ALFexpress® II - GE Healthcare Life Sciences
Contents
1. ALFexpress AutoR ead Sequencing Kit for routine sequencing of both single and double stranded templates with T7 DNA polymerase which produces very even peak heights band intensities e DyeAmidite O ligoPrep for purification of primers labelled or unlabelled e ALFexpress dATP Labelling mix for sequencing with DyeA midite dATP and unlabelled primer e ALFexpress Sizer 50 500 A ladder of labelled fragments with increments of 50 bp from 50 bp to 500 bp e Individual sizers 50 100 150 200 250 and 300 bp for use as internal markers Refer to the web page www amershambiosciences com for more information about new kits 1 4 Gels Introduction 1 Amersham Biosciences provides several gel chemicals for various applications both convenient ready to use gel mixes and stand alone gel components for increased flexibility For automated DNA analysis using the ALF family instrument we recommend our UV polymerised gels e ReproGel High Resolution suitable for applications such as heterozygote detection ReproGel Long Read for general sequencing purposes These gels together with the UV box ReproSet offers a convenient easy and reproducible way of making gels ready for immediate use after only 10 minutes Gel casting and polymerisation with R eproSet in addition to recommended running conditions are described in Chapter 4 of this User M anual 1 5 Information of the Web Detailed protocols2. Figure 3 1 Indicator lamps and symbols on the front of the instrument 3 1 3 Description of ALFexpress Il 3 2 3 1 2 Lid Thelid opens upwards Fig 3 2 The lid should be closed gently Figure 3 2 ALFexpress Il instrument with open lid 3 1 3 Internal lamp The internal lamp in the electrophoresis instrument automatically illuminates when the control software is running and the lid to the instrument is opened If the cassette is not inserted the lamp remains lit for 30 seconds if no further operation is performed When the lid is closed the lamp switches off automatically 3 1 4 Thermostatic circulator The thermostatic circulator holds approx 0 8 litres of deionized or M illiQ water The water level is automatically checked within the instrument and a warning message is displayed on the computer screen if the level is getting low If the water level becomes too low the appropriate warning indicator light on the instrument will illuminate see Section 5 3 together with a message on the screen Top up the water level according to the instruction in Section 5 1 2 It is recommended that the water be emptied when the instrument is moved and changed if the water does not appear to be fresh see Description of ALFexpress Il 3 Section 5 1 3 It is not normally necessary to add an antimicrobial agent Two self sealing push fit couplings situated at the left and right hand sides of the front panel connect the thermostat
3. Operation 4 to separate them Be careful not to use excessive force on the glass plate Carefully remove the glass spacer by sliding the thin plastic spacer strip under one end of the spacer and gradually moving underneath its length Do not use a razor blade or other sharp implement or place the plastic strip under the middle of the glass spacer or bend the glass spacer Usea piece of soft plastic material to scrape the gel from the plates Do not usea razor blade or other sharp instrument Place the plates on the cushion in the sink and clean them as described in Section 4 2 1 Gel residue should be scraped off the plate in the sink under running tepid approx 30 C water Use the scraper which is supplied with the instrument Afterwards gently scrub the plate with a soft nylon brush and washing liquid avoid scratching Ensure that all gel residues are removed especially around the upper 5 cm where the plates were treated with Bind Silane Clean the glass spacers immediately in water and place back in the plastic spacer box 4 15 4 Operation Maintenance 5 5 Maintenance 5 1 Routine Maintenance 5 1 1 Cleaningthe detector unit and the instrument Clean the detectors with soft lint free paper tissue dampened with M illiQ water Clean the instrument with a moist cloth 5 1 2 Filling up the water circulator Ensure that the water circulator remains topped up between the minimum and maximum levels 1 Pull out the w
4. stored in a retrieval system or transmitted in any form by any means without permission in written form from the Contents Contents 1 Introduction 1 1 1 1 Electrophoresis and detection system 1 2 1 2 Software 1 3 1 3 Labelling and kits 1 4 1 4 Gels 1 5 1 5 Information of the Web 1 5 2 Important safety information 2 1 2 1 Electrical safety 2 1 2 2 Laser safety 2 1 2 2 1 Laser safety labels 2 3 2 3 Safe handling of chemicals 2 3 3 Description of ALFexpress Il 3 1 3 1 Electrophoresis instrument 3 1 3 1 1 Indicator lamps 3 1 3 1 2 Lid 3 2 3 1 3 Internal lamp 3 2 3 1 4 Thermostatic circulator 3 2 3 1 5 Laser 3 3 3 1 6 O ptical unit 3 3 3 1 7 Detector unit 3 4 3 1 8 Safety devices 3 4 3 1 9 Electrode connections 3 4 3 1 10 Power supply 3 4 3 2 Computer and software 3 4 3 3 Instrument accessories 3 5 3 3 1 Thermoplate 3 5 3 3 2 Glass plate 3 6 3 3 3 Glass spacers 3 6 Contents 4 Operation 5 Maintenance 3 3 4 Gel clips 3 3 5 Buffer reservoirs 3 3 6 Electrodes 3 3 7 Comb 4 1 Lab equipment 4 2 Assembling the gel cassette 4 2 1 Preassembly cleaning 4 2 2 Bind Silane treatment 4 2 3 Assembling the gel cassette 4 3 Casting the gel 4 4 Preparing for a run 4 4 1 Setting the running conditions 4 4 2 Fitting the cassette into the instrument 4 5 Loading the samples and starting the run 4 5 1 Loading the samples and starting 4 5 2 Starting the run 4 6 Ending the run 4 6 1 Disassembly of the gel cassette 5 1
5. Routine Maintenance 5 1 1 Cleaning the detector unit and the instrument 5 1 2 Filling up the water circulator 5 1 3 Changing water in the thermostatic circulator 5 1 4 Changing dust filters 5 1 5 Lubricating the water connectors 5 1 6 Changing a fuse 5 2 Moving the instrument 5 3 Hardware warning messages www 00 00 NN 4 9 4 9 4 1 4 12 4 12 4 13 4 14 4 14 al i mb Mm i AA q a pb YUNRPRRR Contents 6 Ordering Information and Technical Data 6 1 6 1 Ordering information 6 1 6 2 Technical data 6 3 Contents Introduction 1 1 Introduction ALFexpress Il DNA Analyser is designed for automated detection of fluorescently labelled DN A molecules separated by electrophoresis For sequencing reactions the basic difference from the traditional Sanger dideoxy sequencing method isthe use of a fluorescently labelled primer or fluorescently labelled dN TP instead of radioactive deoxynucleotides For fragment analysis a labelled primer is used in the amplification step Alternatively labelled dN TPs can be used for end labelling restriction fragments ALFexpress Il DNA Analyser system consists of e An electrophoresis and laser fluorescent detection system An external computer with a separately available software for control of electrophoresis run data storage and analysis All necessary accessories to operate the system e Optional items such as sequencing reagents and hardware and software
