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Gene Navigator™ System User manual

1. 33 6 Programming the controller 34 W hen you have indicated and entered the phase time for the final phase of your procedure press ESC instead of entering again Now theLCD will indicate the procedure number the total run time or sum of all the phase times in the procedure and the total number of phases as shown below Procedure 1 Runtime 007 30 Phases 3 IMPORTANT Theprocedure you have just created will be erased if you push ESC to return to the main menu before first having stored the procedure To store the procedure press ESC once moreto return to the edit menu see Edit store section below 6 5 3 Edit pulses Edit1 gt Setup Input PULSES Store Delete Check number of pulses Use jor gt to movethe cursor to pulses and press ENT Now the screen will display the number of pulses that will result from the pulse and run TIM E values entered in the first phase of the procedure you are editing This display may look like this Procedure 1 Phase Pulses lof3 600 Use or gt to move between all phasesin the procedure GN Controller will calculate and display the number of pulses that will occur in each phase of the scheduled run Thenumber of pulses can be adjusted prior to the run by returning to edit input see Section 6 5 2 and changing the pulse and run time values as de
2. Programming the phases EDIT mode Display Comments GN control unit Press a key to continue Press a key Main gt BASIC Edit Load Setup Highlight EDIT then enter by using and ENT Edit gt Select procedure numbrer Example Press 1 Edit 1 gt SETUP Input Store Delete Press gt ENT to reach IN PUT mode and the programming of the different phases SET UP can also be be programmed before IN PUT Phase N S e w phase time PHASE must first be 1of1 000 0 000 0 00 00 00 highlighted then press ENT to reach next menu Programming each phaseislike programming in BASIC mode Phase Select action Insert Press ENT to 1of1 None INSERT Delete enter phase 1 into the working memory By inserting phase 1 the operator automatically moves to the programming of the next phase NONE or DELETE is used when you S steps 2 3 4 5 6 revise a program Note To end use ESC ESC not INSERT 58 10 Short instructions Procedure 1 Run time 25 00 00 Phases 6 ESC Edit gt Setup INPUT Store Delete Edit 1 gt Store as procedure 1 9 ESC M ain gt Basic EDIT Load Setup Both the total run time of all phases and thenumber of programmed phases are displayed Pre
3. of the comb touch the bottom of thetray Check that the clips are fixed on the edge of the frame see Fig 6 Leave the clips in position on the comb remove the comb and set it aside momentarily Pour the gel into the frame making sure that the holes or depressions inside the frame are filled Fig 5 If necessary suck out air bubbles with a Pasteur pipette 13 5 Operation 14 Fig 5 Pouring gel into the rubber casting frame which is positioned for separations using the HEX electrode Placethe comb in the gel The plastic clips should now rest on the top of the gel casting frame holding the comb firmly in place Fig 6 below If you use point electrodes Fig 3 place the comb parallel to the side of the gel that will be facing the southeast corner nearest right corner of the tank the walls of the tank are marked N orth South East and W est and about 2 cm away from the edge of the gel Fig 6 Setting the comb into the gel Note the placement of the clips 5 Operation 5 4 Sample loading Liquid samples should be loaded after the gel tray and electrode s have been placed in thetank asis also the case with submarine gels Sample blocks should be loaded before the gel is submerged The blocks can be removed from the sample tube by using a flame sterilized Pasteur pipette with a bent tip Thetip can easily be bent over a flame Blocks should be cut on a sterile surface for e
4. 4155 4169 Spithill T W Samaras N PFG L eishmania M igration properties of circular DN As using orthogonal field alternation gel electrophoresis Electrophoresis 10 1989 283 290 Hightower R C Santi D V OFAGE circular plasmids supercoiled plasmids Leishmania Chromosomes of Plasmodium falciparum Papau N ew Guinea M eil 29 1986 95 101 Corcoran L M Kemp D J PFG Plasmodium Size variation in chromosomes from independent cultured isolates of Plasmodium falciparum N ature 315 1985 347 350 Kemp DJ PFG Plasmodium Chromosome size polymorphisms in Plasmodium falciparum involve deletions and are frequent in natural parasite populations CELL 44 1986 87 95 Corcoran L M etal PFG Application M alaria Plasmodium Resolution of Acanthamoeba castellanii chromosomes by pulsed field gel electrophoresis and construction of the initial linkage map Chromosoma Berlin W est Germany 1988 Vol 97 no 31 p219 223 Rimm D L Pollard T D H ieter Karyotyping protozoan A cantamoeba O FAG E M olecular karyotyping Parasitology today 1 1985 64 65 Gibson W C PFG Trypanosoma H eat schock genes regulatory role for differentiation in parasitic protozoa Science 228 1985 1443 1446 Van der Ploeg L H T etal PFG Trypanosoma L eishmania 63 10 Reference list 64 Reference 27 Author Keyword Reference 28 Author Keyword Reference 29 Author Keyword Reference 3
5. Author Keyword Reference 57 Author Keyword Reference 58 Author Keyword Reference 59 Author Keyword Reference 60 Author Keyword Reference 61 Author Keyword Reference 62 Author Keyword Long range restriction site mapping of mammalian genomic DNA Nature 322 1896 477 481 Brown W R A Bird P PFG M apping M ammalian Direct construction of a chromosome specific N ot l linking library from flow sorted chromosomes N ucleic Acids Res 1989 Vol 17 no 4 p1665 1678 Wallace M R Fountain J W Brereton A M Collins F S A largeinverted duplication allows homologous recombination between chromosomes heterozygous for the proximal T complex inversion CELL 48 1987 813 825 Herrmann B G etal PFG M apping M ouse Chromosome 17 M apping of the class Il region of the human major histocompatibility complex by pulsed field gel electrophoresis N ature 323 1986 453 455 Hardy D A etal PFG M H C M apping H istocompability complex Long range restriction map around the Duchenne M uscular Dystrophy gene N ature 324 1986 582 585 Burmeister M Lehrach H PFG Gene mapping D M D Duchenne H uman Long range structure of H ras 1 selected transgenomes Somatic Cell M ol Genet 1989 Vol 15 no 3 p229 236 Bickmore W A et al human chromosome 11 oncogene M olecular analysis of the D uchennes muscular dystrophy region using pulsed field gel electrophoresis CELL 48 1987
6. Fig 9 Setting gel support tray in tank The gel support tray can be placed in only one way due to its cut out corner Fig 10 Position of HEX electrode and gel support tray in the electrophoresis tank 17 5 Operation 18 Lower thetray carefully until it is resting securely on theinner edge of the tank If the hinged gate over the pump is open when you lower the gel support tray the gate will close automatically To start circulating the buffer plug the pump into the mains power supply outlet Always make sure that there is buffer in the tank when the pump is running If you are using the H EX electrode make surethat the hook on the electrode rests on the center of the east or right hand power rail when you placethe electrode in thetank Then press each of the 3 power connectors that are attached to the electrode into place on the north south and west power rails When theH EX electrode is used there are no separate electrodes to position All are fixed within the H EX electrode W hen you use the point electrodes however you must press them over the power railsin the following manner The point electrode configuration is known as the double inhomo geneous D1 DI Configuration North wall 1 anode orange in position 90 West wall 1 anode orange in position 90 South wall 3 cathodes black in positions 5 95 180 East wall 3 cathodes black in positions
7. Gibson W C Miles M A PFG T rypanosoma Pulsed field gradient electrophoresis of DNA digested in agarose allows the sizing of the large duplication unit of a surface antigen gene in trypanosomes Gene 42 1986 313 322 Bernards A etal PFG T rypanosomes Chromsomes of Kinetoplastida Embo J 3 1984 3109 3115 van der Ploeg L H T et al PFG Trypanosomes C rithidia 10 Reference list Reference 36 Authur Keyword Reference 37 Author Keyword Reference 38 Author Keyword Reference 39 Author Keyword Reference 40 Author Keyword Reference 41 Author Keyword Reference 42 Author Keyword Reference 43 Author Keyword Reference 44 Author Keyword M igration properties of circular DN As using orthogonal field alternation gel electrophoresis Electrophoresis 10 1989 283 290 Robin C Hightower and Daniel V Santi OFAGE circular plasmids supercoiled plasmids Leishmania Transposition of structural genes to an expression sequence on a linear plasmid causes antigenic variation in the bacterium Borrelia hermsii N ature 318 1985 257 263 Plasterk R H A etal PFG Borrelia Plasmid A physical map of the Escherichia coli K12 genome Science 236 1987 1448 1453 Smith C L etal Pulsaphor PFG N ot 1 E coli Pulsed Field Gel Electrophoresis of circular DNA N ucleic Acids Res 1989 Vol 17 no 11 p4359 4366 Simske J S Scherer S circular D N A supercoil
8. e used as a stand alone unit or connected to other CE labelled Amersham Biosciences AB instruments or other products as recommended This products meets the requirements of the Low Voltage Directive LVD 73 23 EEC through the harmonised standards EN61010 1 10 Short instructions 10 1 Gene Navigator System 10 Short instructions and reference list The short instructions consists of two parts Gene N avigator short instruction which makes it possible for a person who is not experienced with pulsed field electrophoresis to perform a run and GN Controller short instruction which explains the programming of the controller in a way that is easy to understand R ead them carefully to avoid any mistakes For further instructions please read chapters 5 and 6 This short instruction applies primarily to separations using the H EX electrode L Materials required Chemicals 250 ml 0 5M 5x TBE buffer 1 3 g agarose N A 17 0554 01 02 03 Sample DN A immobilized in gel inserts 2x5x10 mm Ethidium bromide EtBr solution 0 5 ml 1 0 mg ml Pasteur pipettes 3 pcs CV IES Equipment 6 GeneNavigator with HEX electrode kit 18 1019 20 consisting of H exagonal electrode array 15x15 cm gel support tray 15x15 cm gel casting frame and three double combs 7 Disposable pipette tips 5 blue 8 Gilson pipette P1000 9 GN control unit 18 1026 17 10 Hoseclamps 10 pcs 18 1104 27 included in Gene N avigator electro
9. ria 21 6 1 GN Controller dado 21 6 2 Contrast and reset commands 23 0 3 BASICIMO Gear 24 64 SUP EE 26 0 9 EMO tala 30 6 6 Loading a procedure 37 7 Evaluation and presentation of data u nr 39 7 1 Electrical poarameters 39 7 2 Relationship between pulse time and resol Man 40 7 3 System MESSAGES ia 43 8 Maintenance and troubleshooting 0 0 45 8 1 Manten Cessna 45 8 2 Troubleshooting 2 eegteeensg eege e 45 9 Ordering information and technical data 49 9 1 Ordering information 49 9 2 Technical data ateos 50 10 Short instructions and reference list uu 51 10 1 GeneN avigator system AA 51 10 2 GN Controller EE 56 10 3 Referencelist E 60 1 Introduction 1 Introduction Congratulations Y ou are about to use GeneN avigator system from Amersham Biosciences GeneN avigator represents a state of the art system and is designed to provide safe reliable performance of pulsed field gel electrophoresis PFGE Gene N avigator is also flexible Point electrodes generating non homogeneous fields and HEX electrodes generating homogeneous fields can be used with optimal resolution GeneN avigator permits radical extension of the molecular weight separation range by enhancing the effective use of the PFGE technique Asa result very large DN A molecules can be separated Thelargest chromosome separated with Gene N avigator
