Ettan™ DALTsix and Ettan DALTtwelve Systems
Contents
1. Programming Ettan DALTsix Turn on the power supply control unit according to the user documentation provided with the equipment Refer also to the user documentation for programming instructions For recommended electrophoresis conditions see Electrophoresis conditions with precast gels on page 34 Programming Ettan DALTtwelve If not already turned on turn on the power supply control unit by pressing the Power switch to the I position When the unit is turned on the default settings of constant power continuous run auto pump and 25 C are shown on the display see Figure 4 2 The control unit has four programmable parameters Run Type Timer Pump and Temperature see Figure 4 2 Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA 31 4 Operation 4 2 32 Preparations before start 1 Const Watt 6W 2 Time 1 1 00hrs 3 Pump Auto 4 Temperature 22 C 5 6 6 7 8 Description Item Description 1 Run type 5 HV ON indicator 2 Timer 6 Arrow buttons 3 Pump 7 set read button 4 Temperature 8 start stop button Figure 4 2 Controller interface in programming mode The set read button toggles the controller between the set and read modes The start stop button starts and stops the execution of the programmed electrophoresis run The lt and P gt buttons move the cursor between run parameters and the A and W buttons change the settings of the parameters Below2. 4 Operation Preparations before start 4 2 Note The gel face of the strip must not touch the opposite glass plate Melt an aliquot of agarose sealing solution see Appendix B 4 in a heating block or boiling water bath for each Immobiline DryStrips Allow the agarose to cool slightly and slowly pipette the molten agarose solution along the upper surface of the gel up to the top of the glass plate Take care not to introduce bubbles Do not allow the agarose to solidify Once the agarose solution has completely set the gel should be run in the second dimension as soon as practically possible Inserting gels into Ettan DALT electrophoresis units 30 WARNING Disconnect the unit from the power supply when inserting gels Inserting gels into Ettan DALTsix When the electrophoresis buffer has reached the desired temperature insert the loaded Gel Cassettes with the Immobiline DryStrip Gels in place 1 Wet the UBC sealings with cathode buffer or 0 1 SDS immerse the sealings in solution or spray the sealings of the UBC using a plant sprayer and carefully slide the UBC over the gel cassettes Note Do not move the UBC repeatedly up and down as this will reduce the sealing effect In a separate container add 2 x 125 ml 2 bottles of cathode buffer Rinse the bottles and fill up with distilled or deionized water to 1 2 Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA Operati
3. full range of proteins Vertical protein streaks IPG strip not properly placed on gel surface Make sure IPG strip uniformly contacts the gel surface along itsentire length Avoid gouging the surface of the separating gel Spots skewed or distorted Gels run too fast uneven migration Run at a lower power setting Use a two step program Start at a low power setting until the proteins enter the gel then increase the power for the remainder of the run Uneven gel surface Overlay the running gel with water saturated butanol before polymerization begins to avoid forming an uneven gel surface Uneven gel polymerization or gradient formation Heavy background after silver staining Use reagents of the highest purity preferably electrophoresis grade Use deionized double distilled water 48 Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA Symptom Distortion in the 2 D pattern Possible cause Bubbles or liquid between the gel and the glass plate Troubleshooting 6 Stained gels 6 3 Corrective action Use the roller to remove any bubbles or excess liquid between the gel and the glass plate Ensure that no visible bubbles remain and that the gel adheres firmly to the glass and resists movement Interfering substances in the first dimension Contaminants in the sample can cause distortions or swollen regions in the IPG stri
4. DALTtwelve Systems are intended for research use only and shall not be used in any clinical procedures or for diagnostic purposes Safety notices These Operating Instructions contain WARNINGS CAUTIONS and NOTICES concerning the use of the product with meanings as defined below WARNING WARNING indicates a hazardous situation which if not avoided could result in death or serious injury It is important not to proceed until all stated conditions are met and clearly understood CAUTION CAUTION indicates a hazardous situation which if not avoided could result in minor or moderate injury It is important not to proceed until all stated conditions are met and clearly understood NOTICE NOTICE indicates instructions that must be followed to avoid damage to the product or other equipment 6 Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA Introduction 1 Regulatory information 1 2 Notes and tips Note A Note is used to indicate information that is important for trouble free and optimal use of the product Tip A tip contains useful information that can improve or optimize your procedures Typographical conventions Software texts and commands are identified by bold italic text A colon is used to separate menu levels e g File Open refers to the Open option in the File menu 1 2 Regulatory information This section lists the directives and standards that are fulf
5. XXXXXXXX Voltage Serial No XXXXXXX Frequency Mfg Year 2009 Power max Fuse N12406 A A D C TNT 751 84 Uppsala Sweden 2895233600 Made in Sweden GE Healthcare Bio Sciences AB Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA 19 2 Safety instructions 2 2 Labels Symbols used in safety labels Label Meaning Warning Read the user documentation before using the system Do not open any covers or replace parts unless specifically stated in the user documentation Indicates that hazardous voltages are generated inside the instrument The system complies with the requirements for electromagnetic compliance EMC in Australia and New Zealand CE The system complies with applicable European directives Labels concerning hazardous substances Label Meaning This symbol indicates that the waste of electrical and electronic equipment must not be disposed as unsorted municipal waste and must be collected separately Please contact an authorized representative of the manufacturer for information concerning the decommissioning of equipment 20 Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA Safety instructions 2 Emergency procedures 2 3 Meaning This symbol indicates that the product contains hazardous materials in excess of the limits established by the Chinese standard SJ T11363 2006 Requi
6. a homogeneous 12 5 polyacrylamide gel It is intended to be used in Ettan DALTsix or Ettan DALTtwelve systems together with the DALT Buffer Kit see 2 D Electrophoresis Principles and Methods DALT Pre cast Gel Cassettes The DALT Gel 12 5 pre cast gels are inserted into a specially designed reusable cassette see below and run vertically in the Ettan DALTsix and Ettan DALTtwelve Systems Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA Introduction 1 Instrument 1 3 Gel casting equipment Gel Caster The Ettan DALTsix and Ettan DALTtwelve Gel Casters can hold a different number of gel cassettes with separator sheets for casting homogenous or gradient gels Cassette capacity Cassette capacity System 1 0 mm gels 1 5 mm gels Ettan DALTsix Gel Caster 14 13 Fewer gels can be cast at one time by using blank cassette inserts to occupy unneeded volume For more information on gel casting see Ettan DALTsix and Ettan DALTtwelve User Manuals Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA 13 1 13 14 Introduction Instrument Gel Casting Cassettes The Ettan DALTsix and Ettan DALTtwelve use the same cassettes The gel casting cassettes are pre assembled Two glass plates are joined along one edge by a hinge strip of silicone rubber and the vinyl side spacers 1 0 mm or 1 5 mm thick are glued in place To complete assembly cl