6. called RO Ps Recommended O perating Procedure have been rigorously tested to cover the most common operating procedures and methods used with ALFexpress Il RO Ps are available for e cassette handling and starting a run e gel recipes and operating procedures e methods for SSCP microsatellite analysis and more ROPs other methods information of the use of gels monomer solutions chemicals and pre made mixes can be obtained at the web site WWW amershambiosciences com 1 5 1 Introduction 1 6 Important safety information 2 2 Important safety information ALFexpress II DNA Analyser has been designed with your safety in mind If handled correctly according to the instructions in this section of the manual ALFexpress Il will work reliably and safely in your laboratory 2 1 Electrical safety A 2 2 Laser safety The safety devices and circuits built into the instrument are designed to prevent electrical related harm A safety interlock switch in the lid of ALFexpress Il prevents the high voltage being applied when the lid isopen Theground leakage detection system gt 0 5 mA automatically responds by turning off the high voltage supply when ground leakage is detected WARNING The voltage supplied by the high voltage power supply is capable of delivering a lethal electrical shock Please take the following precautions when operating the instrument Ensure that the mains power cable is plugged into an undamage
7. clothing Do not breathe vapour Keep in a tightly closed container Use only in a fume hood Always use disposable gloves a laboratory coat and goggles Acetic acid Flammable causes severe burns Avoid breathing vapour Avoid contact with the skin and eyes Always use gloves and goggles All handling of lyophilised chemicals or chemicals in solution especially during Bind Silane treatment and gel casting should be performed in a fume hood Always wear disposable gloves and safety goggles Description of ALFexpress Il 3 3 Description of ALFexpress II ALFexpress Il DNA Analyser consists of the electrophoresis instrument and a computer to control the instrument You can separately obtain the appropriate accessories from Amersham Biosciences to accommodate your analysis needs including the standard and short gel cassettes with accessories gels and software 3 1 Electrophoresis instrument The components of the electrophoresis instrument are described below 3 1 1 Indicator lamps There are five indicator lamps on the bottom left hand side of thefront panel of the electrophoresis instrument From left to right i a green lamp indicating power supply and computer connection ii a red lamp indicating errors iii a yellow lamp associated with the laser iv a yellow lamp associated with the thermostatic control and v a yellow lamp associated with the high voltage The status of the lamps is described in Section 5 3
8. lamp is steady The actual water temperature may be monitored or changed in the control software module Note The water preheat function is automatic with a default of 40 C unless anew temperature value has been entered in the control software The temperature can be selected from the operating range of 20 C above ambient up to 60 C Operation 4 Figure 4 8 ALFexpress Il instrument 1 Fixed screws used to locate the placement of the standard gel cassette into the instrument 2 The lower buffer reservoir By closing and opening thelid the laser is automatically activated to find the optimum operating position which usually takes about one minute and is indicated by the blinking yellow laser on the instrument When the laser has located the optimum operating position the yellow lamp remains on To realign the laser you can click on the Realign Laser button in the Manual window of the ALFwin Control software W hen therunning temperature has been reached removethecomb by gently pulling it upwards from the thermoplate Rinse the wells with running buffer using a 50 ml syringe fitted with a 0 6 or 0 8 mm needle Examine the wells carefully for unevenness at the base and for damage to the walls between wells etc 4 Operation 4 5 Loading the samples and starting the run This section of the manual gives a brief description of how to load the samples and start the electrophoresis run For a more information about the
9. this may cause unsafe activation of the laser beam e Thereisno manual adjustment needed for the laser optical system Do not attempt to remove the back side or top panels Service of the instrument must only be carried out by Amersham Biosciences authorised personnel WARNING TheHelium N eon laser has a power of 2 5 mW and the unprotected eye can be permanently damaged if exposed to a direct or reflected laser beam Important safety information 2 2 2 1 Laser safety labels Please note the following labels and their locations on the instrument Label located inside the lid of the electrophoresis chamber DANGER LASER RADIATION WHEN OPEN AND INTERLOCK DEFEATED AVOID DIRECT EXPOSURE TO BEAM Label and certification text located at the back of the instrument CLASS 1 LASER PRODUCT IEC 825 1 1993 Amersham Biosciences AB certifies that this product complies with 21CFR 1040 10 CAUTION LASER RADIATION WHEN OPEN AVOID EXPOSURE TO BEAM The following statement is required by federal regulations United States only Caution Useof controls or adjustment of performanceor procedures other than those specified herein may result in hazardous radiation exposure Electromagnetic C ompatibility Caution Thisis a Class A product In a domestic or similar environment this product may cause radio interface The user may be required to take adequate measures 2 3 Safe handling of chemicals A CAUTION Several of the chemi
10. uk pubseq DNAsis The dna format http www hitachi soft com gs of DN Asis a sequence analysis program from Hitachi Software Genetics Systems Group 1 3 1 Introduction For more information about the software products available please contact your local Amersham Biosciences representative and also look in our internet home side www amershambiosciences com 1 3 Labelling and kits 1 4 ALFexpress Il is designed to separate fluorescently labelled dideoxy chain terminated fragments by electrophoresis In addition to sequencing ALFexpress Il can be used for electrophoretic analysis of end labelled amplified DN A fragments fill in labelled fragments following restriction cleavage and other suitably sized DN A fragments This non radiochemical approach to DNA sequencing is based on primers and dNTPs which carry a DyeAmidite label Using standard dideoxy sequencing methods a DyeA midite 667 labelled primer unlabelled primer is used for DyeA midite 6 dN TP is annealed to a template and divided into four separate tubes each containing a different dideoxynucleotide in addition to the deoxynucleotides with DyeAmidite9 dN TPs a labelling step is performed prior to the elongation termination step After extension of the primer the chain terminated reaction products are electrophoresed in four adjacent lanes on a sequencing gel Convenient kits are available for performing sequencing reactions and sizing fragments