10. 31 6 Programming the controller 32 6 5 2 Edit input Editl gt SETUP Input Pulses Store Delete Setup procedure Select IN PUT by pressing gt and ENT Now thefollowing screen appears phase N S e w phase time lof1 000 0 000 0 000 00 N ow you are positioned to indicate the parameter values desired for thefirst phase of the first procedure or run T he phase field which now indicates phase 1 of 1 shows how many phases you have created and which one you residein at the moment The cursor begins on the N S pulse value Usethe number keys to indicate the desired N S pulsetime we shall use 60 seconds for purposes of illustration then press ENT or gt to moveto the E W pulse parameter N ow indicate 60 0 seconds for example The digits move from right to left as they are indicated In this setup scale 000 0 seconds you press 6 10 10 for 60 seconds Y ou can also change the default scale and customize it for each procedure if desired by selecting SETUP from the edit menu as noted earlier in this section W hen the E W pulse time has been entered indicate the desired time for the first phase If for example you indicate 2 hours and 30 minutes the cursor will move to the phase field as below when the phase time has been indicated and entered e PHASE n s e w phase time lof1 060 0 060 0 002 30 If you wi
11. 351 357 Kenwrick S etal PFG DM D M apping Duchenne H uman Construction of a general human chromosome jumping library with application to cystic fibrosis Science 235 1987 1046 1049 Collins F S et al OFAGE M apping PFG umping Patterns of polymorphism and linkage disequilibrium for cystic fibrosis Genomics 1 1987 257 263 Estivill X et al Gene mapping C F R FLP C ystic fibrosis 67 10 Reference list 68 Reference 63 Author Keyword Reference 64 Author Keyword Reference 65 Author Keyword Reference 66 Author Keyword Reference 67 Author Keyword Reference 68 Author Keyword Reference 69 Author Keyword Pulsed Field Gel Electrophoresis identifies a high degree of variability in the number of tandem 21 hydroxylase and complement C 4 gene repeats in 21 hydroxylase deficiency haplotypes EM BO J 1989 vol 8 no 5 p 1393 1402 Collier S etal human mapping blotting An extended long range restriction map of the human sex determining region on Y P including ZFY finds marked homology on X P and no detectable Y sequencesin an X X male American J of Human Genetics US 1989 vol 44 no 5 p 756 765 Verga V Erickson R Y chromosomef translocation break points Chromosome 7 long arm deletion breakpoints in preleukemia mapping by pulsed field gel electrophoresis N ucleic Acids Res 1989 Vol 17 no 4 p1511 1520 Kere J H uman lymphocytes mappin
12. 5 95 180 Straighter lanes can be achieved by either moving the electrodes to other positions or putting a fourth cathode inside the unit Place the safety lid carefully on the unit making sure that the pins on the west left side of the unit arein position before you lower the east right side and secure the safety electrical coupling T he electrical connection to the electrophoresis unit will break when you lift the latching mechanism on the right hand side east wall of the lid N evertheless for absolute safety you should always turn off the power supply before removing the lid Also make sure that the maximum values set for the power supply do not exceed 450 V and 500 mA before you plug the controller s DC inlet banana plugs into the power supply When using GN Controller you can either store all run parameters in a memory in the controller or work in BASIC mode where the procedure cannot bestored Programming GN Controller is described in detail in Section 6 5 Operation 5 6 Removing the gel Before starting a run you should check the performance of the electrodes and the levelling of the electrophoresis unit This is most easily done by using BASIC mode Program 4 0 sin N S and E W and set the voltage Activate the power supply and read the current in each field Thecurrent difference should not exceed morethan 10 mA at approximately 200 V or 3 mA at approximately 30 V This is called current levelling and
13. a fuse may blow out Placethe EPS 600 or EPS 200 power supply on the bench or on a shelf close to Gene N avigator electrophoresis unit If you usea different power supply check that its ground current leakage circuit is compatible with the controller To do this run an application and check if the power supply cuts the current Carefully unpack GN control unit and placeit on a work bench so that you can look at the rear panel 4 Installation 10 MAINS ATTENTION a CAUTION m MAINS M I em EH MAINS SE S 4 Fig 2 Rear panel To begin set the mains selector switch at the correct voltage 115 V or 220 V Check that the power rating on the power supply is appropriate Screw in the correct fuseto match the selected voltage Plugthemains power cable into both the mains power source and the mainsinlet socket on the rear panel It may bea good idea to conduct a trial run through the control unit s operational functions and programming steps once before connecting the separation unit s 4 pole plug or the power supply s inlet plugs see Section 10 2 To perform this trial run turn GN Controller On by pushing the mains toggle switch located just above the mains inlet plug Proceed to section 10 2 Y ou can practice programming GN Controller and become familiar with the various operational modes and procedures without activating the procedure that you have programmed W hen
14. different procedures or sets of customized run parameters each with up to 6 phases that can bestored and quickly and easily repeated IN PUT is a command that makes it possible to either create or call a procedure for revision Thefigure C above represents a blinking cursor on yourLCD Create a procedure by pressing a number between 0 and 9 Zero will always take you to the next available new procedure in this case number 1 If you press the 0 key followed by ENT the following LCD message will appear Edit1 gt SETUP Input Pulses Store Delete Check or change procedure modes 6 Programming the controller 6 5 1 Edit setup As mentioned in Section 6 4 you can enter the setup from the main menu to change the default connection stepping or interpolation and time parameter scale units Unless changed these defaults will appear in both the BASIC and EDIT modes The only exception is interpolation which can only exist in EDIT mode In the EDIT mode however the default setup can be changed or customized for each procedure To do this press ENT on SETUP from the above Edit menu Now you can customize both the connection and scale for this particular procedure For the scale follow the same steps outlined in the SETUP mode Interpolation will only affect procedures containing more than one phase thus it is only used in the EDIT mode Setup connection Stepping STEPPING I
15. is the easiest way to check the system When both electrical fields are working correctly you can also seethe electrodes firing by observing bubbles To makea run using only one phase which is similar to one set of pulse times and run times use either BASIC or EDIT To program several phases use EDIT mode where you can store your run procedure To stop the run manually presstheO N OFF button on the front panel of GN Controller WARNING Turn the power supply OFF before opening thelid To remove the gel slide a razor blade or a microscope coverslip beneath it to cut off the agarose in the holes and release the gel from the bottom of thetray then slide the gel into a staining dish Some users think that it is easier however to first slide the gel onto a plastic staining tray delivered with the electrophoresis unit which is 20 x 25 cm long and then place this tray directly into the staining dish This plastic tray is also used to pour off excessive buffer from the gels The old buffer from the tank can now be reused for staining To pump the buffer from the tank into a container lift the left west side of the tank slightly and raise the collapsible leg Let the tank rest on this leg thereby tilting it so that the buffer shifts toward the right side wherethe pump hole is located Use the piece of silicone tubing that you cut earlier see Section 4 1 to lift up the hinged gate on the right side of the ta
16. previous procedure with the newly edited version by storing the edited version under the same procedure number Y ou also have the option however of keeping the original procedure while creating a revised version of it with a new procedure number 6 5 5 Edit delete Any procedurethat has been stored in GN Controller can easily be deleted from its memory Thisis donefrom the edit menu compared to deleting a phase which is donefrom the edit input menu M ovethe cursor to DELETE and push ENT Thenumbers of all stored procedures will appear following Existing procedures W hen you press one of these numbers on the keypad followed by ENT that procedure will be permanently removed from the memory Example Edit procedure W hen several procedures have been created IN PUT and stored STORE you may find it faster to edit an existing stored procedure than to delete an existing one and begin creating a new one again The following example describes how EDIT can be used to quickly change to whatever extent is required a procedure that has been previously stored Let us say you have created and stored 9 different procedures Y ou cannot therefore create another procedure unless you first delete or edit an existing one Instead of deleting a procedure however you can revise one of these 9 Y ou decideto editthe procedurethat you have earlier created and stored as number 3 It contains 6 phases and wa
17. product has been installed and used according to the instructions supplied by Amersham Biosciences AB Amersham Biosciences AB shall in no event be liable for incidental or consequential damages including without limitation lost profits loss of income loss of business opportunities loss of use and other related exposures however caused arising from the faulty and incorrect use of the product Trade marks GeneN avigator is the exclusive trade mark of Amersham Biosciences AB In view of the risk of trade mark degeneration it is respectfully suggested that authors wishing to use these designations refer to their trademark status at least oncein each article Copyright 1997 Amersham Biosciences AB All rights reserved N o part of this publication may be reproduced stored in a retrieval system or transmitted in any form by any means without permission in written form from the company 1 Contents Contents kk Introduction ira 3 2 Important safety information ri 5 3 Description of the System 7 4 Taste i ada 9 4 1 GeneN avigator electrophoresis unit 9 4 2 GN Controller siii 9 5 o Operatiohi icu ia 11 5 1 Preparing the tank 11 5 2 Preparingthe ga sirva 11 5 3 Casting OES n 12 5 4 Sampleloading EE 15 Dis RUNNING tds 16 5 6 Removing the gel ccccccceesessseeeeeeeeseeees 19 5 7 Photographing stained oels 20 6 Programming the controller
18. program and store a run procedureto use later select the Edit mode see Section 6 5 The upper case letters indicate that the screen cursor ison BASIC If the cursor indication isnot in the BASIC position use an arrow key to move the cursor to BASIC Then moveinto BASIC by pressing the ENT key Asan alternative you may use the numeric keys to move directly into the main menu choices If you do so you will not need to press ENT Press 1 to moveinto BASIC 2 to moveinto EDIT 3 to move into LOAD 4 to move into SETUP mode W hen you have entered the BASIC mode the following default parameter settings will be displayed on the LCD N 5000 0 e w 000 0 time000 00 Start by ON Freeze by OFF End by ESC 6 Programming the controller Now you must fill in the numbers for the N orth South N S and East W est E W pulse times as well as the total run time T he default scale unit for pulse time is seconds SSS s and for run time HHH M M When you start a run with the run time set at zero the run will continue until you press off or until the power supply turns off As you press the digits on the numerical keyboard the parameter numbers will move from right to left For a North South pulse time of one minute 60 0 seconds therefore you press 6 0 10 on the numerical keypad if default units are used SSS s see Section 6 4 or for
19. the Stepping mode or 5 phases in the Interpolation mode W hen you have entered all of the parameter values for the final phase of this procedure press ESC from the phase field and review the total run time Then press ESC again to return to the edit menu from which you must moveto STORE to save the new procedure All of the phases just created will be erased if you return to the main menu beforethe procedure has been stored Phase Select action Insert lofl NONE Insert Delete Instead of INSERT you may select NONE from the above phase menu to move back into the phase parameter field you just left Y ou can then check and change any or all of the parameter settings previously entered TheN ONE option takes you back into the previous phase analogous to the ESC key that moves you back to a previous menu in the menu structure W hen editing a procedure ESC moves you out of the procedure and back to the edit menu From the phase menu you may also chooseto delete a phase Phase Select action Insert lofl None Insert DELETE W hen a number of phases have been entered move quickly between them using and gt Push ENT on any phase field PH ASE capitalized to bring up the phase menu as shown above Then use to movethe cursor to DELETE When ENT is pressed the indicated phase will be instantly deleted