7. alone unit or e connected to other CE marked instruments or e connected to other products recommended or described in the user documentation and e used in the same state as it was delivered from GE Healthcare except for alterations described in the user documentation or explicitly authorized by GE Healthcare Regulatory compliance of connected equipment Any equipment connected to Ettan DALTsix and Ettan DALTtwelve Systems should meet the safety requirements of EN 61010 1 IEC61010 1 or relevant harmonized standards Within the European Union connected equipment must be CE marked 8 Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA Introduction 1 Instrument 1 3 1 3 Instrument This chapter contains a general description of Ettan DALTsix and Ettan DALTtwelve Systems General description In 2 D electrophoresis proteins are separated according to isoelectric point by isoelectric focusing most reliably on immobilized pH gradient IPG strips using the IPGphor IEF System The second dimension electrophoresis separates the proteins on the basis of their molecular mass using sodium dodecyl sulphate polyacrylamide gel electrophoresis SDS PAGE The Ettan DALTsix and Ettan DALTtwelve Systems are designed to handle multiple large second dimension gels in a simple efficient and reproducible manner A sample focused in an IPG strip is placed on the cathodic upper surface of a slab gel and sealed
8. be heard as air is drawn into the lower chamber For runs of six or fewer gels alternating Gel Cassettes with blank cassettes also eases the removal of the Gel Cassettes at the end of a run Leave enough of the electrophoresis buffer in the tank to act as a lubricant between the glass cassettes and the buffer seal There are two methods for removing the first cassettes from the unit using the cassette removal tool or the hands e Using the cassette removal tool Carefully insert the cassette removal tool between the cassette and the buffer seal with the folded tip facing the cassette until the tip is beneath the bottom edge of the cassette Verify that the tool is caught on the bottom edge of the cassette then lift it out slowly with the tool Figure 4 3 Figure 4 3 Using the cassette removal tools Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA Operation 4 Procedures afterarun 4 4 e Using the hands By hand apply upward pressure alternately to each side of the cassette gradually shifting it up until you can grasp it and remove it Figure 4 4 Figure 4 4 Removing cassettes by hand 2 Open the cassettes using a Wonder Wedge and carefully transfer the gels to a staining tray for example Take care to ensure that the gel does not adhere to the spacers Note Vinyl gloves are less sticky than latex gloves and make it easier to handle large gels Detection Scan the gels as soon as po
9. bowing should not affect the performance of the seal To remove the anode plate or any of the other internal components follow the directions below 1 Insert the buffer seal removal tools through the fifth or sixth slot with the wider end underneath the tubes Turn the tools 90 and place as close to the end blocks as 42 Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA Maintenance 5 Component maintenance 5 3 possible Carefully pull upward on the buffer seal until it is removed from the unit see Fig 5 1 Figure 5 1 Using the buffer seal removal tools 2 Slide the two baffle plates upward and out of the unit 3 Remove the two flow guides by pulling out the retaining pins and lift the blocks out 4 Remove the anode plate by unscrewing the flathead nylon screw and lifting the plate out 5 To reassemble the unit replace the anode plate and screw making sure that the sealing sleeve is in place Spread a small amount of GelSeal compound on the plug with a swab applicator to prevent corrosion 6 Replace the flow guides and baffle plates 7 Puta light film of GelSeal compound on the gasket of the buffer seal and reinsert it into the unit using even pressure Make sure that it is fully seated before using the unit Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA 43 5 Maintenance 5 3 Component maintenance 44 Ettan DALTsix and Ettan DALTtwelve Systems Operat
10. can cause fatal electrical shock if the safety features are disabled The safety lid must be securely closed before starting a protocol WARNING The instrument covers must not be opened by the user It contains electrical circuits which can give a lethal electric shock Service and planned maintenance should be performed by personnel authorized by GE Healthcare WARNING Disconnect all units from mains power supply before removing the safety lid unloading the gels draining and cleaning the electrophoresis unit gt gt PIP Pe 16 Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA Safety instructions 2 Safety precautions 2 1 Using flammable liquids When using flammable liquids with the Ettan DALTsix and Ettan DALTtwelve Systems system follow these precautions to avoid any risk of fire or explosion WARNING Fire Hazard Before starting the system make sure that there is no leakage WARNING A fume hood or similar ventilation system shall be installed when flammable or noxious substances are used gt gt Personal protection WARNING Always use appropriate personal protective equipment during operation and maintenance of Ettan DALTsix and Ettan DALTtwelve Systems WARNING Hazardous substances When using hazardous chemical and biological agents take all suitable protective measures such as wearing protective glasses and gloves resistant to t
11. in place with agarose The Gel Cassette is then inserted into the electrophoresis unit through the buffer seal slots flanked by rubber gaskets Any unused slots are filled with the blank cassette inserts The buffer seal is an effective current and liquid barrier For more information see Chapter 4 System components The following components are included in Ettan DALTsix and Ettan DALTtwelve Systems e Electrophoresis unit e Power Supply Control Unit e Gel Caster e Gel Casting Cassettes e Gradient maker Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA 9 1 Introduction 1 3 Instrument The Electrophoresis units Description of Ettan DALTsix Electrophoresis unit 1 2 3 Part Description Part Description 1 Safety interlock 5 Upper buffer chamber 2 Anode assembly cassette 6 Lower buffer chamber carrier 3 Buffer circulation pump 7 Ceramic heat exchanger 4 Lid with leads 10 Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA Introduction 1 Instrument 1 3 Description of Ettan DALTtwelve Electrophoresis unit 5 1 a 2 3 q 7 4 Part Description Part Description 1 Lid with leads cathode 5 Safety interlock 2 Upper buffer chamber 6 Rubber gasket 3 Lower buffer chamber 7 Ceramic heat exchanger 4 Buffer circulation pump Electrophoresis unit data The Ettan DALTsix and Ettan DALTtwelve can a
12. is a description of the programmable parameters see also Table 4 2 e Run Type determines the method of power regulation The options for run type are constant power constant current constant voltage and crossover mode In crossover mode the voltage and current limits for the run are set manually instead of using the instrument defaults of 600 V and 1 000 mA As the run progresses the power supply operates in the mode that is limiting Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA Operation 4 Preparations before start 4 2 Timer controls the duration of the electrophoresis run The options are continuous run timed step timed and hold and volt hours Up to three timed steps of up to 100 h each can be programmed using the timed step mode With timed step mode all the steps must have the same run type Pump controls buffer circulation through the separation unit The options for the pump are On Off and Auto Auto mode activates the pump only when power is applied Temperature controls the cooling or heating of the buffer in the separation unit The temperature range is from 10 C to 50 C On the display cooling is indicated by and heating is indicated by 4 The pump must be on to properly cool or heat buffer in the tank The lower temperature limit is a function of the ambient temperature and the power reading To reach the lower temperature limit 10 C or in laboratory environments where t