11. v 250 pl Note This quantity of Bind Silane solution is sufficient for several gels If you do not use all of the Bind Silane solution in one day discard it and prepare fresh solution the next time you require it 3 Sparingly apply a few drops of the Bind Silane solution onto the upper 2 3 cm of the thermoplate and polish dry with lint free tissue Additionally apply the solution to the upper 5 cm of the glass plate on the side without the logo Fig 4 1 Polish dry with a lint free tissue Ensure that the Bind Silane treatment does not extend further down the glass than 5 cm Stand the clean glass plate in a safe dust free environment within the fume hood 4 Soak a lint free tissue with distilled water and gently wipe the treated area Polish again with a dry lint free tissue 5 Moisten a lint free tissue with non denatured alcohol and gently wipe the thermoplate and glass plate avoiding the Bind Silane treated area Thisis achieved by starting at the bottom of the plates and polishing upwards towards the Bind Silane treated area 6 Discard the disposable gloves safely 4 2 3 Assembling the gel cassette This section describes how to assemble the gel cassette Further details can be found in the Recommended O perating Procedure ROP 56 3061 88 Assembly of the cassette and casting the gel which is available at the Internet web site www amershambiosciences com Use a new pair of disposable gloves when assembling the
12. ALFexpress Il User Manual 18 1127 13 Amersham Edition AA e Y Biosciences Important user information Reading this entire manual is recommended for full understanding of the use of this product Should you have any comments on this manual wewill be pleased to receive them at Amersham Biosciences AB SE 751 84 Uppsala Sweden Warranty and Liability All goods and services are sold subject to the terms and condi tions of sale of the company within the Amersham Biosciences group which supplies them A copy of these terms and conditions of sale is available on request Trademarks ALFexpress ALF ALFwin ReproGel ReproSet AlleleLinks HLA SequiTyper are trademarks of Amersham Biosciences Limited or its subsidiaries In view of the risk of trademark degeneration it is respectfully suggested that authors wishing to use these designations refer to their trademark status at least once in each article The following designations are trademarks owned by other companies Pentium is a registered trademark of Intel Corporation Windows95 is a registered tradmark of M icrosoft Corporation DNAsis is a trademark of Hitachi Software Genetics Systems Group The Wisconsin Package GCG is a trademark of Genetics Computer Group a subsidary of Oxford M olecular Group Inc Copyright Amersham Biosciences UK Limited 1998 All rights reserved No part of this publication may be repro duced
13. Lower buffer reservoir 2 Upper electrode socket standard cassette 3 Upper buffer reservoir 4 Upper electrode socket socket short cassette 5 Thermocirculator attachment to the thermoplate 6 Lower electrode socket 3 3 1 Thermoplate The thermoplate is one of the components used to assemble a gel cassette together with a glass plate glass spacers and gel clips A thermoplate is formed from two planes of glass separated by glass bars and sealed at the edges with a frame The plate facing the gel is manufactured from glass which is planar and exhibits very low fluorescence Attached to thethermoplate is the upper buffer reservoir see Section 3 3 5 and a location spring for correct positioning of the gel cassette into the instrument see Section 4 4 A blackened region on the thermoplate helps eliminate reflected light Two sizes of thermoplate are available i a standard thermoplate with a 200 mm separation distance and ii a short thermoplate with an 80 mm separation distance The standard and short thermoplates respectively contain approximately 0 15 and 0 08 litres of water They can withstand a pressure of up to 30 kPa 0 3 kg cm 3 5 3 Description of ALFexpress Il 3 6 Figure 3 5 Thermoplate and glass plate for the standard cassette left and the short cassette right 3 3 2 Glass plate The glass plate is one of the components used to assemble a gel cassette together with a thermoplate glass spacers an
14. accessories Figure 1 1 ALFexpress Il DNA Instrument 1 1 1 Introduction 1 1 Electrophoresis and detection system Electrophoresis is carried out in an off vertical gel cassette specially designed for easy and safe gel casting Temperature control of the gel is achieved by circulating water from the integral water circulator through one of the plates which form the cassette Samples are loaded into wells at the top of the gel During electrophoresis the fluorescently labelled fragments in each lane migrate downwards through the gel The fixed laser beam passes through the glass spacer located between the glass plate and the thermoplate of the gel cassette The beam is directed into the gel perpendicular to the direction of band migration The laser beam excites the fluorescently labelled DN A bands and the light emitted is detected by photodetectors located behind the gel There are 40 photodetectors one for each lane W ith four lanes being used for each sequencing clone ten clones can berun simultaneously Alternatively 40 samples of fragments can be loaded Figure 1 2 ALFexpress Il detection principle The photodetector signals are collected and sent to the computer for storage and processing During electrophoresis the raw data is displayed as a chromatogram in real time on the computer screen For sequencing four different colours are used to display the four bases with each peak representing one nucleotide in the s
15. al Amersham Biosciences representative to obtain a list of recommended printers to use with the specific software and computer Index Index A accessories 3 5 ALFexpress Il cleaning the instrument 5 1 description 1 1 moving the instrument 5 4 ALFwin Fragment Analyser 1 00 1 3 ALFwin Sequence Analyser 2 00 1 3 bind silane treatment 4 3 bubbles in the gel 4 8 buffer reservoirs 3 7 Cc casting the gel 4 7 changing the fuses 5 3 changing the water thermostatic circulator 5 1 chemical handling safety 2 3 chemicals and reagents 6 2 cleaning detergent 4 2 cleaning the detector unit 5 1 cleaning the gel cassette 4 2 cleaning the instrument 5 1 clips 3 7 comb 3 8 computer control 1 3 3 4 control software 3 4 ALFwin Fragment Analyser 1 00 1 3 ALFwin Sequence Analyser 2 00 1 3 D description of ALFexpress Il 1 1 detection principle 1 2 detector unit 3 4 cleaning 5 1 disassembling the gel cassette 4 14 DN Asis export format 1 3 dust filters replacing 5 2 DyeA midite 667 1 4 Index E electrical safety 2 1 electrode connections 3 4 electrode placement 4 13 electrodes 3 8 electrophoresis detection principle 1 2 ending the run 4 14 error indicator lamp 3 1 5 5 error lamps 5 5 F filling the water circulator 5 1 fuse replacement 5 3 G GCG export format 1 3 gel cassette assembly 4 2 4 4 cleaning 4 2 disassembling 4 14 fitting into the instrument 4 10 mounting into the instrument 4 10 gel cast
16. and free from residues before being assembled for use The surfaces of the plates and the glass spacer must be free of scratches Note Always wear disposable gloves when handling the parts of the gel cassette Rinse the gloves under running tap water to remove any talcum powder prior to use 1 Line the bottom of a laboratory sink with the cushion supplied Place the thermoplate and glass plate one at a time in the sink Allow the plates to remain under running tepid approx 30 C water for a few minutes Thetwo plates can then be thoroughly cleaned using a soft nylon brush and standard non fluorescing laboratory detergent Clean the spacers in the same way using tepid approx 30 C water only The glass spacers are fragile and are therefore best washed carefully between the fingers Alternatively place the spacer on a flat surface and carefully wash with the brush Note Do not let thethermoplate soak in detergent solution longer than the time taken to clean it If soaking is necessary use water only Do not let the thermoplates soak over night 2 After cleaning rinsethe items thoroughly under running tap water to ensure that all traces of detergent have been removed Then rinse each item with M illiQ water Note A thorough cleaning procedure is essential since the highly sensitive detection system of ALF express Il DN A Analyser is capable of detecting trace contaminants such as detergent 3 Wipe each item dry using lin