20. well Use a bent glass Pasteur pipette or a syringe to suck the inserts down into the well 7 Buffer breakdown Change according to recommendation 8 Distorted wells Cast a new gel No bands 1 Check samples poor lysis of cells will result in stained material only in the wells 2 The DNA is severely degraded due to wrong treatment or storage buffer Separation has gone too far Abnormal mobility 1 Wrong buffer concentration 2 You forgot to turn on the buffer circulation or pump is not working due to formation of salt crystals in the pump 3 Voltage current too high High currents cf table 1 1 Check buffer concentration and level it should be 2 5 litres of 0 15x or 0 5x or 1 0x TBE Check the actual amperage 2 Check electrodes 47 48 9 Ordering information and technical data 9 1 Ordering information 9 Ordering information and technical data Accessories Product Code no EPS 600 power supply 19 0600 00 EPS 200 power supply 19 0200 00 MultiTemp III thermostatic circulator for 100 120 V 60 Hz 18 1102 77 or for 220 240 V 50 Hz 18 1102 78 MacroVue UV 20 Transilluminator 115 V 80 6245 11 or 230 V 80 6245 30 Image Master VDS DU 115 VAC 60 Hz 80 6246 82 DE 230 VAC 50 Hz 80 6247 01 DJ 100 VAC 50 60 Hz 80 6247 20 Image Master VDS Analysis Kit 80 6309 71 Levelling kit including horizontal table and spirit level 18 1016 88 Staining kit 25 x 35 cm 18 1018 09
21. wider size rangethan one phase can provide If several phases are used for example 180 minutes pulsetime and 75 minutes pulsetime the order in which they are applied becomes important If you start with the longer pulse time the resolution window for the overall separation shiftsto a higher molecule size compared to a start made with a shorter pulse time M inute or second cannot be greater than 59 Press a key W hen time values are being entered the minutes and seconds cannot exceed 59 If you enter a higher number in such a field the above message will appear after you press ENT Just return to the field and enter a corrected value O dd milliseconds are rounded to even Press a key This indicates that you entered a value of for example 0 001 ms When this scale is used for either a pulse or time field the last figure must be an even number 43 7 Evaluation and presentation of data 44 T otal run time too large D ecrease phase times Press a key This indicates that the sum of all the phase times in the procedure exceeds 48 days 23 hours and 59 minutes Y ou will not be able to return to EDIT and store the procedure until the total run time is reduced Some phase times must be shorter N S Pulse delta of Phase x too small Increase the N S Pulse time of Phase x This message only appears when INTERPOLATION is being used and not until you atte
22. 0 Author Keyword Reference 31 Author Keyword Reference 32 Author Keyword Reference 33 Author Keyword Reference 34 Author Keyword Reference 35 Author Keyword Improved separation of chromosome sized DN A from Trypanosoma brucei stock 427 60 N ucleic Acids Res 1989 Vol 17 no 8 p3217 3227 Van der Ploeg L H T Smith C L Polvere R Goltesdiener K M Trypanosoma Antigenic varation in Trypanosoma brucei analyzed by electrophoretic separation of chromosome sized DNA molecules CELL 37 1984 77 84 van der Ploeg L H T et al PFG T rypanosomes Thecontribution of chromosomal translocations to antigenic variationin Trypanosoma brucei Phil Trans R Soc Lond 307 1984 13 26 van der Ploeg L H T Cornelissen A W C A PFG T rypanosoma Theroleof mini chromosomes and gene translation in the expression and evolution of VSG genes Gene Espression 1983 413 435 Borst P et al PFG T ryponosomes V SG Size fractionation of the small chromosomes of Trypanozoon and N annomonas trypanosomes by pulsed field gradient gel electrophoresis M ol Biochem Parasitol 18 1986 127 140 Gibson W C Borst P PFG T rypanosomes N annomonas Trypanosomes of subgenusT rypanozoon are diploid for housekeeping genes M ol Biochem Parasitol 16 1985 231 242 Gibson W C Gibson W C etal PFG T rypanosomes Thekarotype and ploidy of Trypanosoma cruzi Embo J 5 1986 1299 1305 Gibson W C
23. 0 Short instructions HEX electrode Gel support tray Electrophoresis unit Fig 20 Gene Navigator assembly 55 10 Short instructions 10 2GN Controller 56 Menu structure Input Pulses Store Basic Delete Main 4 Edit Sec Load Setup e Stepping Connect T Interpolation Scale a Run time Pulse time nl J Contrast Function GG Function is the same as the F button 10 Short instructions Programming the BASIC mode All parameters can be reset by pressing the F button followed by gt and ENT Display An alternative is F 2 ENTI GN control unit Press a key to continue M ain gt BASIC Edit Load Setup N S000 0 ew 000 0 time 00 00 00 Start by ON Freezeby OFF End by ESC n s090 0 E W 090 0 time 00 00 00 n s090 0 e w 090 0 TIM E 00 00 00 N S090 0 e w 090 0 time 24 00 00 Comments Press a key BA SIC is already lighted Just press ENTI Enter pulsetime 90 s N S by pressing 9 0 0 ENT Enter the same value for E W asfor N Sby pressing ENT Enter time 24 hrsb ressing 2 4 0 0 0 0 ENT on Press off to start interrupt or continue the run Pressing ESC endstherun 57 10 Short instructions
24. 6 23 Insert size standard 100 ul 2x5x10mm Thisisoneof thelayouts weuse for protocols It is especially good for trouble shooting Pulsed field gel electrophoresis protocol Box Run date Run no Gel Agarose Cooler Temp C Buffer 0 5x 0 15 x TBE Gel 15x15 20x20 Comb 1mm 2mm prep Pulse time Fresh buffer Phase N S E W Run time hrs Run time Current Electrodes Hexagonal Electrode no Point electrodes Position EE sr ites nent Voltage V Start Current E mA Current levelling Y N Final Currentl Laia mA Sample Insert Lane Type Date Size Amt Enzyme Comments 123456 23 Insert size standard 100 ul 2x5x10mm 71 72 Notes Amersham e e Biosciences Printed in Sweden by TK i Uppsala AB June 1997
25. Agarose Prep low melting point at ca 40 C to keep the insertsin place during handling of the gel Fill the GeneN avigator with 2 51 0 5M 0 5x TBE 10 Short instructions 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Carefully place the gel support tray with the attached gel into the tank M ake sure that the gel wells are on the right side see Fig 10 Start circulating the buffer by connecting the buffer circulation pump to the mains Position the hexagonal electrode array as shown in Fig 11 The hexagonal electrode holder connector is hooked to the same wall that holds the lid power connection Connect the three remaining connectors to the walls and place the lid on the unit Set the voltage limit appropriately for resolution of the desired fragment size see section 7 1 table 2 and the current limit to a maximum of 500 mA Read section 10 2 of the short instruction for GN control unit Program the control unit Suitable run times and pulse times for different samples are presented in Tables 2 3 and 4 On the power supply switch high voltage on Then start the run by pressing off on the controller To interrupt the run press the key again Another press continues therun When therun time has elapsed turn off the power on the power supply and press f on the controller if the time has not run out before removing thelid WARNIN
26. G Beforetaking off thelid always cut the current by pressing aff on the controller if the run is not yet finished Remove the gel support tray To remove the gel cut through the agarose in the tiny holes by sliding a razor blade under the edges of the gel The buffer used during the run can also be used for staining To empty the tank raise the leg on the bottom left side of the unit Fix a piece of silicone tubing tightly to the outlet from the buffer circulation pump and fill the staining box with approximately 1 litre The rest of the buffer can be emptied into a separate bottle for destaining Add 0 5 ml Ethidium bromide stock solution 1 mg ml water stored at 4 C in a dark bottle to the buffer solution in the staining box M ix WARNING Ethidium bromide is a carcinogen Use gloves protective clothing Stain the gel for 30 minutes Destain if necessary for 30 minutes to several hours 53 10 Short instructions Recipes for stock solutions A 0 5M 5x TBE buffer pH ca 8 3 Tris 500 g 17 1321 01 Boric acid 17 1322 01 Na EDTA 37 2 gor 200 ml 0 5m pH 8 0 Dissolve in distilled water and make up to 101 B 1 0 mg ml Ethidium bromide Dissolve 10 mg Ethidium bromide 17 1328 01 in 10 ml distilled water and storein a dark bottle at 4 C Warning Ethidium bromideisa carcinogen so wear gloves at all times and wash off any solution which comes in contact with your skin 54 1
27. Gel support tray for 15 x 15 cm gels 18 1019 21 Gel casting frame for 15 x 15 cm gels 18 1019 24 Double sided comb making 12 or 23 wells 1 mm thick 18 1019 25 for 15 x 15 cm gels Double sided comb making 12 or 23 wells 2 mm thick 18 1019 26 for 15 x 15 cm gels Double sided preparative comb making one or two 18 1019 27 preparative wells HEX electrode 18 1019 22 Gel casting frame for 20 x 20 cm gels 80 1251 34 Comb with 22 wells 2 mm thick for 20 x 20 cm gels 80 1102 56 Point electrode cathode 18 1019 28 Point electrode anode 18 1019 29 Tube connector set 18 1104 26 CAUTION Only spare parts approved or supplied by Amersham Biosciences AB may beused for maintaining and servicing of GeneN avigator System Chemicals and consumables Agarose NA 10 100 1000 g 17 0554 01 02 03 Agarose Prep 50 g low melting agarose 80 1130 07 Suitable to make inserts EDTA 100g 17 1324 01 Boric acid 500 g 17 1322 01 Bromophenol Blue 10 g 17 1329 01 Tris 500 g 17 1321 01 Ethidium bromide solution 10 ml 17 1328 01 For other Pulse Field Gel Electrophoresis products see the Amersham Biosciences BioD irectory 49 9 Ordering information and technical data 9 2 Technical data 50 Gene Navigator electrophoresis unit Electrical requirements Power consumption Cooling requirement Maximum input Dimensions W x D x H Weight Environment Chemical Safety standards GN control unit El
28. Gene Navigator System User manual 56 1089 80 Amersham Edition AF e Biosciences Important user information WA Reading this entire manual is recommended for full understanding of the use of this product M eaning Consult the instruction manual to avoid personal injury or damageto the product or other equipment WARNING The Warning sign is used to call attention to the necessity to follow an instruction in detail to avoid personal injury Be sure not to proceed until the instructions are clearly understood and all stated conditions are met CAUTION The Caution sign is used to call attention to instructions or conditions that shall be followed to avoid damage to the product or other equipment Be sure not to proceed until the instructions are clearly understood and all stated conditions are met Note TheN otesign is used to indicate information im portant for trouble free or optimal use of the product Should you have any comments on this manual we will be pleased to receive them at Amersham Biosciences AB S 751 82 Uppsala Sweden Amersham Biosciences AB reserves the right to make changes in the specifications without prior notice Warranty and Liability Amersham Biosciences AB guarantees that the product delivered has been thoroughly tested to ensure that it meets its published specifications The warranty included in the conditions of delivery is valid only if the
29. L Cantor C R Supercoiled DN A circular DN A OFAGE Restriction analysis of chromosomal DNA in a size range up to two million base pairs by pulsed field gradient electrophoresis H uman genetic diseases 1986 113 133 van O mmen G J B Verkerk J M H PFG Review Preparation of plant DNA for separation by Pulsed Field Gel Electrophoresis Electrophoresis 1989 vol 10 no 4 p 267 268 Devos K M Vercruysse D ewitte D Plant Pulsed Field Gel Electrophoresis and physical mappingof largeDNA fragmentsin thetm 2a region of chromosome 9 in tomato M olecular General Genetics W est Germany 1989 vol 218 no 3 p 395 400 Ganal M W etal Tomato TM W RFLP plant DNA diagnosis of human leishmaniasis Parasitology Today 3 1987 177 184 Barker D C PFG Leishmania 10 Reference list Reference 18 Author Keyword Reference 19 Author Keyword Reference 20 Authur Keyword Reference 21 Author Keyword Reference 22 Author Keyword Reference 23 Author Keyword Reference 24 Author Keyword Reference 25 Author Keyword Reference 26 Author Keyword M olecular karyotype of species and subspecies of Leishmania M olecular and Biochemical Parasitology 20 1986 279 293 Scholler J K et al PFG Leishmania The molecular karotype of L eishmania major and mapping of alpha and beta tubulin gene families to multiple unlinked chromosomal loci N ucleic Acids Research 13 1985