13. respective owners Bj rkgatan 30 2009 General Electric Company All rights reserved 751 84 Uppsala First published Nov 2009 Swede n All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them A copy of these terms and conditions is available on request Contact your local GE Healthcare representative for the most current information www gelifesciences com 2DE GE Healthcare UK Ltd Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Bio Sciences Corp 800 Centennial Avenue P O Box 1327 Piscataway NJ 08855 1327 USA GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare Japan Corporation Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan imagination at work 28 9640 96 AA 11 2009
14. the separation tank with distilled or deionized water is sufficient If desired the internal components of the separation unit can be removed for a more thorough cleaning see below The unit can also be periodically cleaned with a dilute solution of a mild detergent Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA 41 5 Maintenance 5 3 Component maintenance Clean the gel casting cassettes and pre cast gel cassettes with a dilute solution of a laboratory cleanser such as RBS 35 from Pierce Chemical Company Rinse the cassettes thoroughly with distilled or deionized water NOTICE Do not autoclave or heat any part above 50 C NOTICE Do not expose the unit or its parts to organic solvents including gt 20 ethanol NOTICE If using radioactive reagents decontaminate the unit with a cleaning agent such as CONTRAD 70 or Decon 90 from Decon Laboratories Inc 0 0 0 5 3 Component maintenance WARNING Electrical Shock Hazard Do not use the equipment if any part is cracked or broken gt If any of the components become cracked or broken it should be replaced The upper buffer chamber UBC should be replaced if any of the rubber flaps become cut or torn To prevent damage to the flaps ensure that all sharp edges on glass plates are smoothed and exercise care when using the cassette removal tool With normal use the UBC ribs may become bowed upward this
15. unit is 400 mA 100 W and 600 V For overnight runs we recommend to set the temperature to 15 C Alternatively set the temperature to 22 C For further run conditions see the DIGE Gels and DIGE Buffer Kit Short Instructions in the DIGE Buffer Kit Run conditions Day run Set the MultiTemp temperature to 22 C Step mA gel Voltage V W gel Time hours mins 1 10 80 1 1 0 2 50 500 17 4 00 5 00 The maximum rated input power for the electrophoresis unit is 100 W Continue the electrophoresis until the bromophenol blue reaches the end of the gel Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA Operation 4 Preparations before start 4 2 Run Conditions Overnight Set the MultiTemp temperature to 15 C Step mA gel Voltage V W gel Time hours mins 1 12 150 15 15 00 17 00 Continue the electrophoresis until the bromophenol blue reaches the end of the gel Recommended running conditions for DIGE Gels Ettan DALTtwelve Program the Ettan DALTtwelve electrophoresis unit to deliver the following protocol Constant power Temperature 22 C Step Power W gel Duration 1 1 1 hour 2 17 max 180 Until the bromophenol dye front reaches the bottom of the gel approximately 4 5 hours for a full set of 12 gels For overnight runs the power is set to 1 W gel and the recommended temperature is 15 C Alternatively set the temperature to 22 C Run for 1 hou
16. 115115112112 6 2 Pre cast GEIS eeeeeessessssssssssteesssessssscenceeeeececeeeeceeeeeeestteeet 6 3 Stained Jels srriicseipe nesia 7 Reference information csecceceeeeeees TI SPOGCIIGATIONS sssscccscersasscxscensccsccascacszssszescattassascsauscncsteece TZ LALOR Essin 7 3 Ordering INFOMATION eeesssssssseeeseseststtttneeenenneeseeee Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA Table of Contents Appendix A_ Electrical and communication connections 53 A 1 Front of Ettan DALTtwelve Power Supply Control unit uu 53 A 2 Rear of Ettan DALTtwelve Power Supply Control Unit wes 54 Appendix B RecipeS ssssesssssssesssssressssssressssssesseresssessssseeesesse 55 B 1 SDS equilibration buffer solution sss11s11ssisss1ssrssrrrrssrisnrsssrssrrenrns 55 B 2 4x resolving gel buffer solution B 3 SDS electrophoresis buffer oo ceeccccccsssssssssssseesssessccssssssssssseesessesscsessseess B 4 Agarose SCALING SOLUTION oo cecssssssssssssssssssssseesssesscssssssssssssesessessesessseess 4 Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA Introduction 1 Important user information 1 1 1 Introduction Purpose of the Operating Instructions The Operating Instructions provide you with the instructions needed to handle Ettan DALTsix and Ettan DALTtwelve Systems in a safe way Prerequisites In order to operate Ettan DALTsix and Ettan D
17. 2 C for a day run and to 15 C for an overnight run Equilibrate the buffer to 15 C before starting an overnight run Refer to the MultiTemp Ill User Manual for more information Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA Operation 4 Preparations before start 4 2 4 Set aside upper chamber The upper chamber is prepared once the gel has been inserted into Ettan DALTsix See Inserting gels into Ettan DALT electrophoresis units on page 30 For more detailed instructions refer to Ettan DALTsix Electrophoresis System User Manual Preparing Ettan DALTtwelve 1 Prepare cathode buffer In a separate container dilute the concentrated cathode buffer included in the DIGE Buffer Kit to working strength by adding four bottles of DIGE cathode buffer total volume 500 ml and fill up with distilled or deionized water to a total volume of 2 25 2 Prepare anode buffer Ensure that the valve on the separation unit is set to circulate Add the entire contents of two bottles of DIGE anode buffer stock solution included in the DIGE Buffer Kit into the tank Rinse the bottles with distilled or deionized water and pour it into the tank Fill the tank to the 7 5 fill line with distilled or deionized water in this way washing the DIGE anode buffer from the buffer seal Note Avoid pouring the DIGE buffer on the tubing by spreading the tubing slightly with one hand while pouring the solution with the other Figure
18. 2 kPa 12 PSI WARNING Be careful when lifting and moving the electrophoresis unit It is best to move the unit when empty gt PP PPE System operation WARNING Hazardous biological agents during run When using hazardous biological agents flush the entire system tubing with bacteriostatic solution followed by a neutral buffer and finally distilled water before service and maintenance gt 18 Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA Safety instructions 2 Labels 2 2 WARNING Avoid spilling liquids on the body of the instrument If large volumes of liquid have penetrated the casing of the instrument and come into contact with the electrical components immediately switch off the instrument and contact an authorized service technician Maintenance WARNING Do not remove the main cover There are no userserviceable components inside and you can be exposed to high voltage 2 2 Labels This section describes the safety labels and labels concerning hazardous substances that are attached to the Ettan DALTsix and Ettan DALTtwelve Systems For information about marking of the computer equipment refer to the manufacturer s instructions Labels on the instrument The illustration below shows an example of the identification label that is attached to the Ettan DALTsix and Ettan DALTtwelve Systems XX XXXK XX Ettan DALTxxxx Code No