17. anual window of ALFwin Control The laser will automatically readjust itself as indicated by the initial blinking and then steady illumination of the yellow laser lamp Note If the laser lamp goes out restart the laser adjustment process by closing the instrument lid and then opening it again 4 Close the lid of the instrument 5 Start the run in the control software 4 6 Ending the run On completion of therun the laser beam thermostatic circulator and high voltage switch off automatically If everything has worked well there will be no error messages reported to the control software The instrument may be switched off It is also possible at any time to manually end a run in the control software 4 6 1 Disassembly of the gel cassette 1 Open the lid of the instrument 2 Disconnect and remove the electrodes from the buffer reservoirs 3 Disconnect the thermostatic circulator connections of the gel cassette 4 Allow the gel cassette to equilibrate to room temperature before cleaning 5 Remove the room temperature equilibrated gel cassette and pour out the buffer from the connected upper reservoir Place the gel cassette in the sink and remove the gel clips 6 Pour out the buffer from the lower reservoir 7 Inserta 0 2 mm plastic spacer strip or a similar thin strip of plastic into the top of the gel cassette between the glass plate and the thermoplate Slide it gently along the entirelength of the two plates
18. ater filling tubing on the right side of the instrument and insert a funnel 2 Add MilliQ water up to the maximum mark 5 1 3 Changing water in the thermostatic circulator The water should be emptied if you are intending to move the instrument see Section 5 2 or the water does not appear to be clean To empty and refill the water circulator do the following 1 Pull out the water filling tubing on the right side of the instrument and put the end in a bucket or sink at a lower level than the instrument 2 If not already done uncouple the thermostatic circulator connectors from the gel cassette 3 Connect the angled quickfit connection nipple of the provided blowing tube to one of the thermostatic circulator connections Blow carefully to empty the water including that contained within the tubing of the instrument Connect the blowing pipe to the second thermostatic circulator connection and repeat the procedure 5 1 D nanenane 5 2 4 To refill the circulator e Attach the drain tubing to the drain outlet and place the free end of the tubing into a sink or small container approximately 50 to 100 ml will be drained Figure 5 1 Filling the water circulator CAUTION Thefree end of the drain tubing must not be blocked or be put beneath water as this will cause water spillage within the instrument during filling of the circulation system e Placea funnel at the top of the water filling tubing e Hold the wa
19. cals used with ALFexpress Il DNA Analyser can be hazardous if not handled with care The type of hazard is printed on the labels of the individual chemicals Some of these chemicals and their hazards are summarised below 2 3 2 Important safety information 2 4 Bind Silane Toxic and highly volatile use only in a fume hood Prevent contact with skin and eyes always use disposable gloves and goggles Acrylamide and ready made gel solutions Potent cumulative neurotoxin To prevent contact with skin and inhalation of dust and vapours all procedures involving unpolymerised acrylamide should be performed in a fume hood Always use disposable gloves laboratory coat and goggles N N methylenebisacrylamide Potent cumulative neurotoxin To prevent contact with skin and inhalation of dust and vapours all procedures involving N N methylenebisacrylamide should be performed in a fume hood Always use disposable gloves laboratory coat and goggles Contact with combustible material can cause fire Avoid contact with the skin eyes and clothing TEMED N N N N T etramethyl ethylenediamine Extremely flammable keep away from heat sparks and flame Avoid contact with eyes skin and clothing Do not breathe vapour Keep in a tightly closed container Use only in a fume hood always use disposable gloves laboratory coat and goggles Formamide T eratogenic harmful to a developing foetus Avoid contact with skin eyes and
20. cassette Rinse the gloves under running tap water to remove any talcum powder before handling any part of the gel cassette 1 Taketwo glass spacers of the correct thickness from the spacer box and insert them into the indented edges on the left and right sides of the thermoplate Push the glass spacers against the edges of the plate and apply gentle even pressure to secure them to the silicon rubber seals Fig 4 2 Operation 4 Figure 4 2 Positioning the glass spacers on the thermoplate H olding the upper edge nearest edge of the glass plate place its bottom edge furthest edge against the supports on the thermoplate Carefully lower the glass plate onto the thermoplate and check that the glass spacers remain in the correct position Fig 4 3 Figure 4 3 Placement of the glass plate onto the thermoplate The logo is printed on the outside of the glass plate 4 5 4 Operation 4 6 3 Place four pairs of gel clips along the left and right sides of the standard cassette or two gel clips on the sides of the short cassette N ote that clip pair no 2 must be positioned a few cm above the laser entry point into the cassette For more information obtain a copy of the Instructions for the placement of gel clips order no 71 5005 91 attheweb site www amershambiosciences com Figure 4 4 Placement of gel clips on the standard cassette Slidethe comb directly in between thethermoplate and glass plate at the top
21. computer Check that the control soft ware is loaded and running Restart the computer and instrument Yellow lamp Instrument is Wait until the lamp blinking adjusting to the becomes steady set experimental conditions Yellow lamp Instrument has None steady adjusted to the set experimental conditions Yellow laser Adjustment Retry laser adjustment lamp and red failed timeout error lamp both Try new gel cassette blinking Contact your local A mer sham Biosciences service representative Yellow thermo static circulator lamp and red error lamp both blinking Water level too low Check the water level Maintenance 5 Yellow voltage lamp and red error lamp both blinking If the gel cassette is in the instru ment there may be connection problems If the gel cassette is not in the instrument there may be an inter nal communica tion problem Check the electrode con nections Try new running buffer Try a new gel cassette Restart the instrument Contact your local Amer sham Biosciences service representative Restart the instrument with the cassette in the instru ment 5 7 Lo ica 5 8 Ordering information and technical data 6 6 Ordering Information and Technical Data 6 1 Ordering information ALFexpress Il CodeNo 18 1125 07 For specific version details contact your Amersham Biosciences representative Accessories and spare parts Thermoplate s