30. a 22 well comb 2 mm thick M ultiT emp III thermostatic circulator for 100 120 V 60 Hz 18 1102 77 or for 220 240 V 50 H z 18 1102 78 4 Installation 4 1 Gene Navigator electrophoresis unit 4 2 GN Controller 4 Installation Unpack the instruments and place GeneN avigator electrophoresis unit on a bench so that the cooling coil connections are at the rear and the handle on thelid ison theright M akesurethat the unit is equipped with the properly rated pump for the mains power source by checking the label on the pump Note Do not plug in the pump until you have buffer in the electrophoresis unit Cut off a 0 5 meter long section of silicone tubing and set it aside Y ou will need it to empty the buffer at the end of arun Seesection 5 7 of this manual Attach short pieces of silicone tubing to the cooling coils Placea male connector on oneend of tubing and a female connector on the other end Use the hose clamps to secure the connections To connect M ultiT emp III thermostatic circulator repeat the above procedure but use longer pieces of tubing When you have made all the necessary connections adjust the level of the coolant usually tap water to about 1 cm below the lid of M ultiT emp III WARNING Thecontact points on the right east wall of the electrophoresis unit and the corresponding right side of the safety lid should be kept dry If they are wet when the safety lid isinstalled see below
31. a long time it should first be rinsed several times with tap water then rinsed twice with distilled water and finally placed upside down and left to dry thoroughly Dueto the unusually high current which is used the platinum wires must eventually be replaced In continuous operation the anodes will last about six months and the cathodes for about 18 months The condition of the electrodes can be revealed by visual inspection Problem Correction Current differs by more than 1 The electrophoresis unit is not 10 mA between the two fields levelled and Curved results are obtained 2 Check that the tray is resting using HEX electrode properly in the tank 3 The electrodes are burned out or faulty Check that all electrodes fire watch for bubbles 4 The electrodes are not properly hooked onto the wall of the electrophoresis unit 5 Check circulation of the coolant and buffer Remove and clean circulation pump if necessary One or both fields are not 1 Check the reading on power supply working check that the two fields are firing i e bubbles are formed at the electrode If the electrode wiring is worn out parts of the wiring is extremely thin or cut off Exchange electrode 2 Check that the contacts work between lid and elpho unit Check the metal tongues 45 8 Maintenance and trouble shooting Problem Correction Circulation pump does not work 1 The electrophoresis unit has been l
32. able For proper buffer circulation the slits must be parallel to the East right and West left walls of the electrophoresis chamber If you will be using the point electrodes the 20 x 20 cm or 15 x 15 cm casting frame should be positioned diagonally with the corners pointing toward the middle of the sides of the running tray see Fig 3 below Fig 3 Gel casting frame position for separations with point electrodes If you will beusing the H EX electrodethe 15 x 15 cm frame should be placed on the red square on therunning tray Fig 4 5 Operation Fig 4 Gel frame position for HEX electrode The rubber casting frame should be placed on the red square marked on the running tray In either arrangement the conical holes or depressions at the bottom of the tray must be clearly inside not immediately underneath the gel casting frame Thered square will help you position theframe correctly M akesurethe ridged side of the rubber frame is placed down resting on thetray and that the comb position marks areon top Theridgeis designed to make the frame self sealing and no clamping is needed The position of the comb is marked by the thick red lineinsidethe square on the gel support tray Before pouring the gel into the frame place the plastic clips and then the comb inside the rubber framein the same position that will be used when the gel isin place Be sure that the teeth
33. c expired Restart by On End by ESC Note T his buzzer will continue to sound at 10 minute intervals until any key except the on and off key is pressed Pressing the on and off key again will restart the the sequence SETUP is used to set default values for either connect the stepping or interpolation run mode or the pulse and run time scale units From the main menu M ain gt Basic Edit Load SETUP Check or change instrument modes Select SETUP and one of the following configurations will appear Setup gt CONNECT Scale Select how to connect programmed points 6 Programming the controller Setup gt Connect SCALE Set time scales 6 4 1 Connection Pressthe ENT button to select CONNECT and oneof the following configurations will appear on your LCD Setup connection Stepping STEPPING Interpolation Setup connection Interpolation Stepping INTERPOLATION Use the and arrow keys to select either STEPPING or INTERPOLATION Press EN T to execute your choice and you will return to the SETUP menu Press ESC to return to the main menu N ote that the system is delivered with STEPPIN G as default It will remain so unless you change it Y ou cannot use interpolation when you arerunningin BASIC mode because interpolation requires at least two phases and the BASIC modecan only consist of one phase Y ou can useinterpolation when work
34. centration of 1 2 1 5 has been found to give better resolution than a 1 concentration without significantly increasing therun time Concentrations around 2 improve resolution even more but at the cost of the speed of separation Changes in thetemperature of the running buffer will affect the separation pattern The pulse times shown in Table 2 will produce a resolution window within the range specified at the head of each column but one pulse time usually does not cover the entire size range in a sample depending on its diversity of molecules Theinformation in Table 2 is valid when the running buffer is 0 5 x TBE if the buffer concentration used is lowered the resolution window will be shifted to slightly lower sizes i e optimal resolution will appear in a slightly lower size range The pulse times below are based on the suggested experimental conditions and electrical parameters outlined in Section 6 Ifthefield strength is changed the pulse time should be scaled accordingly For an efficient separation range of 100 kb for example this means that if the voltage is reduced by 50 the pulse time should be increased by a factor of 2 For separation of very large DN A gt 2000 kb it is important to lower the voltage Recommended running parameters are shown in T able 2 For molecules below 100 kb higher voltages can be used without problems Table 3 Relationship between pulse time and resolution for a HEX electrode Pu
35. e LCD press ESC to indicate a no response Up and down arrow keys 1 and are used to move to the previous phase or thenext phasein a procedure When revising a previously stored procedureit is very useful to either INSERT or DELETE phases help provides further instructions Press help a second time or the ESC key to return to the position in the menu at which help was requested The menu structure described in Section 6 5 Edit mode will clarify how different commands are related to each other The menu structure is further explained in Fig 16 Please note that the SET UP command is general when used from main menu whereas it is only valid for the particular procedure of interest when used from the EDIT menu Input Pulses Store Bie pelete Main q Edit SE o Stepping i N ce A Interpolation Load Scale Setup Ss Run time Pulse time tion 1 4 Contrast Function Je Fig 16 Menu structure for GN Controller ESC lets you return to a previous higher menu 6 Programming the controller 6 2 Contrast and reset commands In the display the choosen command is written in capital letters 6 2 1 Contrast When you turn the unit on with the switch on the rear panel for the first time theLCD will indicatethe software versio
36. e 9 Author Keyword Pulsed field electrophoresis application of a computer model to the separation of largeDN A molecules Proc N atl A cad Sci 84 1987 8011 8015 Lalande M PFG T heory M ethod M olecular karotypes separating chromosomes on gels Bioessays 3 no 6 1986 269 271 Corcoran L M PFG Review M apping Separations of open circular DN A using pulsed field electrophoresis Biochemistry 84 1987 4054 4057 Levene S D Zimm B H PFG Circular DN A T opology Preparation and manipulation of largeDNA molecules advances and applications TIBS 12 1987 284 287 Smith C L Cantor R C PFG Pulsaphor Preparation M ethod Pulsed field gel electrophoresis and the technology of large DNA molecules G enome Analysis a practical approach Ed Kay Davies IRL PressInc M cLean Va Smith C L et al PFG M ethod Pulsaphor Pulsed field gel electrophoresis of large DN A molecules N ature 319 1986 701 702 Smith C L Cantor C R PFG Preparation M acrorestriction mapping by pulsed field gel electrophoresis Applications of DNA probes 1986 87 92 Smith C L Cantor C R PFG M apping O ptimized conditions for pulsed field electro phoretic separations of DNA Nucleic Acids Research 16 no 15 1986 Birren B W etal PFG M ethod Estimation of circular DN A size using gamma irradiation and Pulsed Field Gel Electrophoresis Analytical Biochem US 1989 vol 177 no 1 p 110 114 Be
37. e the gel casting frame on the tray The conical holes or depressions at the bottom of the tray must be clearly inside not immediately underneath the gel casting frame The red square will help you position the frame correctly Fig 4 Theridged side of the frame must be turned downwards W hen the temperature of the gel solution has decreased to 60 C pour the gel and make surethat the holes are filled If necessary use a Pasteur pipetteto suck air bubbles from the holes and in the gel The gel should be about 5 mm thick Place the comb parallel to a slotted side on the gel support tray see Fig 6 The position of the comb is marked by the thick red line inside the square on the gel support tray Allow the gel to set for 30 minutes Read Gene N avigator short instructions while the gel is setting Remove thecomb by tilting it backward and forward to freethe comb from the agarose then lifting it at one end Carefully pull the rubber frame free from the gel and removeit Start M ultiT emp III with a set value of 9 C Inserts can either be cut on a sterile surface or on a nuclease free plastic film M icroscope coverslips are preferred for cutting see Fig 8 but a scalpel can be used instead Place the inserts in contact with the front wall of the well to get sharper bands A flamesterilized Pasteur pipette with a bent tip is recommended for loading the inserts before submerging them in the gel Seal the loaded wells with 0 5
38. ectrical requirements Power consumption Maximum input Dimensions W x D x H Weight Environment EMC standards Safety standards 100 130 V 200 260 V 50 or 60 Hz 4VA Minimum 6 l min 450 V 500 mA 395 x 380 x 196 mm 5 5 kg 4 40 C 20 95 relative humidity The wetted parts are resistant to solvents commonly used in electrophoresis and solutions containing inorganic and organic acids alkalis and alcohol s This products meets the requirements of the Low Voltage Directive LVD 73 23 EEC through the harmonised standards EN61010 1 Note The declaration of conformity is valid for the instrument when it is e used in laboratory locations e used in the same state as it was delivered from Amersham Biosciences AB except for alterations described in the user manual e used as a stand alone unit or connected to other CE labelled Amersham Biosciences AB instruments or other products as recommended 100 130 V 200 260 V 50 or 60 Hz 30 VA 450 V 500 mA 364 x 280 x 110 mm 5kg 4 40 C 20 95 relative humidity This product meets the requirement of the EMC directive 89 336 EEC through the harmonised standards EN 50081 1 emission and EN 50082 1 immunity Note The declaration of conformity is valid for the instrument when it is e used in laboratory locations e used in the same state as it was delivered from Amersham Biosciences AB except for alterations described in the user manual