19. 4 1 Figure 4 1 Spreading the tubing elements apart with one hand while pouring the solution with the other to avoid pouring the DIGE buffer onto the tubing 3 Turnon the electrophoresis unit Turn on the electrophoresis unit by pressing the Power switch to the I position Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA 27 4 Operation 4 2 Preparations before start 4 Start the pump Start the pump to mix the buffers and set the separation unit to desired temperature A temperature of 22 C is recommended for day runs and 15 C for overnight runs Note A temperature of 25 C is recommended for electrophoresis For more detailed instructions refer to Ettan DALTtwelve System User Manual Equilibrating Immobiline DryStrip Gels 28 Equilibration is carried out in a two step process to reduce vertical streaking in the second dimension The first step saturates the IPG strip with the SDS system and the second step blocks the protein thiol groups The equilibration solution contains buffer urea glycerol reductant SDS and dye Tubes and volume of equilibration solution to use are specified in Table 4 1 Prepare equilibration solution 1 Prepare SDS equilibration buffer see Appendix B 1 This is a stock solution 2 Just prior to use add 50 mg DTT per 10 ml SDS equilibration buffer 0 5 w v Equilibration 1 Place the IPG strips in individual tubes with the support film toward the tube wa
20. 96 AA 25 4 Operation 4 1 Operation overview 4 1 Operation overview The typical workflow in Ettan DALT system when using precast gels can be divided into the following steps Step Action Page 1 Preparing Ettan DALT system for electrophoresis using precast gels 26 3 Equilibrating Immobiline DryStrip Gels 28 4 Applying equilibrated Immobiline DryStrip Gels 29 5 Inserting gels into Ettan DALT electrophoresis units 30 6 Programming the power supply control unit 31 7 Starting the electrophoresis 37 8 Unloading the gels from the Ettan DALT electrophoresis units 37 Note Precast DIGE Gels have been used as an example throughout this chapter 4 2 Preparations before start Preparing Ettan DALT system for electrophoresis using precast gels Preparing Ettan DALTsix 1 26 Prepare anode assembly Insert the anode assembly cassette carrier into the tank The anode assembly is molded so thot it can only be inserted in one orientation The side edge of the assembly should fit into the slot in the side of the tank Fill with anode buffer Add one bottle 125 ml of DIGE Anode Buffer stock solution included in the DIGE Buffer Kit into the tank Fill the electrophoresis unit to the 4 5 fill line with distilled or deionized water Connect the pump to mains power supply Turn on the temperature controller Turn on the MultiTemp IIl temperature controller or similar and adjust the temperature to 2
21. ALTtwelve Systems safely and according to the intended purpose the following prerequisites must be met e You should be acquainted with the use of general laboratory equipment and with handling of biological materials e You should understand the concepts of electrophoresis e You must read the Safety Instructions in Chapter 2 of these Operating Instructions e The system should be installed according to the instructions in Chapter 3 of these Operating Instructions In this chapter This chapter contains important user information and a general description of Ettan DALTsix and Ettan DALTtwelve Systems and their intended use 1 1 Important user information Read this before using Ettan DALTsix and Ettan DALTtwelve Systems All users must read the Safety Instructions in Chapter 2 of these Operating Instructions before installing using or maintaining the instrument Do not operate Ettan DALTsix and Ettan DALTtwelve Systems in any other way than described in the user documentation If you do you may be exposed to hazards that can lead to personal injury and you may cause damage to the equipment Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA 5 1 Introduction 1 1 Important user information Intended use The Ettan DALTsix and Ettan DALTtwelve Systems are designed to handle multiple large second dimension electrophoresis gels in a simple efficient and reproducible manner Ettan DALTsix and Ettan
22. GE Healthcare Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions Original instructions Ettan Table of Contents T REO CUCU ascites 1 1 Important user Information eseeesesssssssseceeeeeeeee 1 2 Regulatory information LS WASUMUMS WG sscessssadeensztcctencpestassictecncastiarencssetectaeonncestints 2 Safety instructionS seessseessessssessssesesses 2 1 Safety precautions eeeeeeesssssssssssseeseesesesettttteetenesseesee Z2 LADES nenn ek 2 3 Emergency procedures 24 Recycling INFOrmation oe eeeesssssssssceeeecesesesettsttteneeneeeeee Table of Contents 3 MASCOT ON nnana 23 3 1 Site requirements 3 2 Unpacking 3 3 Placement 34 TRONS POM se csacracseressss sasncsscessasesiateetaniessmeencesensioanvacectaveie BBs ASSEDI resen S 3 6 CONNECTIONS eeeeecssssscssssseeeee 3 7 Spare parts and accessories 4 Operati M cisdiseissascsaiscessiasessstiasctectisssasesssess 4 1 Operation Overview aoeeeescccssssssssesseccccsssssneeeseceesssnneees 4 2 Preparations before start 4 3 Performing a run GA Procedures OFF g rUn arassssiiiiriresiiiis GS MaimtenaNCE sissies 5 1 User maintenance schedule 52 CIC CIAUIAG secasscassadsssasecsvtsnccsonssceonesccadecsedeatonscsavessanconnsbnnetot 5 3 Component MAINTENANCE reeeeeeessssessssseeseeeeeeeeeceeee 6 Troubleshooting siisisccsisiccsescsscssssiaissosccardisencs 6 1 Electrical and mechanical s ss ss15115115
23. Second dimension separation proceeds slowly with high current All of the slots in the buffer seal are not occupied by either gel cassettes or blank cassettes Ensure that all 12 slots in the buffer seal are occupied Do not pour more than the suggested volume 7 5 into the lower reservoir Anodic buffer has mixed with cathodic buffer from overfilling of either the cathodic or the anodic reservoir Ensure that the level of the anode lower buffer does not come above buffer seal when the separation unit is fully loaded Remove any excess anode buffer from the upper reservoir Ensure that the level of cathode buffer is not above the air vents in the upper reservoir Dye front is irregular The top surface of the gel has been damaged during application of the IPG strip Take care during application of the IPG strip that neither gel is damaged Bubbles or liquid between the gel and the glass plate Use the roller to remove any bubbles or excess liquid between the gel and the glass plate Ensure that no visible bubbles remain and that the gel adheres firmly to the glass and resists movement Interfering substances in the first dimension Contaminants in the sample can cause distortions or swollen regions in the IPG strip following IEF Modify sample preparation to limit these contaminants Refer to 2 D Electrophoresis Principles and Methods Pronounced downward curving of the dye front
24. ccommodate a different number of gels either 1 mm or 1 5 mm thick in a common tank under identical conditions Electrophoresis system Gel size Gel capacity Ettan DALTsix 25 5 x 20 5 cm 6 Ettan DALTtwelve 25 5 x 20 5 cm 12 Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA 11 1 13 Introduction Instrument Power supply control unit Electrophoresis system Power supply unit Requirements Ettan DALTsix EPS 601 Power Supply or 600 V 400 mA or 100 W any other power supply with similar specifications Ettan DALTtwelve Ettan DALTtwelve Power 600 V 1A or 200 W Supply Control Unit Pre cast Gels and Gel cassettes 12 Pre cast gels If fluorescent staining labeling techniques will be used do not run gels cast on plastic backing as it can pose a problem of high background with some dyes during subsequent analysis Instead a DIGE Gel can be used A DIGE Gel is a 12 5 precast polyacrylamide gel in a low fluorescent glass cassette for second dimension 2 D electrophoresis and specially developed for 2 D DIGE analysis It is intended to be used in Ettan DALTsix or Ettan DALTtwelve systems together with the DIGE Buffer Kit For more information on DIGE Gels see Ettan DIGE System User Manual DALT Gel 12 5 is a precast polyacrylamide gel 25 5 x 19 6 cm 1 mm thick for the second dimension of 2 D electrophoresis The gel is provided already cast onto a plastic support film The gel is