22. control software refer to either the ALFwin Sequence Analyser 2 00 User M anual or ALFwin Fragment Analyser 1 00 User M anual for sequencing or fragment separation runs respectively 4 5 1 Loading the samples and starting 1 Check in the control software that the preset temperature of the gel has been reached The set and actual water temperatures can be viewed Ensure that the samples have been denatured according to the sequencing protocol Load the samples for sequencing or fragments analysis accordingly a For sequencing Starting at the left load 4 10 ul of each sample depending on the gel thickness into the wells in the following suggested order A C G and T Up to 10 clones can bedonein this way Loading stop solution or dummy samples in the outermost wells will also improve results b For fragment analysis Starting at the left load samples into the wells numbered from 1 to 40 Fig 4 9 Ignore the markings used for sequencing i e A C G and T and the 10 clone groupings It is recommended to load samples in adjacent lanes and not to leave empty wells between clones Loading stop solution or dummy samples in the outermost wells will also improve results c For AutoLoad combs Fill the wells with 10 ul of loading dye W hen the gel temperature has reached 55 C insert the comb and leave for 10 minutes to allow denaturation of the samples Carefully remove the comb Note The markings in the instrument are mea
23. d and fully grounded mains outlet Ensure that the instrument lid is closed before starting the run e Regularly check all external cables for damage to the insulation For your safety do not remove the hood of the instrument Service of ALFexpress Il must only be carried out by Amersham Biosciences authorised personnel Two safety interlock switches Fig 2 1 behind the gel cassette prevent laser operation when the gel cassette is not fitted in place The laser can only be turned on if the cassette is first mounted and the lid then closed The lid may afterwards be opened and the laser will stay on until the cassette is removed With the gel cassette in position the laser is only visible as it passes through the gel cassette T his scattered laser radiation which is emitted from the gel cassette is harmless 2 1 2 Important safety information 2 2 fa ie Bl oe mila da al a Figure 2 1 Safety interlock switches for the laser There are several features on the instrument to ensure smooth and safe operation of the laser Please follow the guidelines for proper use e The gel cassette is easy to fit into the instrument in the correct position without the need for readjustment O n correct insertion the laser beam is automatically switched on due to the depression of two safety interlock switches located behind the gel cassette Fig 2 1 CAUTION Never attempt to override any of the safety interlock switches as
24. d gel clips The glass plate is manufactured from glass which exhibits very low fluorescence and is planar Cut into each of two opposite sides of the glass plate is a groove These grooves prevent the formation of urea crystals on the glass spacer at the point where the laser is directed into the gel The face of the glass plate without the logo is that which will be in contact with the gel 3 3 3 Glass spacers The glass spacers are essential components used to assemble a gel cassette together with a thermoplate glass plate and gel clips The glass spacers determine the thickness of the polyacrylamide gel and guides the laser beam through the gel Two glass spacers are used in the preparation of each cassette Glass spacers of two different lengths are available longer spacers for the standard cassette size and shorter spacers for the short cassette assembly Two different thicknesses 0 5 mm and 0 3 mm are available for each length Description of ALFexpress Il 3 3 3 4 Gel clips The gel clips are essential components used to assemble a gel cassette together with a thermoplate glass plate and glass spacers Eight gel clips hold together the glass plate spacers and thermoplateto form the standard gel cassette or only four clips for the short cassette Figure 3 6 Placement of gel clips onto the gel cassette Care must be taken when placing the pairs of clips onto the gel cassette Please refer to the Instructions for placem
25. ea lus holders in tha power ar n modula A 4 Close fhe lid ard check Mal A Ihe correct voltage setting can ba sean in tha window W Figure 5 2 How to change a fuse 1 Locate the power entry module on the back panel of the instrument Remove the power cable from the module 5 3 D nanenane gt gt 2 Open thelid of the module with a screwdriver 3 Insert fuses in the two fuse holders T6 3A for all voltage selections 4 Insert thetwo fuseholdersin the power entry moduleand closethe lid CAUTION If a fuse after replacement blows again switch off the instrument and disconnect the Power supply Cord Please contact your Amersham Biosciences representative CAUTION Do not change the setting on the voltage selector drum The correct voltage should be seen in the window of the closed lid 5 2 Moving the instrument A A Should you wish to move the ALFexpress Il instrument from its original installation position there are a few simple rules to follow WARNING 1 Turnoffthepowerto the instrument and computer and disconnect them from the mains Also disconnect the cables between the computer and the instrument CAUTION 2 Drain the water out of the thermostatic circulator as described in Section 5 1 3 Steps 1 to 3 3 Prepare the new area of lab bench where the instrument is to be installed The lab bench should be level and stable 4 Transfer the instrument and computer to a troll
26. edge nearest leading edge of the gel cassette Ensure that the comb rests flush with the left hand side of the gel cassette Fig 4 5 Usethe correct thickness of comb either 0 3 or 0 5 mm corresponding to the glass spacer thickness Figure 4 5 Placement of the comb into the gel cassette Operation 4 4 3 Casting the gel This section describes how to cast UV polymerised gels such as ReproGel High Resolution and ReproGel Long Read 1 Place the cassette in the centre of the ReproSet using the positioning guide marks on the protection pad _ Lell 3 fi N 1 E Figure 4 6 Placing the ALFexpress Il cassette in ReproSet 2 ReproGel solution is packed in two bottles one large bottle and one small Uncap both bottles and pour the solution from the smaller bottle into the larger A WARNING Use gloves and avoid skin contact with the solutions 3 Pour some of the solutions back and forth between the bottles to recover all of the solution from the small bottle 4 Cutthenozzle screw cap that is provided in the package and screw the nozzle onto the larger bottle 5 Put the nozzle lid on and mix the solutions gently to prevent bubble formation by inverting the bottle several times If bubbles form let the bottle stand for a few minutes before casting the gel Note Thelarger bottle is transparent and exposure of the mixed gel solutions to UV light or sunlight will lead to