39. ed plasmids Tracking bacterial DNA replication forks in vivo by pulsed gel electrophoresis N ucleic Acids Res 1989 Vol 17 no 9 p3479 3490 Ohki M Smith C L E coli replication forks radionucleotide N ot 1 Separation of yeast chromosome sized DN As by pulsed field gradient gel elctrophoresis CELL 37 1984 67 75 Schwarz D C Cantor C R PFG Y east Localization of Y east Glucoamylase genes by PFGE and OFAGE Curr Genet 1988 Vol 14 no 1 p9 14 Pretorius l S M armur J Saccharomyces mapping O FAGE Separation of chromosomal DN A molecules from C albicans by pulsed field gel elctrophoresis N ucleic Acids R esearch 14 1986 4401 4406 Snell R G Wilkins R J PFG Candida Chromosomal variations in Candida albicans N ucleic Acids Research 15 no 8 1987 3625 Snell R G etal PFG FIGE Applications C andida 65 10 Reference list 66 Reference 45 Author Keyword Reference 46 Author Keyword Reference 47 Author Keyword Reference 48 Author Keyword Reference 49 Author Keyword Reference 50 Author Keyword Reference 51 Author Keyword Reference 52 Author Keyword Reference 53 Author Keyword Comparison of the separation of Candida albicans chromozome sized DN A by Pulsed Field Gel Electrophoresis technique N ucleic Acid Research 1989 vol 17 no 10 p 3783 3794 Lasker B A Carle G F Kobayashi G S Candida mapping karyotyping An elect
40. eft with buffer in the pump the pump does not work due to crystallization Check the pump housing and remove any particles rinse with a mild detergent anda soft brush WARNING Changing of the pump unit must always be done by Amersham Biosciences AB Service Engineer High background 1 Insufficient washing of samples 2 Sample contaminated with other material such as RNA or endonucleases 3 Overloading of samples 4 Incomplete destaining Smeary lanes 1 Improper preparation of DNA and bad resolution samples Protein has not been dialyzed away from the DNA samples The sample contains too much RNA RNAse the sample inserts 2 Restriction enzyme digestion of DNA samples is incomplete because Proteinase K was not completely inactivated 3 Too much sample was loaded Load thinner slices to improve resolution 4 The voltage current used was too high 5 Gel concentration too low 6 Impure enzymes Amersham Biosciences enzymes recommended Bad resolution 1 Check samples they may be contaminated with endonucleases from hands or other sources 2 You are not using the optimal pulse time 3 The voltage is too high 4 The insert or insert slices were not loaded flat against the wall in the well 46 8 Maintenance and trouble shooting Problem Correction Bad resolution cont 5 Air bubbles in the well 6 The insert was crushed when it was pushed into the
41. example 00 01 00 if the HH M M SS setup is used If you typea N S pulse time of 60 seconds and then either press EN T or Jto moveto theE W pulsetime the LCD will appear as below default setup n s060 0 E W 060 0 time000 00 Start by ON Freeze by O FF End by ESC Thetime entered for the N S Pulse will automatically be repeated as the E W pulsetime If you want both pulse times to be the same press ENT again to moveto the run TIM E parameter setting Now indicate the desired run TIM E H hh mm If it is 20 hours for example press 2 0 10 10 Then executewith ENT Your BASIC run parameters are now set and you are ready to begin a run If you type a desired time and then change your mind before moving to the next field you can clear the field by pressing CE Then you can start anew Once you have pressed the ENT or any arrow key the CE key will no longer clear the field W hen the time parameters are correctly indicated in all three fields press ENT before starting the run Note The pulse and run time parameter SCALE units can be changed from the default shown above Y ou can choose between five different SCALE units H h mm ss Ssss Sss s Ss ss and S sss for pulse time setting and three different SCALE units Days hh mm H hh hh and H h mm ss for run TIM E SCALE SET UP To change the default SCALE press ESC to go to the main menu and
42. exposure time W hen photographing a separation of very large molecules for example N eurospora crassa chromosomes you may occasionally need to use a magnifying lens to get the best possible picture A gel documentation system such as the Amersham Biosciences ImageM aster V DS System can also be used for documentation and analysis A WARNING When photographing gels stained with ethidium bromide use gloves protective clothing and face shield Ethidium bromide is a carcinogen UV irradiation enhances transfer during vacuum and Southern blotting but do not irradiate a gel more than necessary if you want to store it for along time or if UV nicking is undesirable 20 6 Programming the controller 6 1 GN Controller 6 Programming the controller Thefront panel of GN Controller consists of Fig 13 Front panel A Liquid Crystal Display LCD which shows the menu selections and program parameters that have been chosen The contrast of the LCD can be adjusted by using the F Key See Section 6 2 Contrast and reset commands Number keys 0 through 9 which are used to enter run parameters such as run time and pulse times or to number procedures when they are called for IN PUT or stored STORE The N umber Keys can also be used instead of Arrow K eys to reach a command in a menu The commands in a menu are numbered from left to right i e the leftmost command is called number 1 e
43. g R PLP M apping of xp21 translocation breakpoints in and around the Duchenne M uscular Dystrophy gene by pulsed field gel electrophoresis Genomics 1988 Vol 3 no 4 p315 322 M eitinger T Boyd Y Anand R Craig I W H uman chromosomal rearrangement hybridization mapping Clones from an 840 kb fragment containing the 5 region of the DM D locus enriched by pulsed field gel electrophoresis Genomics 1988 Vol 3 no 3 p177 186 Anand R H oneycombe J Whittaker P A Elder J K Southern E Duchenne mapping Chromosome jumping from d4s10 g8 toward the H untington disease gene Proceedings of the N ational Academy of Sciences of the United States of America US 1988 Vol 85 no 17 p6437 6441 Richards J E Gilliam T C Cole J L Drumm M L Wasmuth J Gusella J F Collins F S H uman chromosome 4 Physical mapping of the Cystic Fibrosis region by pulsed field gel electrophoresis Genomics 1988 Vol 2 no 4 p 346 354 Drumm M L Smith C L Dean M Cole J L lanuzzi M C Collins F S H uman chromosome 7 cystic fibrosis O FAGE 10 Reference list Reference 70 Author Keyword Reference 71 Author Keyword Reference 72 Author Keyword Reference 73 Author Keywords A long range restriction map of the pseudoauto somal region by partial PFGE analysis from the telomere N ucleic Acids Res 1988 Vol 16 no 12 p5361 5378 Rappold G A Lehrach H human x and y chrom
44. g a 1 mm comb instead of 2 mm comb if the sample is sufficiently concentrated To seal the wells you may use a little low melting point agarose Agarose Prep 80 1130 07 This step may not be absolutely necessary but it will ensurethat the blocks or block slices stay in position during subsequent handling of the gel This is especially important when thin slices are loaded in the gel W erecommend that newcomers to PFGE use Amersham Biosciences markers These are delivered with a Certificate of Analysis and have been thoroughly tested with GeneN avigator system Place the gel support tray with the gel attached into the tank M ake sure that the cut out corner of the gel support tray is placed in the upper left corner of the electrophoresis tank To avoid entrapping air bubbles beneath the gel support tray place one side of the tray down first and then lower the remainder carefully into place If the buffer does not cover the gel add alittle more Yeast DNA PFGE markers Order no 27 4520 01 5 blocks Lambda DNA PFGE markers Order no 27 4530 01 5 blocks 5 Operation If you are using the point electrodes the gel will bein a diagonal position In this case the wells should be nearest the southeast front right corner of the tank see Fig 3 When you use the H EX electrode however the gel will bein a squared position and the sample should be nearest the east side Figs 9 and 10 below
45. ing in the EDIT mode because up to 6 different phases each with its own pulse time and phase time values can be programmed In the EDIT mode the total run time becomes the sum of the programmed phase time values If you work mostly in the EDIT mode and wish to use interpolation most of the time you can save time by changing the default SETUP SET UP in the main menu to INTERPOLATION Empty procedures in EDIT mode will indicatethe INTERPOLATION asthe default If you change SETUP intheEDIT mode you will only alter the setup for that particular running procedure When you are working with several phases in the EDIT mode stepping gives a stepwise increase or decrease in pulse time O ne phase isone step In the BASIC mode the pulse time values are constant throughout the run 27 6 Programming the controller 28 Pulse times N S EW No of Pulses Fig 14 Stepping If STEPPING is chosen in the EDIT mode each phase of the run can have different constant values This will not influence the first phase Thecurve would still appear as illustrated above A multi phase run using INTERPOLATION modeasksGN Controller to interpolate the set pulse times during the run In this mode the following phase will influence the separation performance during the previous phase as the two pulse times will be interpolated Thecontrol unit interpolates the difference between the N S a
46. ions The pulse time makes it possible therefore to focus on the size range of interest This size range is called the resolution window The range in the resolution window isa function of field strength current and pulse time Changes in run time can also affect the degree of separation Generally longer than overnight runs are required to separate large fragments see Table 4 The design of GeneN avigator makes it possible to generate almost any field shape The H EX electrode gives a homogenous field resulting in straight lanes when the pulsed field technique is used The diode equipped point electrodes reduce undesirable field distortions The DI configuration works well for a broad range of separations To create more unusual field shapes DI configuration both the anode and cathode electrodes can be set at other positions on the north and south sides For maximum resolution up to 2000 kb allow at least 17 hours of run time Larger DN A s such as the chromosomes of Schizosaccharo myces pombe need longer run times For most purposes however an overnight run will provide satisfactory resolution W hen determining the quality of sample preparation begin with a short run When it is clear that the samples are of good quality you can begin fine tuning the separation for the best possible result Fig 17 Two different fields are applied using the HEX electrode The figures show isofield lines a No
47. irst phase of that procedure Select IN PUT from the edit menu to get back into the phases of the stored procedure just selected W hen you have re entered an existing procedure use up and down arrow keysto move quickly from phaseto phase Y ou can enter any changes in phase parametersthat you wish Once more if you return to the main menu without having stored the changes they will be erased Y ou will still have saved the old information but not the changes To insert a phase into an existing procedure via the IN PUT command moveto the phase that will be placed beforethe one you will add Capitalize the word PHASE by using the arrow keys and press ENT Y ou will be asked if the new phase shall be inserted IN SERT the old phase shall be deleted DELETE or if you want to go back to a previous phase N ON E Press ENT on INSERT To delete an existing phase you must again move to the phase field PH ASE capitalized and press ENT Now move the cursor position to DELETE and press ENT again That phase will be deleted W hen editing a procedure therefore you can add phases delete phases or change the parameters in any or all phases When the editing is complete you must escape to the edit menu only and then press ENT on STORE before returning to the main menu 35 6 Programming the controller 36 By doing this you will replace the
