocol mee Ne cz 2 Protocol steps i agh 6 Editing of step values 3 Wash Figure 11 Evolve Protocol Editor window PamGene International B V Page 15 of 64 Version 3 2009 Evolve12 User Manual Open saving a protocol Located at the top middle of the protocol editor screen see figure 12 are five buttons With these buttons it is possible to start a completely new protocol open an existing one save the current protocol and save the current protocol with a different name save as After saving a protocol and clicking anywhere on the screen a fifth button will appear This so called running man run button will provide easy access to the protocol runner n Open a completely new protocol Open an existing protocol a Save current protocol LI mi Save current protocol as e Fa Running man run button provides access to the protocol runner Figure 12 Open saving protocol Protocol steps On the left side of the protocol editor screen are 9 symbols These symbols represent the different steps of the protocol These steps can be added to form the desired protocol The steps are described in a short reference table and in more depth in chapter 5 1 3 Description of protocol steps 5 1 3 Description of protocol steps Short descriptions of the seven protocol steps are given in the reference table below Also a recommended value for each step is given for optimal results of the assay Plea
39. ing of step values If a step is added to the protocol list the options variables of that step will be shown in the editing window on the right side of the protocol screen By clicking on an option value this can be changed Every step has different options and variables these are described in chapter 5 1 3 Description of steps After changing an option or variable click on the update button located on the bottom right side of the screen 5 1 7 Advanced protocol parameters Use the Tools button to select the protocol to be edited This button is located at the top of the screen in the centre iT In the advanced protocol parameters it is possible to set some of the instrument operating parameters It is advised not to change these setting unless suggested to do so by PamGene personnel The advanced protocol settings are saved with the protocol Protocol Editor MEE i D i UD we dT SR RRE ee Advanced Aratecal Parameters a Pa rPressureUnderlimit 03 barg Aspirate Dispense rPressureUpperLimit 7 0e 002 barg uAspirateTubeCleanDelay wm msec uAspPressBuildupDelay w msec rPrimeAmount 70 H E rRetractAmount A T Puis uRetractDelay sm msec rDefaultOverPressure B barg B rDefaultUnderPressure 93 barg mex rinitWagonTemperature 3 aC rSafeUnloadTemperature m x UFluidFlowDelay 20 msec uMaximumWaitingTime 100000 msec uPressure SettlingDelay sm msec uValveOpenTime W msec rBrokenMembraneP
40. ith the fluid barrier oolution There are multiple causes for this error Broken Use the unload option to inspect the array that gives an error microarray No fluids Use the unload option to inspect the array that gives an error Fluid barrier Use the unload option to move wagon outside Open wagon lid Remove disposable Inspect fluid barrier colour change from blue to white Change fluid barrier Call Support Note Broken microarray Edit current protocol remove all steps except load before the failed step Edit current protocol step if needed Set load step temperature at desired assay temperature Open the new protocol and run the new protocol without the broken microarray No fluids on the microarray Use the unload step to move wagon outside Add fluids manually Resume the protocol Edit the protocol for next the assay Fluid barrier Change the fluid barrier Edit protocol if needed Run edit protocol or resume current protocol E HW 2504 Message Disposable s not present Category Error Number 2504 Explanation No all disposables specified at the beginning of the run are present at the correct positions oolution Disposable s Use the unload option to change the disposable position not present PamGene International B V Page 45 of 64 Version 3 2009 Evolve12 User Manual LEE P amier E HW 3201 Message Wagon temperature above safety limit Category Error Number 3301 Explanation Ther
41. l see section 8 and consult ICT used to inform the user to setup the system of the processing status of the instrument PamGene International B V Page 17 of 64 Version 3 2009 Evolve12 User Manual Pe Loading and unloading disposable Every protocol starts with a load disposable load PamChip step This step initialises the instrument to a set pressure and predetermined variable temperature The temperature variable is between 20 and 85 degrees Celsius During this step the instrument will ask the operator to press the load button located on the left bottom of the screen After pressing this button the wagon will move outside the instrument and the operator can place the PamChip into the incubator The green button turns into a red stop button Every protocol ends with an Unload disposable step During this step it is possible and recommended to remove the disposable from the instrument METNE X ae Unload Disposable a Temperature 30 20 85 Load Disposable Figure 13 Load and Unload window Aspiration step The aspiration step removes liquids from the arrays manually or by use of the aspiration needle By selecting the manual check box the wagon will move outside and the operator can remove the fluid manually The default option is to remove fluids by use of the aspiration needle The aspiration time is the time it takes the aspiration needle removes fluids from the array The time needed depends