27. ent or clips 56 3069 34 provided with the clips 3 3 5 Buffer reservoirs The upper buffer reservoir is already connected to thethermoplate and thus forms an integral part of the assembled gel cassette The assembled gel cassette hangs in thelower buffer reservoir which is itself placed into the instrument as a separate component Figure 3 7 The lower buffer reservoir high voltage electrodes comb and gel clips 3 7 3 Description of ALFexpress Il 3 8 3 3 6 Electrodes Thetwo electrodes are inserted into the upper and lower buffer reservoirs and act as lids to prevent evaporation T he cathode is placed in the upper buffer reservoir and the cable connected to the black female socket on the right side of the front panel The anode is placed in the lower buffer reservoir and the cable is connected to the red female socket on the right side of the front panel If the short cassette is being used a separate black female socket is located on theright side of the front panel at the same level as the cathode see Fig 3 4 3 3 7 Comb The42 well comb is equipped with a stop to ensure a reproduciblewell depth Two extra wells give the possibility to load stop solution or dummy samples in order to further reduce smiling Combs are available in two thicknesses 0 3 mm and 0 5 mm Operation 4 4 Operation This section isa detailed guide to the complete operating procedure It is divided into the following sections 4 1 Lab e
28. equence The labelled fragments can thus be visually interpreted on the screen at the same time as the bands pass the detectors 1 2 1 2 Software Introduction 1 ALFexpress Il is controlled by the ALFwin Control module within the application software ALFwin Sequence Analyser 2 00 or ALFwin Fragment Analyser 1 00 available from Amersham Biosciences The choice of software product is determined by your own needs either for sequence determination or fragment separation respectively Each software allows you to control the run based on specific casebooks that contain all of the experimental run conditions sample information and post run actions Results can be real time monitored on the screen and subsequently subjected to intuitive and graphically interactive analysis Processed data produced by the software can be utilised by different DNA analysis software from Amersham Biosciences e g M utation Analyser AlleleLinks and HLA SequiT yper and also other analysis software e g Format Description Further information GCG The seq format http www gcg com for GCG the sequence analysis program from Wisconsin Univer sity STADEN SCF The scf format Staden Rodger 1982 An interac for the Staden tive graphics program for com sequence analysis paring and aligning nucleic acid package SCF for and amino acid sequences N ucl matincludescurve Acids Res 10 9 2951 2961 data http www mrc Imb cam ac
29. ey and transport them to their new location Note Thecentreof gravity of the instrument is at the rear of the instrument so take care when lifting it 5 If necessary readjust the feet of the instrument so that its sits level on the lab bench Maintenance 5 6 Reconnectthe cables to the mains and between the computer and instrument 7 Fill up the water circulator with water see Section 5 1 3 Step 4 8 Youcan now turn the instrument power on You are now ready to operate the ALFexpress Il instrument again 5 3 Hardware warning messages If there are any problems with the instrument hardware due to operator error or otherwise various warning signals will be given Warning signals comein theform of indicator lampson the front panel of the ALFexpress Il instrument see Fig 5 3 and also as messages displayed on the computer screen Figure 5 3 Indicator lamps and symbols on the front of the instrument The table below describes the various patterns of indicator lamps and identifies the possible problems Any warning on the instrument should be used in combination with the message displayed on the computer to identify the specific problem 5 5 D cds 5 6 Lamp status Warning signal C onsequence action Green lamp only Instrument turned on and connected to computer None Green lamp and Instrument not Check the computer con red lamp only connected to nection to the instrument
30. hate 25 g TEMED 25ml Bind Silane 25 ml Amberlite IRN 150L ReproGel high resolution ReproGel long read Other related product ALFwin Sequence Analyser 2 00 software ALFwin Fragment Analyser 1 00 software M utation Analyser 1 00 software HLA SequiT yper 2 00 software AlleleLinks 1 00 software 27 2690 02 27 2693 02 27 1801 01 27 2694 01 27 4539 01 27 4533 01 27 4534 01 27 4535 01 27 4536 01 27 4537 01 27 4531 01 17 1035 01 17 1307 01 17 1300 01 17 1304 01 17 0889 01 17 1321 01 17 1322 01 17 1324 01 17 1311 01 17 1312 01 17 1330 01 17 1326 01 17 6001 08 17 6001 09 18 1125 88 18 1125 92 18 1118 77 18 1118 79 18 1118 74 Ordering information and technical data 6 6 2 Technical data Electrical requirements Power consumption Dimensions W x D x H Weight Ambient operating temperature Ambient humidity Gel dimensions L x W standard short Sample capacity Thermostatic circulator volume Thermostat equilibration time O perating range Electrophoresis Power supply output Laser specifications Dynamic range EM C Standards 100 120 230 240 V 50 60 Hz 500 VA see Section 4 490 x 535 x 550 800 with lid up mm 33 kg 4 to 40 C M ax 80 270 x 310 mm 200 mm separation distance 150 x 310 mm 80 mm separation distance 40 sample wells for analysis of 10 sequencing clones or 40 fragment samples 0 8 litres 25 30 minutes from 20 to 55 C From 20 C above amb
31. ic circulator to the thermoplate Fig 3 3 The operating temperature range is from 20 C above the ambient temperature up to 60 C 3 1 5 Laser TheHelium N eon laser operates at a wavelength of 632 8 nm A fixed laser power value of 2 5 mW is used which ensures a stable long lifetime for the laser unit The aperture for the laser beam is indicated in Figure 3 3 3 1 6 Optical unit The optical unit inside the instrument consists of a mirror and lens system which guides and focuses the laser beam through the glass spacer into the gel Figure 3 3 The interior of the instrument 1 Gel cassette attachment screws 2 Thread screws 3 Safety interlock switch laser 4 Laser beam aperture 5 Lower buffer reservoir 6 Safety interlock switch high voltage 7 Electrode connectors 8 Detectors 9 Connectors for the thermoplate 3 3 3 Description of ALFexpress Il 3 1 7 Detector unit Thedetector unit includes 40 photodetectors one for each sample lane in the gel Each photodetector is located behind its individual aperture The light emitted when the laser beam excites the DyeA midite 667 label on the DNA molecule is detected as it passes in front of the aperture In front of the detectors are optical filters to eliminate background and stray light 3 1 8 Safety devices There are two safety interlock switches which protect the operator from the laser beam These are described in detail in Section 2 and are also indicated
32. ient room temperature ambient 20 C up to 60 C 35 2500 V 1 100 W 1 150 mA H elium N eon 2 5 mW output 632 8 nm 15 000 hours lifetime 50 amol 45 fmol This product meets the requirement of the EM C Directive 89 336 EEC through the harmonized standards EN 50081 1 emission and EN 50082 1 immunity N ote The declaration of conformity is valid for the instrument when it is used in laboratory locations used in the same state as it was delivered from Amersham Biosciences AB except for alterations described in the user manual 6 3 Doro information and technical data 6 4 Safety Standards Computer requirements Computer RAM memory Recommended free hard disk space O perating system Graphics Serial ports Printer used asa stand alone unit or connected to other CE labelled Amersham Biosciences AB instruments or other products as recommended This product meets the requirement of the Low Voltage Directive LVD 73 23 EEC through the harmonized standard EN 61010 1 Compaq Pentium 233 M Hz minimum PC with Pentium processor clock speed 90 MHz 64 M B minimum 32 M B 300 MB if running two ALFexpress systems simultaneously Windows 95 international edition including Service release 1 4 00 950b 800 x 600 pixels with 64k colours minimum 800 x 600 SuperVGA graphics adapter 256 colours 2 serial ports are recommended allows two instruments to be run Please contact your loc