25. cription 1 High voltage connector 2 Signal and power cable connector Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA 53 Appendix A Electrical and communication connections A 2 Rear of Ettan DALTtwelve Power Supply Control unit A 2 Rear of Ettan DALTtwelve Power Supply Control unit Part Description Part Description 1 Power switch 2 Power inlet 54 Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA Recipes Appendix B SDS equilibration buffer solution B 1 Appendix B Recipes B 1 SDS equilibration buffer solution 6 M urea 50 mM Tris HCl pH 8 8 30 glycerol 2 SDS 0 001 bromophenol blue 200 ml Final concentration Amount Urea FW 60 06 6M 72 07 9 Tris HCl pH 8 8 see Appendix B 2 50 mM 6 7 ml Glycerol 87 w w 30 v v 69 ml 84 2 g SDS FW 288 38 2 w v 40g 1 Bromophenol blue stock solution 0 001 w v 2 mg Double distilled water to 200 ml 1 This is a stock solution Just prior to use add DTT or iodoacetamide for first or second equilibration respectively as described in the in Equilibrating Immobiline DryStrip Gels on page 28 Store in 40 ml aliquots at 20 C B 2 4x resolving gel buffer solution 1 5 M Tris base pH 8 8 1 Final concentration Amount Tris base FW 121 1 1 5 M 181 7 g Double distilled water 750 ml HCigg Double distilled water adjust to pH 8 8 to1l Ettan DALT
26. e placed on a stable support such as a bench or sturdy table A length of rubber or vinyl tubing sufficiently long to reach a sink should be attached to the drain port on the back of the unit before use The unit should not be placed on bench paper or any other material that might be pulled in by the air intake fans as any hindrance to air circulation will reduce the cooling capacity Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA 23 3 Installation 3 4 Transport For Ettan DALTsix The tubing leading to and from the heat exchanger should be connected to a circulating water bath such as the MultiTemp III the heat exchanger should not be connected to a water tap or any other coolant supply that lacks pressure regulation The Power Supply should be placed conveniently near the electrophoresis unit in a dry place without risk for liquid exposure 3 4 Transport Before moving the system e remove liquids and gels from the separation unit e disconnect all cables e lift the instrument by the base unit 3 5 Assembly The following parts must be added to the Ettan DALTsix and Ettan DALTtwelve instruments before they can be used e IPG strips e Gel Cassettes containing gels e Buffer liquids 3 6 Connections Communication Connect Ettan DALTtwelve according to the electrical drawings in Appendix A Connect Ettan DALTsix according to the user documentation supplied with the Power Supply Electrical
27. etting Problems with first dimension Refer to troubleshooting guides for IPGphor or Multiphor units or 2 D Electrophoresis Principles and Methods Smeared or comet shaped spots Ettan DALTsix only Buffer problems Check pH of cathode buffer Should be between 8 3 and 8 8 Make sure that 2x Laemmli buffer is used in the upper cathode chamber Buffer or SDS depleted Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA 47 6 Troubleshooting 6 3 Stained gels Symptom Protein spots are near the buffer front Protein spots have not entered the gel when buffer front has reached the bottom of the gel Possible cause Incorrect pore size Corrective action Pore size of the gel is too large Increase the T Degraded proteins during sample preparation Proteins degraded during sample preparation Add protease inhibitors Incorrect pH Incorrect pore size Incorrect pH The gel pore size is too small Check the pH of the 4x gel buffer It should be pH 8 8 Proteins will migrate faster below pH 8 8 Decrease the T Check the pH of the 4x gel buffer It should be pH 8 8 Proteins will migrate more slowly above pH 8 8 Protein spots are at both extremes but not in center The molecular weight range of the sample requires an acrylamide concentration gradient to resolve the
28. he ambient temperature is elevated it may be necessary to place the unit in a cold box or cold room Table 4 2 Parameter options Parameter Option Display Range Run Type Constant power Const Watt 1 to 200 W Constant current Const Amp 1 to 1000 mA Constant voltage Const Volt 1 to 600 V Crossover mode V amp mA 1 to 600 V 1 to 1000 mA Timer Timed step Time 1 Time 3 00 01 to 99 59 hrs Time and hold T Hold 00 01 to 99 59 hrs Volt hours Stop at 1 to 9999 Vhr Continous run Continous run Pump On On off off Auto Auto Temperature Temperature 10 to 50 C Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA 33 4 Operation 4 2 Preparations before start See Table 4 3 for a sample program Twelve gels are electrophoresed at 180 W constant power and 25 C with a 5 W gel entry phase The second phase is extended longer than required to ensure that the dye front runs off the gel Step Parameter Value 1 Const Watt 60 W Time 1 00 45 hrs Pump Auto Temperature 25 C 2 Const Watt 180 W Time 2 04 00 hrs Pump Auto Temperature 25 C Table 4 3 Sample program For recommended electrophoresis conditions see Electrophoresis conditions with precast gels on page 34 Electrophoresis conditions with precast gels 34 Recommended running conditions for DIGE gels Ettan DALTsix The maximum rated electrical input for the electrophoresis
29. he substances used Follow local and or national regulations for safe operation and maintenance of the system s WARNING Spread of biological agents The operator has to take all necessary actions to avoid spreading hazardous biological agents in the vicinity of the instrument The facility should comply with the national code of practice for biosafety gt PP Installing and moving the instrument WARNING Supply voltage Make sure that the supply voltage at the wall outlet corresponds to the marking on the instrument before connecting the power cord Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA 17 2 Safety instructions 2 1 Safety precautions WARNING Protective ground The Ettan DALTsix and Ettan DALTtwelve Systems must always be connected to a grounded power outlet WARNING Power cord Only use power cords delivered or approved by GE Healthcare WARNING Access to power switch and power cord Do not block the rear and side panel of the instrument The Power switch must always be easy to access The power cord must always be easy to disconnect WARNING Use EPS 601 as power supply for Ettan DALTsix WARNING Only connect MultiTemp Ill or a circulating water bath with earthed water pipe and low pressure water circulation Do not connect the heat exchanger to a water tap or any other water source with unregulated pressure that may exceed 8
30. ifications 7 1 Parameter Value Ingression protection IP20 Supply voltage 100 to 240 V 50 to 60 Hz Power consumption Ettan DALTsix Ettan DALTtwelve Fuse specifications 100 W 650 W T2 5 A 250V Dimensions H x W x D 160 x 278 x 471 mm Weight 8 3 kg Ettan DALTsix 11 1 kg Ettan DALTtwelve 24 5 kg Ambient temperature 4 to 40 C Relative humidity 0 to 90 tolerance Atmospheric pressure Acoustic noise level 84 to 106 kPa 840 to 1060 mbar lt 70dBA Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA 51 7 Reference information 7 2 Literature 7 2 Literature For further information regarding the Ettan DALTsix and Ettan DALTtwelve Systems refer to the following e Ettan DALTsix Electrophoresis System User Manual e Ettan DALTtwelve System User Manual e Ettan DIGE System User Manual e 2 D Electrophoresis Principles and Methods e DIGE Gels and DIGE Buffer Kit Short Instructions e MultiTemp Ill User Manual 7 3 Ordering information For ordering information visit www gelifesciences com 2DE 52 Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA Electrical and communication connections Appendix A Front of Ettan DALTtwelve Power Supply Control unit A 1 Appendix A Electrical and communication connections A 1 Front of Ettan DALTtwelve Power Supply Control unit Part Description Part Des