48. lse time Efficient Maximum Run Set voltage separation resolution time V 0 3s 1 10kb 1 10kb 1 450 0 15x TBE 0 5s 5 30kb 10 25kb 2 450 0 15x TBE 0 8s 30 50kb 35 50kb 3 450 0 15x TBE 5s 20 100kb 60 90kb 4 300 25s 40 400kb 200 300kb 6 300 45s 40 600kb 400 550kb 10 24 300 100s 40 1000kb 700 900kb 17 40 165 200 125s 100 1600 kb 800 1200kb 17 40 165 200 20 min 1 2 5Mb 1 6 2 5Mb 100 140 165 200 30 min 1 6 3Mb 2 5 3Mb 100 140 165 200 40 min 1 6 6Mb 2 5 6Mb 140 30 75 min 2 9 Mb 3 6Mb 140 170 30 90 min 2 10Mb 6 9Mb 185 30 100 min 3 13Mb 7 10 Mb 190 200 30 0 6 agarose 180 min 3 13Mb 10 13Mb 190 200 27 0 6 agarose The buffer used is 0 5x TBE and agarose concentration is 1 2 unless a different value is stated 7 Evaluation and presentation of data Tables 2 and 3 can be used as rough guidelines for separations in 0 5x TBE and also for separations in 0 15x TBE The figures in Table 3 are sometimes limited by the size of the sample at hand and do not reflect exact limits Run times for certain samples can be reduced considerably depending on how complex the separation pattern is The figures mentioned here are presented only as a guide for the first trial The optimal voltage for each sample size can be found in Table2 InTable3thelower voltages are valid when theH EX electrode is used When a certain pulse time is selected molecules smaller or larger than the chosen range will not get the optimal resolving condit
49. mpt to load the procedure Either increase the indicated pulse time or decrease the phase time of the previous phase Note T his message will appear if the time difference between two subsequent pulses is less than 2 milliseconds i e the time difference is too short for theINTERPOLATION to occur The remedy is to a increase pulse time in the latter phase b de crease the pulse time of phase x or c increase the run time of phase x No phases defined Press a key This message which occursin the EDIT mode indicates that the user has failed to program a phase or pulse time somewhere in the current procedure Usethe up and down arrow keys to move through the phases checking that parameter values have been entered A procedure isrunning in load Press a key When working in the BASIC mode this message indicates that the user has started a run and then tried to return to themain menu without first having stopped the run You must first press the on and off key to stop the run and then press ESC 8 Maintenance and trouble shooting 8 1 Maintenance 8 2 Trouble shooting 8 Maintenance and trouble shooting Werecommend a mild detergent and a soft brush to clean the electrophoresis unit Rinse with distilled water several times before using it Check the pump housing monthly to remove any particles capable of reducing the efficiency of the pump If the unit is to be stored unused for
50. n number Press any key to moveto the main menu which looks likethis M ain gt BASIC Edit Load Setup O peratethe controller in a basic way To adjust the contrast on the LCD press the F key F means function Y ou will see the following on your LCD Function gt CON TRAST R eset Adjust the contrast of the display Theupper case characters in CONTRAST indicate that the cursor isin that position Execute your menu choice by pressing ENT Now the LCD will prompt Function contrast gt Use arrow keys to adjust contrast Adjust the background and foreground clarity of the LCD by repeatedly pressing either the or the gt key Either key will both lighten and darken the LCD if you continue pressing it If you turn the mains switch on and find the LCD blank push followed by 2 and ENT Note This will reset the contrast and erase all previously stored procedures as well as reset all default values If you want to keep your stored procedures type F 1 and ENT Usethe arrow keys When the contrast is satisfactory press ESC twice to return to the main menu Note Y ou can press F which isthe Function key to adjust the contrast from any menu position Y ou need not begin from the main menu asin this example mm 6 2 2 Reset To reset the instrument pre
51. nd E W pulse times of the first phase and those of the next higher phase It then automatically applies as many pulses as are possible within the limits of the phase time that has been programmed The pulse always stops when the programmed phase time ends The separation will continue with the next phase and new pulsetime values T hen GN Controller begins interpolating anew using the programmed values for the next higher phase W hen interpolation is used therefore the pulse time values must always be increasingly higher with each new phase Interpolation means that pulse time is linearly increasing with increased number of pulses see Section 6 5 1 Edit setup Fig 15 below illustrates the results of a 4 phase interpolated run Note During the last phase pulse times are held constant since there is no reference 5th phase for interpolation 6 Programming the controller Pulse times N S E W No of Pulses Fig 15 Interpolation 6 4 2 Scale To change scale in the SET UP mode use gt to movethe cursor to SCALE Setup gt Connect SCALE Set time scales Press ENT to select SCALE and the setup scale menu appears Setup scale gt RUN TIME PulseTime Set run time scales Press ENT when the cursor ison RUN TIM E and the following choices appear Setup scale run time H hh mm Days hh mm HHH MM H h mm ss Thefirst line indicate
52. nk Cut a blue plastic pipette tip 1000 ul from about 1 cm from the wide end and put the widest part of the piecein thetubing This will make it easier to attach the tubing to the outlet of the pump Fig 11 Place the tubing in the outlet The running buffer will now be pumped out of the tank through the tubing and into the staining dish or a bottle Pump out one liter of buffer into the dish then store the remainder into a bottle for destaining Ethidium bromide is used for staining Add 0 5 ml of stock solution 1 mg EtBr ml water stored in a dark bottle at 5 C and agitate to homogeneity Staining takes up to 30 minutes 19 5 Operation Complete destaining will take several hours A WARNING Ethidium bromide is carcinogenic O bserve all safety precautions when using it For information on blotting and hybridization see Amersham Biosciences blotting protocols and especially the protocols for VacuGene XL for information on blotting and hybridization Cut here Fig 11 How to cut a pipette plastic tip to fit the tubing used with the pump of Gene Navigator electrophoresis unit 5 7 Photographing Forroutinework Polaroid 667 film or a similar film can be used stained gels together with a Kodak Wratten No 9 filter Typical exposure times are5 to 10 seconds atf 16 If negatives are required use Polaroid 665 or 55 film If high resolution is required use Polaroid 55 film which has a longer
53. nterpolation When using interpolation GN Controller calculates and inserts pulses based on the difference between the pulse times both N S and E W of the first 1 of 2 phase and those of the second 2 of 2 phase etc Note Runningin INTERPOLATION mode the pulse times of each successive pulse must be at least 2 milliseconds longer than the previous pulse GN Controller automatically calculates the number of pulses to be inserted at equal intervals based on each phase time The result of interpolation is always a straight line that increases over the total number of pulses as illustrated in Fig 15 A new interpolation begins as soon as each phase ends The new interpolation is then based on the difference between the N S and E W pulse times of the second 2 of 3 and the third 3 of 3 phases To end the procedure the final phase time is set with all zeros This means that the final phase of an interpolation is not actually run Asaresult an interpolation run can consist of no more than 5 phases The parameter values for phase 6 will resemble those shown below phase n s e w PHASE TIME 6 of 6 030 0 0 30 0 00 00 00 If for example the total run consists of only 3 phases the run will be terminated by entering a zero phase time in the 4th phase Pulse times will still be needed in the 4th phase When the setup selections haveall been entered press ESC to return to the edit procedure menu
54. osome turner syndrome O FA GE Long range mapping of the Philadelphia chromosome by ulsed field gel electrophoresis Blood US 1988 Vol 71 no 3 p697 702 Westbrook C A Rubin CM Carrino J J Le Beau M M Bernards A Rowley D human chromosome 9 philadelphia chronic myelogenous leukemia Construction of long range restriction maps in human DNA using pulsed field gel electrophoresis GeneAnal Tech 1987 Vol 4 no 6 p119 132 Gemmill R M Coyle M orris J F M cPeek F D Jr Ware Uribe L F H echt F mapping human chromosome 3 R FE M olecular mapping of the human major histo compatibility complex by pulsed field gel electrophoresis Proceedings of the N ational Academy of Sciences of the United States U S 1987 Vol 84 no 20 p7237 7241 Dunham l Sargent C A Trowsdale J Campbell R D M H C human tumor necrosis factor 69 70 Pulsed field gel electrophoresis protocol Box Run date Run no Ee nu Gel Agarose Cooler Temp C Buffer 0 5x 0 15 x TBE Gel 15x15 20x20 Comb 1mm 2mm prep Pulse time Fresh buffer Phase N S E W Run time hrs Run time Current Electrodes Hexagonal Electrode no Point electrodes Position Alia E Voltage V Start Current re mA Current levelling Y N Final Currentl Pista mA Sample Insert Lane Type Date Size Amt Enzyme Comments 12345
55. perature is critical for reproducibility If you havea high ambient temperature it may be wise to cover the cooling tubing with insulation tubing Before you pour the buffer level the electrophoresis unit Adjust the unit by turning the appropriate feet Pour buffer into the tank 2 5 L before inserting the gel support tray Note To make surethat the pump works properly check that the tubing underneath thetank is filled with cooling solution The protocol below describes how to prepare 15 x15 cm or 20 x 20 cm gels The gels are made in the same buffer you use for electrophoresis Amersham Biosciences supplies high gel strength A garose N A 17 0554 01 02 03 and low melting Agarose Prep 80 1130 07 for pulsed field gel electrophoresis See information on chemicals and consumables Section 9 1 M elt the agarose by boiling it in a microwave oven or in a water bath To obtain optimal resolution in reasonable timea 1 2 agarose concentration should be used for most applications Foral5x15cmgel dissolve 1 3 gagarosein 110 ml 0 5x or 0 15x TBE pH 8 3 Fora20x 20 cm gel dissolve 2 4 gof agarosein 200 ml of buffer 0 5x or 0 15x TBE pH 8 3 11 5 Operation 5 3 Casting gels 12 M akesure that the conical holes in your tray are free from agarose W hen the gel is cast the holes will prevent the gel from floating around when the buffer is being circulated during arun Place the gel support tray on a levelling t
56. perature when taking the unit from a colder to a warmer environment WARNING Always check the wires for damage before using the unit WARNING Always check that the electrodes are properly connected before closing thelid WARNING Always connect the lid according to the mounting instruction WARNING Always connect the cables to the power supply BEFORE turning the power supply ON WARNING AlwaysTURN OFF the power supply before removing thelid WARNING DoNOT useconcentrated acids bases or halogenated and aromatic hydrocarbons WARNING N ever exceed maximum allowed voltage current or power WARNING T he instrument must be connected to a grounded mains socket WARNING Changing of the pump unit must always be done by Amersham Biosciences AB Service Engineer The contact points on the right east wall of the electrophoresis unit and the corresponding right side of the safety lid should be kept dry If they are wet when the safety lid is installed see below a fuse may blow out NEVER run with morethan 450 V or 500 mA asthis will burn out the control unit 3 NEVER run thecirculation pump dry NEVER leave buffer in the unit for long periods Evaporation may cause the buffer to crystallize and hinder the smooth operation of the pump If the unit is to be stored unused for a long time it should first be rinsed several times with tap water then rin