33. in Figures 2 1 and 3 3 Thelid of the instrument is connected to a safety interlock switch which prevents the high voltage being applied while the lid is open 3 1 9 Electrode connections The two electrodes act as lids to the buffer reservoirs Each electrode has a cable that is connected to the instrument into female sockets on the right hand side of the front panel Fig 3 4 3 1 10 Power supply A modular power supply built into the rear of the instrument supplies the high voltage for the electrophoresis operations The mains power switch is located at the rear of the instrument on the right hand side A green and a red lamp on the front panel illuminate when the instrument is switched on When the control software is connected to the instrument the red lamp will switch off 3 2 Computer and software 3 4 The instrument is controlled by an external computer running the appropriate control software In addition to controlling the instrument the software also stores and processes the signals from the detectors The experimental conditions for the electrophoresis run and the run log are saved together with the results See the ALFwin Sequence Analyser 2 00 or ALFwin Fragment Analyser 1 00 User M anual as appropriate Description of ALFexpress Il 3 3 3 Instrument accessories The instrument needs several other accessories to function E Figure 3 4 The standard gel cassette placed into the ALFexpress Il instrument 1
34. ing 4 7 gel clips 3 7 gels ReproGe 1 5 glass plate 3 6 glass spacers 3 6 indicator lamps 3 1 5 5 warning 5 5 information on the web 1 5 internal lamp 3 2 L labelling DyeAmidite 667 1 4 laboratory equipment needed 4 1 lamp internal 3 2 laser adjustment 4 14 laser alignment 4 11 laser indicator lamp 3 1 5 5 laser safety 2 1 loading samples 4 12 Index lower buffer reservoir 3 7 O optical unit 3 3 ordering information 6 1 P plastic spring thermostatic circulator 4 10 power supply 3 4 R reagents for sequencing 1 4 ReproGel 4 7 run conditions 4 9 ReproSet 1 5 4 7 reservoirs for buffer 3 7 ROPs on the web 1 5 run conditions 4 9 S safety chemical handling 2 3 electrical 2 1 laser 2 1 laser labels 2 3 safety devices 3 4 samples loading 4 12 sequencing reagents 1 4 size markers 1 4 software ALFwin Fragment Analyser 1 00 1 3 ALFwin Sequence Analyser 2 00 1 3 spacers glass 3 6 spare parts and accessories 6 1 spring thermostatic circulator 4 10 STADEN SCF export format 1 3 starting the run 4 13 T technical specification 6 3 temperature indicator lamp 3 1 5 5 thermocirculator connection 4 10 thermoplate sizes 3 5 thermostatic circulator 3 2 Index thermostatic circulator changing the water 5 1 thermostatic circulator filling 5 1 U upper buffer reservoir 3 7 UV box 1 5 4 7 Vv voltage indicator lamp 3 1 5 5 W warning messages 5 5 water circulator 3 2 water circula
35. nt as a guide only see Fig 4 7 It is important that the gel cassette is correctly positioned to allow the laser entry into the gel Operation 4 Figure 4 9 ALFexpress Il instrument 1 Markings on the instrument used to guide the loading of sequencing samples or fragments 2 Thread screws for vertical repositioning of the gel cassette 4 5 2 Starting the run 1 Place the electrodes into the buffer reservoirs The anode has a groove on each side to accommodate the thermostatic circulator connectors The two electrodes act as a lids to the reservoirs and limit evaporation of the buffer Each electrode has a coloured cable and plug black for cathode and red for anode which is connected to the instrument by insertion into female sockets on the right side of the front panel Check that there are no dust particles in the gel or on the glass in front of the detectors If some dust particles are in evidence the whole cassette can be repositioned in the vertical plane by simultaneous movement of two thread screws on the front panel Fig 4 7 Try and balance by eye the horizontal alignment of the two markings on the thermoplate Fig 4 8 Figure 4 10 Markings on the thermoplate and instrument for correct horizontal alignment 1 Horizontal line on the instrument 2 Horizontal line on the thermoplate 4 13 4 Operation 3 Asa final check that the laser is correctly positioned click on the Realign Laser button in the M
36. per buffer reservoir and hang the whole cassette into the front of theinstrument by locating the two holes at the top of the cassette onto the corresponding fixed screws protruding from the instrument Fig 4 8 The whole cassette should now sit in an off vertical position with the lower edge of the cassette hanging within the volume of the lower buffer reservoir The plastic spring on the right hand thermostatic circulator connector of the thermoplate should have guided the cassette into the correct lateral position within the instrument Note Check that the gel cassette makes proper contact with the safety interlock switches behind If you did not fit the gel clips correctly when assembling the gel cassette the gel cassette will not hang properly to make proper contact 4 Fill the upper and lower buffer reservoirs with the appropriate running buffer to the maximum level marks inside the reservoirs For example the ROP recommends 0 5x TBE buffer from a 10x stock solution 5 Connect the two push fit connectors of the instrument s thermocirculator to the appropriate connections on the thermoplate Depression of the two safety interlock switches behind the gel cassette should automatically allow water to be pumped through the thermoplate on connection to the thermostatic circulator system This is indicated by a blinking yellow lamp for the thermostatic circulator When the running temperature is reached the illumination of the yellow
37. polymerisation 6 Removethe nozzle lid 4 7 4 Operation 4 8 Figure 4 7 Casting the gel Pour thesolution slowly and evenly back and forth along theopen slit between the glass plates at the lower edge of the gel cassette Keep a steady pressure during the procedure Capillary action will draw thesolution front upwards to fill the entire space between the plates If there is a tendency for the gel front to become uneven tap the glass plate lightly with a finger This problem can be due to dirty glass plates and or insufficient polishing with ethanol Note Releasing the pressure on the bottle can let air in and cause 10 11 bubbles on the gel This can be avoided by first turning the nozzle upwards before releasing pressure on the bottle If bubbles remain in the gel they can be removed by using a bubble chaser a 0 2 mm thin strip of plastic spacer with a cut groove supplied with the instrument Let the filled cassette stand for a minute while the capillary action of the gel front ceases Uncap the large bottle with the remaining gel solution and leave it under UV light during exposure of the gel This prevents unpolymerised hazardous gel solutions being left in the bottle Set the R eproSet timer localised on the lower part of the lamp housing lid to 10 minutes default value Pull down the lamp housing lid and push the start button on its front After polymerisation the gel is ready for immediate