42. oad Unload the disposable and shutdown the experiment Shutdown Shutdown the instrument The disposable will stay inside the instrument For the handling of the errors see chapter 7 3 troubleshooting Troubleshooting Error codes Here is a list of error message which can be generated by Evolve12 Each error has a oolution suggestion Check chapter 7 3 2 for solutions related to the errors in this section Aspiration errors E HW 2101 Aspirate For Solution see E HW 2101 pressure not ok CCD Camera errors E HW 2203 CCD get device list error E HW 2204 CCD get device count error Dispense head errors E HW 2401 Syringe almost empty E HW 2402 Syringe not present Disposable errors E HW 2503 Broken membrane s detected E HW 2504 Disposable s not present Wagon errors E HW 3301 Wagon cover not closed E HW 3201 Wagon temperature above safety limit PamGene International B V Page 41 of 64 Version 3 2009 7 3 2 Evolve12 User Manual Can errors E HW 4000 Can errors 4100 Sound error E HW 2400 Sound error in combination with a wait time error Other problems without error numbers No error Pictures are out of focus number Error Handling E HW 2101 Message Aspiration pressure not ok Category Error Number 2101 Explanation The pressure monitored during aspirating is too high or too low Solution There are multiple causes for this error If pressure was If viscose fluids are used increase
43. osoft e Click Continue anyway e Click Finish e Check the list of new COMs available in Device Manager e Change the register barcode entry for Evolve e verify installation by using open the Evolve runner and check for message barcode reader activated 8 6 Setting up the Evolve mail notification service Note an example is given For specific setting contact your local ICT department Step Description Step 1 Click start 22 Run 22 Type in regedit and hit ENTER Step 2 Go to HKEY LOCAL MACHINE gt SOFTWARE gt Pamgene gt Evolve gt Mail Or else create the Mail folder in the register Step 3 In the screen on the right you should see 7 parameters Change the values of the parameters to the values below Default data password xxxxxxx port 25 smtpserver mail pamgene com ssl 0 username XXXXXXX PamGene International B V Page 52 of 64 Version 3 2009 686 Pamee LE E 6 Evolve12 User Manual or add new parameters keys default data add the following as strings password PamGene01 smtpserver mail pamgene com username evolve add the following words port Hex 19 ssl Hex 0 PamGene International B V Page 53 of 64 Version 3 2009 Evolve12 User Manual 9 PAMSTATION 12 SPECIFICATIONS 9 1 Instrument specifications Dimensions and Weight of Instrument 49cm height x 66cm width x 58cm depth Weight 82 kg Optical Properties Filter specific
44. ressure barg Figure 24 Advanced protocol settings PamGene International B V Page 27 of 64 Version 3 2009 Evolve12 User Manual P ameer 5 1 8 Running man run button Located next to the save button at the top middle of the screen is the running man run button This button is only visible if the protocol is saved and when it is selected from the protocol list P By clicking on this button a quick link to the protocol runner is opened so it is not necessary to open the protocol runner separately 5 1 9 Editing an existing protocol Use the open a protocol button to select the protocol to be edited This button is located at the top of the screen in the centre A screen with a list of protocols will open Select the protocol to be edited and press the open button Protocol Editor sol ws 3 dX ELE Undate Add au dts e Load Disposable 30 C Kegiair Tuum y Unload Disposable ae Prime imi El Example P512Protacol My Recent Documents 2 Desktop My Documents My Computer femme 3 Files of type Protocols PS12Protocol Cancel Figure 25 Editing an existing protocol PamGene International B V Page 28 of 64 Version 3 2009 Evolve12 User Manual 66 Po oteps can be added removed and edited as described in chapter 5 1 2 to 5 1 6 The steps can be moved up and down the protocol list by using the positioning buttons as described in 5 1 5 Saving the protocol
45. se note that the terms disposable and PamChip have the same meaning PamGene International B V Page 16 of 64 Version 3 2009 Evolve12 User Manual Pame LEE E Table 3 Description of Protocol Steps Variable Parameters Recommended values Explanation Every protocol starts with a loading step and ends with an unloading step Load disposable Unload disposable 35 75 C preheat temperature ES Aspirate Duration 5 10 sec Removes fluid from Depending on amount arrays Dispense Manual option to add solution Lr MN am 4 Dispenses specified 35 75 C fluid onto the arrays Amount of dispense 95 ul Number of Number of Cycles Number of Cycles Any amount Incubates the array for a specified time and controls pressure 20 60 sec depending on Cycle time amount and viscosity of fluid and application Cycle frequency frequency See above perc read by cycle Assay dependent Filter 1 3 Records images once or kinetically Exposure time mS Assay and filter dependence K Temperature Changes temperature Temperature j Assay dependent of the incubator ngo Dispensing L Wash aspiration and pumping cycles The wash step is a combination of the dispensing pumping and aspiration steps Provides the option to Wait EOD RECON pause in a protocol Prime 25 50 ul Primes a dispense Select Fluid 1 4 needle The mail step can be Mai
46. te buffer Call Support Note If this error is given at the start of a dispense step then the experiment can be resumed If fluid has been dispensed into some of the arrays it has to be removed first Use a new protocol with an aspiration step set the temperature load step at the assay temperature and select only the wetted microarrays After that the original protocol can be resumed E HW 2401 Message Syringe almost empty Category Error Number 2401 Explanation This error is given during a load step when there is between 150 and 100 ul of fluid left in a syringe automated dispense oolution There are multiple causes for this error Syringe empty Remove the syringe and fill it with the appropriate buffer Call Support Note Use the resume protocol option in Evolve E HW 2402 Message Syringe not present Category Error Number 2402 Explanation This error is given when the syringe is not present at the correct position oolution There are multiple causes for this error Syringe not Place the syringe in the correct slot present Call Support Note Use the resume protocol option in Evolve PamGene International B V Page 44 of 64 Version 3 2009 Evolve12 User Manual E HW 2503 Message Broken membrane s detected Category Error Number 2503 Explanation There is an unexplained pressure difference in the given array number s Reasons broken microarray no fluid on given microarray problem w