31. illed by Ettan DALTsix and Ettan DALTtwelve Systems Manufacturing information Requirement Content Name and address of manufacturer GE Healthcare Bio Sciences AB Bj rkgatan 30 SE 751 84 Uppsala Sweden Name and ID of notified body INTERTEK SEMKO AB NB 0413 Place and date of declaration Uppsala Sweden May 2009 Identity of person authorized to sign DoC See EC Declaration of Conformity CE Conformity Directive Title 2006 42 EC Machinery Directive MD 2006 95 EC Low Voltage Directive LVD 2004 108 EC ElectroMagnetic Compatibility EMC Directive Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA 7 1 Introduction 1 2 Regulatory information International standards Standard Description Notes EN 61010 1 Safety requirements for electrical IEC 61010 1 equipment for measurement control CAN CSA C22 2 and laboratory use No 61010 1 EN 61326 1 EMC emissions and immunity Harmonized with requirements for electrical equipment 2004 108 EC measurement control and laboratory use EN ISO 12100 1 Safety of machinery Basic concepts Harmonized with 12100 2 general principles and design 2006 42 EC EN ISO 14121 1 Safety of machinery Principles of risk Harmonized with 14121 2 assessment 2006 42 EC CE marking The CE marking and the corresponding Declaration of Conformity is valid for the instrument when it is e used as a stand
32. ing Instructions 28 9640 96 AA 6 Troubleshooting 6 1 Electrical and mechanical Symptom No current at start of run Possible cause Insufficient volume of buffer in upper reservoir Troubleshooting 6 Electrical and mechanical 6 1 Corrective action Ensure that the unit contains enough buffer to contact the upper electrode Buffer not circulating Pump is not primed Turn circulation valve to Drain to fill with buffer then back to circulate Pump is off or set to Auto Set unit to ON on control unit Pump is broken Contact GE Healthcare Service Display on the control unit blank Ettan DALTtwelve only Unit is not turned on Turn unit on with power switch Unit is not plugged in Display is broken Plug in unit Contact GE Healthcare Service Control unit display malfunction Turn the AC power switch off for a few seconds then on again If the problem persists call GE Healthcare Service Open circuit warning Ettan DALTtwelve only Safety interlocks not engaged Make sure that the lid is completely closed Make sure that the pump valve is turned completely to circulate If this does not correct the error contact GE Healthcare Service Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA 45 6 Troubleshooting 6 2 Pre cast gels 6 2 Pre cast gels Symptom Possible cause Corrective action
33. irculate mode empty again and repeat if necessary Remove the lid from the unit by sliding it to the left and rinse it with distilled or deionized water Slide the lid back on its hinges before using the unit again Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA Maintenance 5 User maintenance schedule 5 1 5 Maintenance WARNING Disconnect power Always disconnect power from the instrument before performing any maintenance task WARNING Hazardous substances When using hazardous chemical and biological agents take all suitable protective measures such as wearing protective glasses and gloves resistant to the substances used Follow local and or national regulations for safe operation and maintenance of the system 5 1 User maintenance schedule Before each run check the following All parts are clean All parts are intact and free of cracks and other damage If a part is damaged replace the part or entire instrument before use The power cord and high voltage cable is intact and free of cracks and other damage If the cable is damaged replace it before use There is no leakage from parts containing buffer or cooling water Moist or liquid must be removed from the equipment before use The circulation pump spins and that there is circulation of liquid 5 2 Cleaning For day to day operation of the unit the cleaning procedure outlined in unit operation thoroughly rinsing
34. ll 2 Add 10 ml DTT containing solution to each tube 3 Place the tubes on a rocker and equilibrate for 15 min Note When using CyDye DIGE saturation dyes Labeling Kit for scarce samples repeat the first equilibration with DTT containing SDS equilibration solution for another 15 min Second equilibration Asecond equilibration is performed with an iodoacetamide solution instead of DTT 1 Prepare a solution of 450 mg iodoacetamide per 10 ml of SDS equilibration buffer 4 5 w v 2 Decant the first equilibration solution and add the same volume of iodoacetamide containing equilibration solution to each tube 3 Place the tubes on a rocker and equilibrate for an additional 15 min Note The subsequent steps of electrophoresis unit preparation insertion of gels and melting of the Sealing Solution can be performed while the IPG strips are equilibrating Note Be consistent with the timing of the equilibration steps Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA Operation 4 Preparations before start 4 2 Table 4 1 Suggested containers and volumes of equilibration solution Strip length Container Equilibration cm solution ml 7 Disposable 15 ml conical tubes 2 5 5 11 25 x 200 mm screw cap culture tubes 5 10 13 25 x 200 mm screw cap culture tubes 5 10 18 25 x 200 mm screw cap culture tubes Equilibration 10 15 24 tubes available from GE Healthcare or Petri dish Equilibration
35. ll the upper buffer chamber upward using the handles at each end As the upper buffer chamber is pulled upward make sure that the cassettes remain in the anode assembly Take out the anode assembly Remove the Gel Cassettes from the anode assembly Open the Gel Cassettes using a Wonder Wedge to separate the plates on the side opposite the hinge Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA 37 4 Operation 4 4 Procedures after a run 38 For lab cast gels run an edge of the Wonder Wedge down each side of the cassette along the spacer and carefully lift the gel out of the cassette For precast gels lift the gel out by grasping the GelBond backing Tilt the entire electrophoresis unit on the side of the unit do NOT tilt to front or back to empty the buffer from the lower buffer chamber into a sink or a waste container See Section 3 3 for instructions of how to place the unit for safe use Rinse all the components with distilled or deionized water Flush the pump with distilled or deionized water by filling the unit and turning the pump on Unloading the gels in Ettan DALTtwelve 1 After the run has been completed remove one or more of the Blank Cassette Inserts or gels and drain enough of the electrophoresis buffer from the tank to expose 2 3 cm of the Gel Cassettes This will ease removing the remaining cassettes When the first cassette either blank or gel is removed a sucking sound will
36. on 4 Preparations before start 4 2 3 Fill the UBC with 1 2 liters of diluted cathode buffer and use a funnel to adjust the buffer level in the lower buffer chamber to the same height as in the UBC by adding water or diluted anode buffer 4 Attach and close the lid Connect the power leads to the power supply Inserting gels into Ettan DALTtwelve When the electrophoresis buffer has reached the desired temperature insert the loaded Gel Cassettes with the Immobiline DryStrip Gels in place Note Gel Cassettes and Blank Cassette Inserts slide much more easily into the unit if they are wet Wetting the cassette with some cathode buffer using a soaked kleenex or alternatively distilled or deionized water from a squirt bottle can be used to wet the cassettes and Blank Cassette Inserts as they are being loaded into the unit 1 Fit Blank Cassette Inserts into any unoccupied slots 2 Load the unit from back to front 3 Whenall 12 slots are occupied the buffer level should be slightly below the level of the gaskets If the buffer level is too low add distilled or deionized water to the lower buffer chamber If excess anode buffer is in the upper reservoir remove it with a pipette Pour the diluted cathode buffer into the tank to the fill line some of this buffer may drip through the gasket and mix with the anode buffer during the run but this will not affect performance or results 4 Close the lid Programming the power supply control unit