57. phoresis unit 11 Tubeconnections male and female 18 1104 26 included in GeneN avigator electrophoresis unit 12 Gel knifefor cutting gels 80 1106 37 or a razor blade 13 Power supply EPS 600 19 0600 00 or EPS 200 19 0200 00 14 MultiTemp III thermostatic circulator 18 1102 78 220 240 V 50 Hz and 18 1102 77 110 V 60 H 2 15 Staining box stainless steel 18 1018 09 16 MacroVueUV 20 Transilluminator 80 6245 11 115 V and 80 6245 30 230 V 17 ImageM aster V DS 80 6246 82 115 VAC 60 Hz 80 6247 01 230 VAC 50 Hz 80 6247 20 100 VAC 50 60 Hz 18 Levelling kit with spirit level 18 1016 88 51 10 Short instructions Gene Navigator gel preparation and electrophoresis using the HEX electrode N otes l Do not plug the buffer circulation pump into the mains before the start of the run Before connecting the controller make sure that the current on the power supply does not exceed 450 V and 500 mA Procedure d 2 10 11 12 52 Check that you have everything on the checklist Dissolve 1 3 gagarosein 110 ml 0 05 M 0 5x TBE buffer M elt the agarose in a water bath or a microwave oven while supervising it to make certain that the solution does not boil over M ix the solution occasionally to avoid local gelling W hile the solution is cooling to 60 C place the gel support tray on the levelling table Level the table by placing a spirit level on the gel support tray Plac
58. rophoretic karyotype of N eurospora crassa M olecular and cellular biology 8 no 4 1988 1469 1473 Orbach M J etal PFG Fungi N eurospora Chlorella viruses contain linear nonpermuted double stranded DNA genomes with covalently closed hairpin ends Virology US 1989 Vol 168 no 2 p363 369 Rohozinski J Girton L E Van Etten J L Terminal repetition virus Jumping libraries and linking libraries the next generation of molecular tools in mammalian genetics Trendsin Genetics 1986 174 179 Poustka A Lehrach H PFG d umping M ammalian Analysis of genome organization and rearrange ments by pulsed field gradient gel electrophoresis Genetic Engineering 8 1986 45 70 Smith C L etal PFG Gene mapping M apping and sequuencing the human genome how to Biotechnology 5 1987 933 939 Smith L Hood L DN A Sequencing G ene mapping H uman Approaches to physical mapping of the human genome Cold Spring H arbor Symp on quantitative biology LI 1986 115 122 Smith C L Cantor C R PFG Gene mapping H uman M egabase scale mapping of the HLA gene complex by pulsed field gel electrophoresis Science 235 1987 1387 1390 Lawrance S K etal PFG Genemapping HLA M apping of HLA genes using pulsed field gradient electrophoresis FEBS Letters 204 1986 1 4 R agoussis J et al PFG Genemapping HLA 10 Reference list Reference 54 Author Keyword Reference 55 Author Reference 56
59. rs 7 Evaluation and presentation of data The parameters for a typical experiment are shown in Table 1 below These parameters will give good overall separation for 40 2000 kb The values described below aretypical when using 2 5 liters 0 5 x TBE buffer Table 1 Typical current values for different electrode configurations Parameter Point electrode HEX electrode electrode 20 x 20 cm gel 15 x 15 cm gel Set voltage 300 330 V 165 170 V Starting 0 5 x TBE current ca 100 140mA 80 100 mA The current usually increases 5 10 during arun If morethan 2 5 liters of TBE buffer are used the current will be higher Similarly other buffers or concentrations will result in different current values depending on conductivity of the buffer Table 2 General run parameters Size Mb 0 01 lt 0 1 0 1 2 2 6 6 12 0 05 agarose 1 2 1 2 1 2 0 6 0 6 Buffer xTBE 0 15 0 15 0 5 0 5 0 5 0 50 Temperature C in MultiTemp II 8 9 8 9 8 9 8 9 8 9 set value in elpho unit 12 14 12 14 12 14 12 14 12 14 Voltage V HEX 450 ca300 165 40 25 50 200 100 Voltage point 450 ca370 300 60 ca 30 330 100 Pulse time s 0 3 1 0 1 10 10 120 3 75 50 100 min min Run times h 1 4 1 6 17 24 24h 3 6 3days days 0 6 agarose will give a faster separation at the cost of resolution 39 7 Evaluation and presentation of data 7 2 Relationship 40 between pulse time and resolution An agarose con
60. rs and recheck them Theup and down arrow Keys move you quickly through the phases Besureto press ENT after all parameter changes you make If no rechecking is required or when all of your value changes have been entered in all phases escape back to the edit menu and press ENT on STORE IMPORTANT Becareful NOT to press ESC onetimetoo many A because you will have erased the above created procedure in theEDIT mode After entering on STORE in the edit menu you have an option If you store the newly created procedure as number 3 you will replacethe previous procedurewith the version you just created If you select another number this procedure will replace the one previously stored on that number To begin the run press ENT on LOAD from the main menu to load a previously stored procedure Note When you load a procedure which is using INTERPOLATION mode error messages will warn you if the time differences between two subsequent pulses are below 2 milliseconds See Section 7 3 for a list of all system error messages 6 6 Loading a From the main menu procedure M ain gt Basic Edit LOAD Setup Load the experimental conditions Select LOAD and press ENT TheLCD now displays the following Load gt Select procedure number 1 9 C Existing procedures 1 2 3 4 37 6 Programming the controller 38 The C above represents a blinking cursor An
61. rth South field b East West field 41 7 Evaluation and presentation of data 42 Fig 18 Two different fields are applied using the point electrodes DI configuration The figures show isofield lines a North South field b East West field Example Finetuning a separation of Saccaromyces cerevisieae chromosomes Y PH 148 In an earlier run a 90 second pulse time with 17 hrs run time resolved all except the two largest bands Another run with 125 s pulse time resolved the larger bands but not all the mid range bands After atrial using 90 sand 125 s pulsetime in a stepped program the optimal program looked likethis Phase Pulse time Run time 1 90s 6 00 hrs 2 105s 5 00 hrs 3 125s 6 00 hrs 17 00 hrs 7 Evaluation and presentation of data 7 3 System messages Table 4 Examples of run parameters DNAsample DNA Pulse Time Run Time Voltage Buffer size range hours HEX xTBE Kb Electr Volts molecular 0 5 50 300 800ms 1 2 450 0 15 weight DNA standards Lambda 50 1100 90s 24 40 165 200 0 5 ladder Saccharo 90 2500 90 105 125s 5 6 6 165 200 0 5 myces 17 cerevisieae Hansenula 1000 3000 40 20 min 80 60 165 0 5 wingei 140 Schizo 3000 6000 75 min 160 35 V 0 5 saccharomyces min run pombe time 80 h Neurospora 4000 12600 180 75 min 100 90 25 30 0 5 crassa 190 Note means that several phases are used in stepping mode Several phases will give a better overall separation over a
62. s created in the STEPPING mode Thenew procedure will beinINTERPOLATION mode and can therefore only havea maximum of 5 phases The 6th phase will be programmed with the requisite higher pulse times so that the interpolated insertions can occur but the phase time will be set at zero Therun procedure will conclude after phase 5 Y ou press ENT on EDIT from the main menu and then press 3 to call up the procedure that was previously stored as number 3 N ow the edit menu will appear as below Edit3 gt SETUP Input Pulses Store Delete Check or change procedure modes M ovethecursor to SETUP and press ENT Now changethe connection setup for this procedure number 3 from ST EPPING to INTERPOLATION Then movethe cursor to IN PUT and press ENT Now you arein position to change any or all of the parameters in phase 1 of 5 When 6 Programming the controller you have pressed ENT on thephaseTIM E parameter you will automatically advance to phase 2 of 5 Continue in this manner until you reach the final phase TIM E parameter in phase 6 of 6 Now you will replace the previous phase TIM E with all zeros and press ENT Y ou arenow in the phase field Phase capitalized Press ENT again to moveto the following menu Phase Select Action Delete 6 of 6 NONE Insert Delete Select NONE only if you want to return to the phase paramete
63. s the default scale units or the previously chosen scale units Use and gt keys to select the run time scale of your choice followed by ENT Return to the setup scale menu Use gt key to movethe cursor to the PULSE TIM E position TheLCD will appear as Setup scale gt Run Time PULSE TIME Set pulse time scales 29 6 Programming the controller 6 5 Edit mode 30 Press ENT when the cursor ison PULSE TIM E and the following will appear Setup scale pulsetime Sss s H h mm ss Ssss SSS S Ss SS S SSSS The first line indicates the existing default pulse time Use the right and left arrow keys to movethe cursor position to the desired scale unit then press ENT When both theCONNECTION and SCALE have been entered press ESC twice to return to the main menu Note T he setup scale that you select from the main menu isa global parameter It will become the default scale for future procedures in both the BASIC and EDIT modes When working in the EDIT mode however you will have the option of customizing the setup for each individual procedure if you wish From the main menu M ain gt Basic EDIT Load Setup Edit the experimental conditions Select EDIT The LCD now displays the following Edit gt Select procedure number 1 9 C Existing procedures Y ou can now create up to 9
64. sed twice with distilled water and finally placed upside down and left to dry thoroughly NEVER useorganic solvents with GeneN avigator electrophoresis unit 3 Description of the system 3 Description of the system GeneN avigator system is made up of the following components Gene N avigator electrophoresis unit for pulsed field gel electrophoresis Includes pump for buffer circulation cooling coil safety lid with cable insert moulds gel staining tray silicone tubing hose connectors and hoseclamps Gene N avigator electrophoresis unit is availablein two versions 100 130V 60Hz 18 1019 18 and 200 260 V 50 Hz 18 1019 19 Note TheH EX electrode kit 18 1019 20 and the point electrode kit 18 1019 23 can both be used with either of the above electrophoresis units GN control unit 18 1026 17 A controller for switching the electric field and programming up to 9 run procedures Includes connecting cable with banana plugs For either 100 130 V 60 H zor 200 260 V 50 Hz Power supply EPS 600 19 0600 00 e Power supply EPS 200 19 0200 00 HEX electrodekit 18 1019 20 Includes a H exagonal electrode gel supporting tray and gel casting frame and three double combs 1 mm 18 1019 25 and 2mm 18 1019 26 thick and a preparativecomb 18 1019 27 e Point electrode kit 18 1019 23 Includes 6 cathode electrodes and 2 anode electrodes gel support tray gel casting frame for 20 x 20 cm gels and
65. select the SETUP procedure See Section 6 4 2 Start the run by pressing the key on the front panel on the control unit This activates the BA SIC mode you have just programmed Thegreen LED indicates that the unitis ON TheLCD will then resemble the screen below with the clock counting elapsed time oo gt Basic N S Runtime 060 0000 00 of 020 00 25 6 Programming the controller 6 4 Setup 26 Fluctuations back and forth between the N S and E W pulses will be displayed during therun Y ou will also beableto read how much of the total run time has elapsed If you press off while a run isin progress the green LED will go off and theLCD will indicate the following Basic frozen Continue by On End by ESC on s a 1 If you press therun will continue from the point at which you stopped Note Ifa power failure should occur during a run when the power returns therun will continue from where it was frozen 2 If you press ESC instead of off the BASIC programming screen will be displayed again n N ow press of again and therun will start from the beginning 3 If you press ESC twice you will return to the main menu W hen the set run time has elapsed a buzzer will sound and theLCD will show Basi
66. sh to have only one phase in this procedure press the key and you will beinformed that procedure 1 consists of 1 phase with a run time of 2 hours and 30 minutes N ow you can press once more and moveto STORE to save the newly created single phase procedure see Store procedure below Note W hen you have programmed the last phase use ESC to store the procedure Do not uselN SERT when you want to finish programming a procedure If you want to create a second phase in the same procedure however press EN T from the phase field instead of ESC and the following phase menu will be displayed 6 Programming the controller Phase Select action Insert lofl None INSERT Delete If you enter on INSERT from the above menu you will move into phase 2 of the same procedure as shown below phase N S e w phase time 2 of 2 060 0 060 0 002 30 All of the values from phase 1 are automatically duplicated but can easily be changed by writing them over if desired J ust use arrow keys to move through the fields as before W hen the desired phase time has been typed in press ENT to move into the phase field PH ASE capitalized Press ENT again and the phase menu see below is displayed again Press ESC again and you will go into phase 2 of 2 Repeat the same procedure each time you want to create a new phase Continuein the same way to create up to 6 phases in