47. tional B V Page 48 of 64 Version 3 2009 Evolve12 User Manual E E Panus 8 1 2 Electrical Compliance The European configuration of the instrument is designed to be compatible with the following standards CE Mark 8 2 Recommended Environmental Conditions Operating temperature o At humidity range 0 54 non condensing 15 30 C o At humidity of range 55 80 non condensing 15 23 C Operation is preferred in an air conditioned environment dust free controlled humidity and temperature Temperature storage 0 40 C Humidity operating and storage maximum 80 relative humidity non condensing Other atmospheric conditions The instrument is for indoor use only Avoid placing in direct sunlight Place the instrument on a level surface without vibration Do not expose to inflammable gases or solvents or to corrosive liquids or vapours Do not place close to devices that emit electrical noise The maximum altitude for use of the instrument is 2000m above sea level 8 2 1 Electrical requirements Place close to a well grounded electrical supply capable of handling at least 1500W Three outlets are required for the instrument and associated computer equipment PamGene International B V Page 49 of 64 Version 3 2009 Evolve12 User Manual D 8 2 2 Space requirements The dimensions of PamStation 12 are approximately 66 cm width x 59 cm depth x 49 cm height On the back sides and top of the
48. tional microarrays the rate limiting factor of the hybridization reaction is diffusion Due to the size of the pores the maximum distance of the target and probe molecules is reduced to 100 nm thereby eliminating diffusion as a factor in the hybridization Furthermore due to the hybridization by pumping a continuous mixing of the sample is achieved Typically hybridization or incubation is achieved in 0 5 2 hours Each PamChip 4 consists of 4 separate microarrays Each array is spotted with up to 400 oligonucleotides or peptides molecules Upon wetting of the surface material of the PamChip microarrays it becomes translucent This allows for imaging during or at the end of hybridisation or incubation using a CCD digital camera of the system Up to three different wavelengths can be selected to suit the dyes used Because the light is of low intensity no bleaching occurs with normal exposure times During the assay the temperature of the reaction can be varied This allows for the generation of melting curves when used in combination with the kinetic read out feature The PamStation 12 allows for aspiration and automated washing of the arrays Up to four different washing buffers can be loaded two of which can be heated to the assay temperature All of these processes are controlled by Evolve12 software PamGene International B V Page 5 of 64 Version 3 2009 Dames PamStation 12 User Manual 3 PAMSTATION 12 SYSTEM 3 1 General prin
49. ton and dragging the step to its desired location in the protocol list A second option is to click on the add button whereupon a scroll down menu with the names of the different steps will appear Click on the name of the preferred step and it will appear in the value editing screen After inserting the relevant values for the preferred step the operator can click on the add button located at the bottom right of the value editing window PamGene International B V Page 25 of 64 Version 3 2009 Evolve12 User Manual 66 86 5 1 4 Protocol list Located in the middle of the screen is the current protocol with a listing of all the current protocol steps By clicking on a step the information relating to that step will be shown in the value editing window at the right hand side of the screen 5 1 5 Positioning of steps Located above the list of protocol steps are 6 small buttons s x Baud These buttons can be used to move a step to a different position in the list of protocol steps or to delete the step if New step X Delete step Move step to the top of the protocol Move step one place up in the protocol ka E Move step one place down in the protocol Move step to the bottom of the protocol If the evolve screen is minimized these buttons can disappear just enlarge the screen and they will reappear PamGene International B V Page 26 of 64 Version 3 2009 Evolve12 User Manual Pamite ma 5 1 6 Edit
50. ycles pT 1 Cycle Time D sec 2 5 Cycle Freq ee l min 1 10 24 0 Figure 16 Pump window PamGene International B V Page 20 of 64 Version 3 2009 Evolve12 User Manual Po Read step The read step is the step that takes the pictures This can be done once or kinetically every few cycles This is determined by checking the box of your choice If the once box is checked the only other option available is the selection of filter or filters and the desired shutter time This selection is done by double clicking on filter 1 A pop up screen with scroll down function will appear In the scroll down function the selection of filters one to three can be found and at the bottom of the pop up screen The desired shutter time can be programmed It is possible to add different filters this is done by pressing the add button shown as a small sun button This will activate the aforementioned pop up menu WR MEI REESE IG Filter Name inet Fikar Marne Esposure Ti EH en 300 Exposure Time 200 msec 1 40000 Seed Filter Alter filters Figure 17 Read window It is not possible at this time to use different shutter times with one filter If this is required use different read steps If a kinetic read is chosen an addition to the variables is shown This addition includes all the variables of the pump step as described in the aforementioned pump step as array as the now open option available when a pictur
Download Pdf Manuals
Related Search