37. on one side of the gel There is an unfilled gap between the gel and one of the spacers When sealing the IPG strip into place ensure that some of the agarose sealing solution flows down any gap that may exist between the gel and the spacer 46 Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA 6 3 Stained gels Symptom Protein spots are diffuse or broader than usual Possible cause Poor quality reagents Troubleshooting 6 Stained gels 6 3 Corrective action Use only the highest quality reagents Incomplete polymerization Make sure that polymerization is complete Incorrect equilibration time Check equilibration time of IPG strips Too long can lead to diffusion and too short can lead to incomplete equilibration Gel surface damaged by IPG strip Problems with first dimension Make sure the IPG strip rests on the slab gel surface without damaging Refer to troubleshooting guides for IPGphor or Multiphor units or 2 D Electrophoresis Principles and Methods Protein spots are poorly resolved Incomplete polymerization Make sure that polymerization is complete Diffusion of proteins Begin electrophoresis as soon as the IPG strips are loaded to prevent diffusion of low molecular weight proteins Running too fast Reduce the power current or voltage Too high temperature Reduce the temperature s
38. ose the two plates like a book and press the plates together over the length of the spacer Gels are removed by opening the book after the run and carefully lifting out the gel slab Gradient maker A Gradient maker is used to make gradient gels using two gel solutions of different acrylamide concentrations and densities a light solution and a heavy solution Below is a description of Ettan DALTtwelve Gradient maker 6 T 1 g Pl IG Pl 5 1 2 in ee m 4 3 Part Description Part Description 1 Feed tube 4 Gradient maker 2 Gel caster 5 Heavy chamber 3 Pump 6 Light chamber For more information on how to cast gradient gels see Ettan DALTsix and Ettan DALTtwelve User Manuals For information on how to use the pump refer to the user documentation supplied with the pump Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA Safety instructions 2 Safety precautions 2 1 2 Safety instructions This chapter describes safety compliance safety labels general safety precautions emergency procedures power failure and recycling of Ettan DALTsix and Ettan DALTtwelve Systems 2 1 Safety precautions Introduction The Ettan DALTsix and Ettan DALTtwelve Systems are powered by mains voltage and handles liquids that may be hazardous Before installing operating or maintaining the system you must be aware of the hazards described in
39. p following IEF These distortions can result in disturbances in the seconddimension Vertical gap in the 2 D pattern Vertical streaking Spots are vertically doubled or twinned Bubble between IPG strip and top surface of second dimension gel Incorrectly prepared equilibration solution Poor transfer of protein from IPG strip to second dimension gel Ensure that no bubbles are trapped between the IPG strip and the top surface of the second dimension gel Prepare equilibration solution according to instructions Use low power for sample entry phase Extend entry phase if necessary Insufficient equilibration IPG strip is not placed properly Prolong equilibration time Ensure that the plastic backing of the IPG strip is against the glass plate of the second dimension cassette Poor representation of higher molecular weight proteins Incorrectly prepared equilibration solution Prepare equilibration solution according to instructions Poor transfer of protein from IPG strip to second dimension gel Use low power for sample entry phase Extend entry phase if necessary Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA 49 6 Troubleshooting 6 3 Stained gels 50 Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA 7 1 Specifications Reference information 7 7 Reference information Spec
40. power Connect the power cord to a grounded power outlet specified in Section 3 1 3 7 Spare parts and accessories For correct up to date information on spare parts and accessories visit www gelifesciences com 2DE 24 Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA Operation 4 4 Operation WARNING Check electrical cables for damage and the electrophoresis unit for damage or leakage before use If damage and or leakage is present do not use the equipment There is a risk for high voltage injuries WARNING The high voltage power supply must always be disconnected when the safety lid of the electrophoresis unit is taken off The high voltage power supply must never be switched on unless the safety lid is on the electrophoresis unit WARNING Do not overfill the tank Spillage can cause short circuiting of internal circuits and the breakdown of electrical isolation which increases the risk of electrical shock Always make sure the equipment is disconnected from power supply when filling the tank WARNING Any liquid on the equipment must be dried off before connecting the power supply WARNING Always wear gloves protective clothing and eye protection when handling DIGE Gels DIGE Buffer IPG strips gel cassettes or any other equipment these items will come into contact with PP Pe gt Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640
41. r and then increase to 1 5 W gel Recommended running conditions for DALT Precast Gels and 1 mm Laemmli gels Ettan DALTsix Run conditions Day run Applicable to 1 mm thick 12 PAA gel and Laemmli Buffer system Set the MultiTemp temperature to 25 C Step mA gel Voltage V W gel Time hours mins 1 10 80 1 1 00 2 40 500 13 4 30 6 00 For 1 5 mm gels increase the current by 50 Continue the electrophoresis until the bromophenol blue reaches the end of the gel Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA 35 4 Operation 4 2 Preparations before start 36 Run Conditions Overnight Set the MultiTemp temperature to 30 C Step mA gel Voltage V W gel Time hours 1 10 80 1 1 2 12 150 2 15 17 1 For 1 5 mm gels increase the current by 50 Continue the electrophoresis until the bromophenol blue reaches the end of the gel Recommended running conditions for DALT Precast Gels and 1 mm Laemmli gels Ettan DALTtwelve Electrophoresis is performed at constant power in two steps Stop electrophoresis when the dye front is approximately 1 mm from the bottom of the gel Temperature control improves gel to gel reproducibility especially if the ambient temperature of the laboratory fluctuates significantly For best results gels should be run at 25 C For overnight runs we recommend to set the temperature to 30 C Step Power Approximate run W gel d
42. rements for Concentration Limits for Certain Hazardous Substances in Electronics 2 3 Emergency procedures This section describes how to do an emergency shutdown of Ettan DALTsix and Ettan DALTtwelve The section also describes the result in the event of power failure Emergency procedures In an emergency situation do as follows to stop the run Step Action 1 Switch off power to the instrument by pressing the Power switch on the external power supply to the 0 position If required disconnect the mains power cord The run is interrupted immediately 2 For Ettan DALTsix If required disconnect the Ettan DALTsix mains power cord Power failure The result of a power failure depends on which unit that is affected Power failure to will result in Ettan DALTsix and Ettan The run is interrupted immediately in an undefined DALTtwelve state Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA 21 2 Safety instructions 2 4 Recycling information 2 4 Recycling information The equipment shall be decontaminated before decommissioning and all local regulations shall be followed with regard to scrapping of the equipment Disposal general instructions When taking Ettan DALTsix and Ettan DALTtwelve out of service the different materials must be separated and recycled according to national and local environmental regulations Recycling of hazardous substances Ettan DALTsix and Et