67. sired in each phase 6 5 4 Edit store Edit1 gt Setup Input STORE Delete Store procedure Use to movethe cursor to STORE and press ENT to bring forward the following 6 Programming the controller Edit 1 gt Store as procedure 1 9 C Existing procedures As before when you entered on INPUT the C above is a blinking cursor If you had previously stored any procedures the numbers that you assigned them would appear after Existing procedures Store the procedure by pressing any number between 1 and 9 since all 9 positions are available W hen the number has been pressed and entered in the example below we pressed number 1 you will return immediately again to the edit menu N ow you can safely escape to the main menu W hen next you re enter the EDIT mode from the main menu the following will be displayed Edit 1 gt Store as procedure 1 9 C Existing procedures 1 N ow you can either re enter the existing procedure by pressing 1 or INPUT anew procedure by pressing another number Remember that pressing 0 always takes you to the next available number in this case 2 If you have previously created and stored several procedures the numbers of the existing procedures will all be displayed If you press 1 to reenter the procedure just stored you will return to the edit menu Select IN PUT to re enter thef
68. ss gt ENT to storethe procedure Enter the number under which you want to storethe procedure by pressing the chosen number followed by ENT To run the program the instrument must beinLOAD mode 59 10 Reference list 10 3 Reference list 60 This list describes some general applications of PFGE In these applications types of PFGE other than HEX and DI arealso used T hese references give you valuable information on sample preparation and running parameters Organism reference Amoeba Bacterial DNA Blotting megabase DNA Candida Circular DNA Crithidia GenomicDNA Human chromosome x and y Human chromosome 3 Human chromosome 4 Human chromosome 7 Human chromosome 9 Human chromosome 11 Human chromosome 17 Leishmania M ethod N eurospora crassa Plant Plasmodium Saccharomyces Schizo saccharomyces Theory Trypanosome Virus 24 37 40 Amersham Biosciences V acuG ene Protocols 1 to 4 43 45 20 36 37 39 35 48 73 64 70 72 68 62 65 69 71 59 55 17 20 26 36 1 14 48 50 54 46 15 16 21 23 41 42 1 2 5 10 12 25 35 47 10 Reference list References Reference 1 Author Keyword Reference 2 Author Keyword Reference 3 Author Keyword Reference 4 Author Keyword Reference 5 Author Keyword Reference 6 Author Keyword Reference 7 Author Keyword Reference 8 Author Keyword Referenc
69. ss the Function key F Now press or 2 to movethe cursor to RESET The following will appear on theLCD Function gt Contrast RESET Reset the instrument 23 6 Programming the controller 6 3 BASIC mode 24 Again the upper case characters indicate the position of the cursor N ow execute RESET by pressing ENT Thefollowing warning message will appear on the LCD WARNING All procedures will be ERASED Press ESC to abort PressENTER to RESET If you press EN T theinstrument will reset itself and return you to the initial screen showing the software version number Strike any key to move into the main menu Asindicated above all procedures that have been created and stored will be erased All set parameters will be changed to default If you press ESC you will return to the Contrast R eset menu Press ESC again to return to the main menu When you turn the unit on with the switch on the rear panel for the first time the LCD will indicate the version number of the installed software Thesame information will be displayed whenever you reset the unit Press any key to move to the main menu which looks like this M ain gt BASIC Edit Load Setup O peratethe controller in a basic way BASIC mode isthe simplest way to program one set of pulse and run time parameters just before starting a run To
70. system so far comes from a fungus and is 12 6 million base pairs in size This manual will help you get the most out of your GeneN avigator system Recommended running parameters and the latest preparation protocols for the most commonly used samples are included for the benefit of newcomers to PFGE Experienced users will find that GeneN avigator is easy to adapt to the running conditions required by different samples The system consists of an electrophoresis unit a power supply a control unit and a cooling bath which enable you to optimizerunning conditions and fine tune all separations Fig 1 Power supply order separately Gene Navigator electrophoresis f Thermostatic cooler unit order separately Fig 1 Gene Navigator system is shown here using two electrophoresis units Please do not attempt to use the system until you have at least read the operating instructionsin Chapter 5 2 Important safety information 2 Important safety information To avoid any risk of injury the instrument should only be operated by properly trained personnel and alwaysin accordance with the instruction provided Read this entire manual before using the instrument WARNING This instrument is designed for indoor use only WARNING Do not operate the instrument in extreme humidity above 95 RH Avoid condensation by equilibrating to ambient tem
71. tc An F ke Function key which permits two functions resetting of all run and pulse time parameters or adjusting the contrast of the LCD See Section 6 2 Contrast and reset commands Left and Right arrow keys and which will movethe cursor horizontally between menu selections on the display LCD After pressing the F key see Example 1 below use Jand gt keys to position the cursor over either CONTRAST or RESET Upper case characters indicate where the cursor is placed on the screen 21 6 Programming the controller 22 The op key is used to start an electrophoresis run after all parameters have been set It is also used to freeze or temporarily halt a run that has begun or to end arun that has been completed when the buzzer sounds ENT is used to enter a command in upper case letters in a menu or a new parameter value W hen questions are prompted on theLCD press ENT to respond yes ENT also moves the cursor one position to the right similar to the right arrow key when you arein the basic or edit input modes CE will clear the last entry and return the old pulse or run time parameter that was typed over This works before ENT has been pressed ESC isused to exit from a sub menu and move back to an earlier menu or to the main menu Also when questions are prompted on th
72. verley S M E coli L eishmania method 61 10 Reference list 62 Reference 10 Author Keyword Reference 11 Author Keyword Reference 12 Author Keyword Reference 13 Author Keyword Reference 14 Author Keyword Reference 15 Author Keyword Reference 16 Author Keyword Reference 17 Author Keyword H igh resolution separation and accurate size determination in pulsed field gel electrophoresis of DNA 1 DNA sizestandards and the effect of agarose and temperature Biochemistry US 1988 Vol 27 no 26 p9204 9210 M athew MK Smith C L Cantor C R bacteriophage DN A gel concentration temperature H igh resolution separation and accurate size determination in pulsed field gel electrophoresis of DNA 2 Effect of pulse time and electric field strength and implications for models of the separation process Biochemistry US 1988 Vol 27 no 26 p9210 9216 M athew M K Smith C L Cantor C R pulsetime field strength H igh resolution separation and accurate size determination in pulsed field electrophoresis of DNA 3 Effect of electrical field shape Biochemistry US 1988 Vol 27 no 26 p9216 9221 Cantor C R Gaal A Smith C L electrode angle separation zones H igh resolution separation and accurate size determination in pulsed field gel electrophoresis of DNA 4 Influenceof DNA topology Biochemistry US 1988 Vol 27 no 26 p9222 9226 M athew MK Hui C F Smith C
73. xample Parafilm preferably with a pair of nuclease free microscope cover slips Fig 7 or a sterilized razor blade If you cool the blocks on ice before cutting them it will be easier to make symmetric slices Note The slice must be slightly narrower than the width of the combs if not reduce the width of the sample blocks This will improve loading Fig 7 To cut sample blocks use microscope cover slips Transfer a slice to the gel while it is attached to one of the glass cover slips Use the other cover slip to push thesliceinto the well If necessary use a bent Pasteur pipette to carefully push the slices farther down or suck them down with a syringe if the slices cover the whole well Fig 8 Theslices should be placed in contact with the front wall of the well The front wall faces the direction in which samples will move This positioning will help produce sharper bands Also make sure that the sample block slices are not wider than the well Parafilm is a trademark of American Can Company 15 5 Operation 5 5 Running 16 Fig 8 Loading sample into wells Three different methods that can also be used in combination Between 0 5 and 1 0 microgram of DNA is usually loaded into each well A higly concentrated sample loaded asa thin slice gives better separation enhanced resolution than a less concentrated sample loaded asa thicker slice Therefore we recommend usin
74. y numbers following the Existing procedures indicate procedures that have been created and STORED To beableto run a procedure load it by simply pressing the appropriate number followed by ENT If any of the programmed pulse or run times now require adjusting a message on the LCD will indicate what changes are required If this occurs note the message carefully and then press ESC If there is more than one error in the procedure a new message may appear Note it and then continue pressing ESC until this menu appears Procedure 1 Start by ON Freeze by O ff End by ESC N ow press ESC onceagain to move back into the EDIT mode where you can re enter the procedure and make the required changes W hen completed return to LOAD and repeat the above steps If all programmed values are acceptable the procedure number will move you directly to the above menu Begin the programmed run by pressing ff key Temporarily stop or freeze the run at any time by pressing the button once more To terminate a run press off and then ESC Y ou can also escapefromLOAD while a procedure is running to edit another procedure or even change the one that is currently running o CO W hen the run is completed you can use the switch on the rear panel to turn off the instrument 7 Evaluation and presentation of data 7 1 Electrical paramete
75. you are ready for an actual run return to the rear panel and complete the hook up as follows 1 Connect the DC inlet plugs to the outlet on your power supply There plug isthe anode and the black plugisthe cathode WARNING NEVER run with morethan 450 V or 500 mA as this will burn out the control unit 2 Connect the 4 pole plug from the electrophoresis unit to the 4 pole socket above the DC inlets Secure the connection by turning the locking ring clockwise 5 Operation 5 1 Preparing the tank 5 2 Preparing the gel 5 Operation Y ou will need 2 5 of buffer for the electrophoresis unit pH ca 8 3 Werecommend using TBE because of its buffering capacity Prepare 10 litres of 5x TBE 0 5M TBE stock by dissolving Tris base 540g Boric acid 2759 EDTA 37 2 gor 200 ml 0 5 M pH 8 0 The buffer will last longer if you autoclave and storeit cold If you storethe diluted buffer a long time some buffer salt may precipitate Werecommend using either 0 15x TBE 0 015M or 0 5x TBE 0 05M as buffer depending on application These buffers differ in migration speed current flow and heat generation For your first trials werecommend 0 5x TBE Start the thermostatic circulator well in advance of the run ca 30 min to cool the circulating buffer To reach a typical running temperature of 12 C to 15 C in the buffer M ultiT emp III thermostatic circulator should be set at 8 to 10 C A uniform tem

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Gene Navigator™ System User manual