38. quipment 4 2 Assembling the gel cassette 4 3 Casting the gel 4 4 Fitting the gel cassette into the instrument 4 5 Loading samples and starting the run 4 6 Ending the run 4 1 Lab equipment The equipment list below will speed up and simplify sample preparation and gel casting M icrocentrifuge for 1 5 ml capacity Eppendorf tubes Balance 1 to 500 g 0 1 g accuracy Heating block or water bath 20 to 90 C Water vacuum pump or equivalent UV box for photo polymerisation Fume hood Freezer 20 C Refrigerator 4 C Eppendorf tube holder Filtration equipment with 0 45 um filter for preparation of the gel solution M easuring cylinders 100 ml 1000 ml and 2000 ml capacity M icropipettes Eppendorf 0 5 to 1 0 ul 10 to 100 ul 100 to 1000 ul Sterile pipette tips Eppendorf tubes M icro Sample Plates Amersham Biosciences Code No 18 1013 69 Lint free medical wipes Kimwipes or similar Disposable gloves Syringe 50 ml N eedles 0 6 mm diameter N eutral laboratory detergent solution e g 5 v v Decon 90 solution 4 1 4 Operation 4 2 Assembling the gel cassette 4 2 This section deals with the cleaning and assembly of the gel cassette Cleaning the gel cassette 4 2 1 Pre assembly cleaning A new gel cassette must be thoroughly cleaned before using it for the first time To avoid uneven band migration and band broadening in subsequent runs it is essential that all parts of the gel cassette are clean
39. t free tissue Operation 4 4 Wet the surface of the thermoplate and the side of the glass plate without the logo with non denatured ethanol Polish dry with lint free tissues 5 Examine each item carefully under reflected light Ensure that there are no residues remaining and that the surfaces are free from scratches and dust Spacers combs and gel clips should be clean dry and free from residues The glass spacers need special attention They should be thoroughly cleaned and have no cracks chips or marks 4 2 2 Bind Silane treatment Bind Silane treatment of the thermoplate and glass plate is recommended WARNING Bind Silane is toxic and highly volatile and should be used only in a fume hood To prevent contact with the skin and eyes always use disposable gloves and safety goggles 1 Working in the fume hood set down the thermoplate so that the side with the upper buffer reservoir is positioned nearest to you Fig 4 1 The thermoplate stands on the upper buffer reservoir and the two other legs on the thermoplate to give level support Figure 4 1 Orientation of the thermoplate and glass plate placed in front of you in the fume hood The top 5 cm of the glass plate and 2 3 cm of the thermoplate have been Bind Silane treated 2 Immediately before use prepare Bind Silane solution by mixing together the following components 4 3 4 Operation 4 4 Absolute ethanol 1 0 ml Bind Silane 3 0 ul 10 Acetic acid v
40. tandard Glass plate standard Thermoplate short Glass plate short Spacer 0 5 mm standard pack of 8 Spacer 0 3 mm standard pack of 8 Spacer 0 5 mm short pack of 8 Spacer 0 3 mm short pack of 8 Comb 0 5 mm Comb 0 3 mm Electrode upper Electrode lower Buffer tank lower Gel clips pack of 8 RS232 cable Plastic spacing spring Standard plate kit Short plate kit Cooler Kit ALFexpress Il ALFexpress Il User M anual M anual box pack of 2 R eproSet Quantity supplied One unless indicated otherwise 18 1123 51 18 1115 01 18 1123 52 18 1115 03 18 1116 14 18 1116 16 18 1116 15 18 1116 17 18 1105 94 18 1105 95 18 1105 96 18 1105 97 18 1105 98 18 1124 20 18 1106 00 18 1106 01 18 1123 59 18 1123 60 18 1125 85 18 1127 13 18 1127 20 18 1125 64 6 1 Doro information and technical data 6 2 Chemicals and reagents ALFexpress AutoR ead Sequencing Kit ALFexpress A utoC ycle Sequencing kit DyeA midite 100 mg ALFexpress dATP Labelling mix ALFexpress Sizer 50 500 bp external ladder ALFexpress Sizer 50 bp ALFexpress Sizer 100 bp ALFexpress Sizer 150 bp ALFexpress Sizer 200 bp ALFexpress Sizer 250 bp ALFexpress Sizer 300 bp ReadyM ix Gel ALF grade 6 X 75 ml alternatively ReadyM ix acrylamide bisacrylamide 196 6 10 4 g Acrylamide 250 g Bisacrylamide 25 g Urea ALF grade 500 g Tris 500 g Boric acid 500 g EDTA disodium salt 100 g Ammonium persulp
41. ter filling tube vertical and pour in approximately 0 8 litres of M illiQ water until water starts coming out of the draining tube The water level should be between M in and M ax when the tubing is in this upright position e Remove the funnel and squeeze back the water filling tubing into its place Remove the drain tubing CAUTION If the instrument has to be transported it is important to empty the water prior to transportation 5 1 4 Changing dust filters There is an air filter on the right side of the instrument that should be changed once per year or more regularly if necessary The air filter prevents dust entering the system that could otherwise interfere with the laser operation Maintenance 5 1 Loosen the four quick release fasteners and remove the panel 2 Remove the old filter and insert a new one 3 Replace the panel and tighten the four fasteners 5 1 5 Lubricating the water connectors There aretwo push fit couplings with O rings on the thermoplate Apply regularly a thin layer of Vaseline on the O rings to lubricate the couplings 5 1 6 Changing a fuse WARNING To changea fuse on the instrument first make sure that the instrument is turned off and the cable removed from the mains outlet ian n n cn TM om Power enty modula 1 Dpen io with i on back panal a Screwed iver A te fi Insert husa in the two fusa la holders Te TEJA lor all vollage selections i ingen tha t
42. tor filling 5 1 water circulatorchanging the water 5 1 water connectors lubricating 5 3 water heating 4 10 web address 1 5 Amersham e Y Biosciences 18 1127 13 Edition AA Printed in Sweden by Tryck Jouren 9803
43. use although can be stored up to one hour at room temperature Operation 4 12 If there are any gel traces on the protection pad remove them with water and detergent For further information regarding the R eproSet please see the ReproSet User M anual order no 18 1125 68 supplied with the ReproSet For further information regarding the gels please see the R eproGel Instruction order no 71 5005 91 provided with the product 4 4 Preparing for a run 4 4 1 Setting the running conditions 1 Turn on the computer and launch the ALFwin Software application 2 StarttheALFwin Control module for the instrument that you are using 3 Openanexisting casebook or create a casebook for your run The following run conditions are recommended for use with UV polymerised gels Condition ReproG el high res ReproGel long read Voltage V 1500 1500 Current MA 60 60 Power W 30 25 Temp C 55 55 Run time min 450 750 4 Save the casebook For further details about making a run from ALFwin Control please refer to ALFwin Sequence Analyser 2 00 User M anual or ALFwin Fragment Analyser 1 00 User M anual 4 9 4 Operation 4 4 2 Fitting the cassette into the instrument 1 Check that the outer surface of the glass plate is completely clean especially around the area of the detectors 2 Place the lower buffer reservoir into the front of the instrument Fig 4 6 3 Lift the gel cassette by the ears of the up
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