43. six and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA 55 Appendix B Recipes B 3 SDS electrophoresis buffer B 3 SDS electrophoresis buffer 25 mM Tris base 192 mM glycine 0 2 SDS 20 I Final concentration Amount Tris base MW 121 14 25 mM 60 5g Glycine MW 75 07 192 mM 288 0 g SDS MW 288 38 0 2 w v 40 0g Double distilled water to 101 1 The pH of this solution should not be adjusted Store at room temperature B 4 Agarose sealing solution 25 mM Tris base 192 mM glycine 0 1 SDS 0 5 agarose 0 002 bromophenol blue 100 ml Final concentration Amount Laemmli SDS electrophoresis buffer 100 ml see Appendix B 3 Agarose NA or M 0 5 0 5g 1 Bromophenol blue stock solution 0 002 w v 200 ul Add all ingredients into a 500 ml Erlenmeyer flask Swirl to disperse Heat in a microwave oven on low or on a heating stirrer until the agarose is completely dissolved Do not allow the solution to boil over Dispense 1 5 ml aliquots into screw cap tubes and store at room temperature 56 Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA For local office contact info rmation visit GE imagination at work and GE monogram are trademarks of General fi Electric Company www gelifesciences com contact 7 CyDye Ettan Immobiline and Typhoon are trademarks of GE Healthcare companies GE Healthcare Bio Sciences AB All third party trademarks are the property of their
44. ssible after the second dimension SDS PAGE is finished in order to minimize protein diffusion Typhoon Variable Mode Imager or Ettan DIGE Imager are recommended for scanning DIGE second dimension SDS PAGE gels Store the gels in a refrigerator in a closed container and keep the gels moist Allow the gels to reach room temperature before scanning Keep the gels in the glass cassettes throughout scanning Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA 39 4 Operation 4 4 Procedures after a run Cleaning the Ettan DALT units after a run 40 Cleaning Ettan DALTsix 1 When all of the gels and Blank Cassette Inserts have been removed tilt the entire electrophoresis unit on the side of the unit do not tilt to front or back to empty the buffer from the lower buffer chamber into a sink or a waste container See Section 3 3 for instructions of how to place the unit for safe use Rinse all the components with distilled or deionized water Flush the pump with distilled or deionized water by filling the unit and turning the pump on Cleaning Ettan DALTtwelve 1 When all of the gels and Blank Cassette Inserts have been removed drain the buffer by turning the pump valve to drain with the pump on Emptying will take about 1 min After the buffer has been removed pour 3 4 of distilled or deionized water into the unit and allow it to drain Rinse the unit with 5 7 of distilled or deionized water in c
45. tan DALTtwelve contain hazardous substances Detailed information is available from your GE Healthcare representative Disposal of electrical components Waste of electrical and electronic equipment must not be disposed as unsorted municipal waste and must be collected separately Please contact an authorized representative of the manufacturer for information concerning the decommissioning of equipment 22 Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA Installation 3 Site requirements 3 1 3 Installation Ettan DALTsix and Ettan DALTtwelve are delivered in protective packing material and shall be unpacked with great care Any equipment connected to Ettan DALTsix and Ettan DALTtwelve must fulfill applicable standards and local regulations For detailed information on Installation see Ettan DALTsix and Ettan DALTtwelve User Manuals 3 1 Site requirements Parameter Requirement Electrical power 220 240 V 50 60 Hz Ambient temperature 4 C to 40 C Placement Stable laboratory bench Humidity 0 to 90 non condensing 3 2 Unpacking e Check the equipment for damage before starting assembly and installation e Document any damage and contact your local GE Healthcare representative Remove straps and packing material and stand equipment upright before starting installation 3 3 Placement Position the electrophoresis unit close to a sink for easy rinsing and draining It must b
46. this manual Follow the instructions provided to avoid personal injury or damage to the equipment The safety precautions in this section are grouped into the following categories General precautions Using flammable liquids Personal protection Installing and moving the instrument System operation Maintenance General precautions WARNING Do not operate the Ettan DALTsix and Ettan DALTtwelve Systems in any other way than described in the Ettan DALTsix and Ettan DALTtwelve Systems manuals WARNING Operation and user maintenance of the Ettan DALTsix and Ettan DALTtwelve Systems should be performed by properly trained personnel only Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA 15 2 Safety instructions 2 1 Safety precautions WARNING Do not use any accessories not supplied or recommended by GE Healthcare WARNING Do not use the Ettan DALTsix and Ettan DALTtwelve Systems if they are not working properly nor if it have suffered any damage for example e damage to the power cord or its plug e damage caused by dropping the equipment e damage caused by splashing liquid onto items not supplied or recommended by GE Healthcare WARNING Operation and user maintenance of the Ettan DALTsix and Ettan DALTtwelve Systems should be performed by properly trained personnel only WARNING Ettan DALTsix and Ettan DALTtwelve Systems are high voltage instruments that
47. tubes available from GE Healthcare 10 15 or Petri dish Note The subsequent steps of gel assembly preparation of electrophoresis unit Applying equilib insertion of the gel into the Precast Gel Cassette and melting of the sealing solution can be performed as the Immobiline DryStrip Gels are equilibrating as long as the time frames above are adhered to rated Immobiline DryStrip Gels CAUTION Handle the gels with care The edges of the glass plates of the gels are sharp Place the gels in the Ettan DALT cassette rack Briefly rinse the Immobiline DryStrips by submerging them in a measuring cylinder containing SDS electrophoresis buffer see Appendix B 3 for Ettan DALT Holding one end of the Immobiline DryStrip with forceps carefully place the Immobiline DryStrip in between the two glass plates of the gel Using a thin plastic spacer push against the plastic backing of the Immobiline DryStrip not the gel itself and slide the strip between the two glass plates until it comes into contact with the surface of the gel Note The strip should just rest on the surface of the gel Avoid trapping air bubbles between strip and the gel and avoid piercing the second dimension gel with the strip Note The acidic end of the Immobiline DryStrip should be on the right side of the gel when the shorter of the two plates is facing the user Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA 29
48. uration h min Day runs set temperature to 25 C 1 mm thick gels 1 2 0 45 lab cast and precast 2 17 max 180 4 00 1 5 mm thick gels 1 5 0 30 2 17 max 180 6 00 Overnight runs set temperature to 30 C power supply for continuous run 1 0 mm thick gels run overnight 1 18 00 1 5 mm thick gels run overnight 15 17 00 1 For the best possible resolution faster separation times should be used Use the faster lt 6 h protocols instead Ettan DALTsix and Ettan DALTtwelve Systems Operating Instructions 28 9640 96 AA Operation 4 Performing arun 4 3 4 3 Performing a run For Ettan DALTsix For instructions on how to start and stop the electrophoresis program refer to the user documentation for the power supply control unit used For Ettan DALTtwelve 1 Press the start stop button on the power supply control unit to start the electrophoresis program To stop the electrophoresis program before it is finished press the start stop button 4 4 Procedures after a run Unloading the gels from the Ettan DALT electrophoresis units WARNING Disconnect all units from mains power supply before removing the safety lid unloading the gels draining and cleaning the electrophoresis unit Unloading the gels in Ettan DALTsix CAUTION Ettan DALTsix electrophoresis unit is heavy especially when filled with buffer Handle the unit with care to avoid personal injury Carefully pu
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