1. Pulse Controller Function and Description
Contents
1. To continue ENTER to end run EXIT To continue the run press the Enter key To cancel the run press the EXIT key a second time Note Always use the EXIT EXIT sequence to cancel a run Turning off the unit does NOT end the run it only interrupts it This feature causes the run to resume once power is restored this is also the case when power is restored after a power failure 5 Once the run is complete the Pulse Controller will sound several beeps and the screen will show the program parameters in effect at the end of the run Note Due to rounding error the elapsed time at the end of a run may be slightly less than the programmed run time This discrepancy can be particularly noticable with very short pulse times 6 Press the EXIT key to return to the Single Main Menu 7 Turn off the Pulse Controller and turn off the power supply Disconnect the power supply leads Disconnect the leads to the electrophoresis unit Amersham Biosciences PC 500 SwitchBack Pulse Controller User Manual 12 6 Multi phase runs As many as five single phase programs may be concatenated to form a multi phase program After programming and saving single phase programs according to the instructions in Section 5 A and 5 B above follow the instructions below to create a multi phase program 6 A Programming a Multi phase run 1 From the Single Main Menu press the MODE key to switch to the Multi Main Menu The scree2. Single Main Menu screen will appear To run the program proceed to Section 5 C below Amersham Biosciences PC 500 SwitchBack Pulse Controller User Manual 11 5 C Performing a Single phase run 1 Make sure the INPUT leads from the SwitchBack Pulse Controller are securely connected to an approved power supply Make sure the leads from your electrophoresis unit are securely connected to the receptacles marked OUTPUT on the front of the Pulse Controller Load the samples turn on the power supply and set the voltage We recommend using constant voltage Maximum voltage is 500 V maximum current 500 mA For suggested running conditions See Section 13 2 To start the run go to the Single Main Menu and press 2 RUN If you have programmed a run in time the Run in screen will appear ERE pa File TEST1 S 1 Elapsed Time 21 15 Program Time 2 2 3 After the run in is complete the run will begin and the Run Screen will appear TEST1 S 1 Run Time D O O 1GE F R 3 1 4 19 OOS Mode REV 1 Sec PT In this example the single phase run file name is TEST1 The single phase run file number is 01 The elapsed time E is 10 seconds The forward reverse ratio F R is 3 1 the set time S is 4 hrs 10 min The unit is running in the reverse field REV mode and the current pulse time PT is 1 second 4 To pause a run press the EXIT key When the run is paused the screen will read Run is paused
3. a value that is outside the programmable range the closest allowable value will automatically display For example if you enter an F R ratio of 1 0 1 the lowest allowable value of 2 0 1 would be set and if you enter an F R ratio of 5 0 1 the highest allowable value of 4 0 1 would be set Press the FWD key to see the programmed value 6 To print this screen press the PRINT key 7 After entering the last value for the F R pulse time ratio and printing the screen optional press the Enter key The Single Main Menu screen will display 8 To save the program press 3 FILER MENU and proceed to Section 5 B Saving a Single phase Program We recommend that you always save the program Otherwise the program will be lost when the SwitchBack is turned off at the end of a run or when the program is aborted 9 To start the run proceed to Section 5 C Performing a Single phase run Amersham Biosciences PC 500 SwitchBack Pulse Controller User Manual 9 5 B Saving a Single phase Program 1 After you have finished entering or editing a program and pressed the Enter key the Single Main Menu screen will display To save the program press 3 FILER MENU The Single Filer Menu screen will appear Filer Menu lt 2 gt Del lt 4 gt Format lt 6 gt Prt Dir 2 The Directory can store up to 30 programs To save a program press 1 SAVE to access the directory Each file in the directory is identified by a number and can be
4. checked prior to initiating any experiment in electrophoresis Important The instrument must always be used with the earth lead of the power cord correctly grounded to earth at the mains outlet Work area The bench and floor should be dry High voltage connections All high voltage leads used should be intact and not frayed All plugs should have a fixed protective plastic sleeves that shields the plug as it is inserted into the power supply or Pulse Controller Exposed plugs are a shock hazard and should be replaced with shielded plugs Stackable leads that connect more than one gel unit to a single outlet are not recommended and should be replaced with shielded style plugs Electrophoresis chambers These should be covered when in use with no openings large enough to allow fingers or other objects to make contact with the electrified buffer or electrodes If this equipment is used in a manner not specified by the manufacturer the protection provided by the equipment may be impaired Only accessories and parts approved or supplied by Amersham Biosciences may be used for operating maintaining and servicing this product Informations importantes Securit Electrique Les points suivant doivent tre v rifi s avant de commencer toute experience lectrophor tique Important l instrument doit toujours tre utilis propremment connect a la prise de terre Place de travail la paillasse et le sol doivent tre
5. end and slowly slide it completely out of the module 4 Pull the fuse out of its cassette and inspect If the fuse element is burned or broken replace the fuse with an identical type If the fuse appears to be intact check it with an multimeter A reading of 10 or less indicates the fuse is still usable 5 After placing a good fuse into the cassette slide it into the power module making sure the arrow on the cassette points to the right in the same direction as the guide arrows on the inside of the compartment door 6 Repeat steps 3 4 and 5 for the second cassette 7 Close the fuse compartment cover and gently press it into the power module until it snaps shut 8 Plug the power cord in and turn the mains power switch on Amersham Biosciences PC 500 SwitchBack Pulse Controller User Manual 20 N FUSE MODULES i N POWER SWITCH gt HIGH VOLTAGE INPUT HIGH VOLTAGE FUSE F 0 5 A 250V PRINTER o i MAINS IN Figure 3 Back panel Figure 4 Mains power module replacing fuses Amersham Biosciences PC 500 SwitchBack Pulse Controller User Manual 21 15 Bibliography Cosmid Mapping Birren B W E Lai L Hood and M I Simon Pulsed field gel electrophoresis techniques for separating 1 to 50 kilobase DNA fragments Anal Biochem 177 282 286 1988 RFLP Inglis B P R Matthews and P R Stewart Induced deletions within a cluster of resistance genes in the mec region of
6. example in which a ten minute run in time was specified Run In File TEST1 S 1 1 5 Elapsed time 88 OO Program time 19 In this example there are five single phase programs concatenated into a multi phase program The name of the multiple file is TEST1 and the single file number currently running is S 1 1 5 indicated at the end of the second line reports that the first of five files is currently running 4 After the run in time has elapsed the run screen will appear TEST1 S 1 Run Time Multi 1 5 1 E F R 3 1 4 03 08 S Mode REV 1 Sec PT In this example the multi phase program name is TEST1 The single phase run in progress is 1 Multi 1 5 indicates that the run in progress is the first of five concatenated runs The elapsed time E is 10 s The forward reverse ratio F R is 3 1 the set time S is 4 hr The unit is running in the reverse field REV mode and the pulse time PT is 1 s 5 To stop a run once it has started press the EXIT keyThe display will show Run is paused To continue ENTER To end run EXIT Amersham Biosciences PC 500 SwitchBack Pulse Controller User Manual 15 6 When the multi phase run is complete the Pulse Controller will sound several beeps and the final screen will show Multiple run is now completed Press any key Press the EXIT key to return to the Multi Main Menu Turn off the Pulse Controller and turn off t
7. named When you are setting up your first file the screen will look like this S 1l empty S 2 empty S 3 empty S 4 empty S 5 empty S 6 empty Store program at S 1 The program defaults to the next empty file for storage If you wish to store the program in a different file use the delete key to delete the default number and then use the numeric keys to enter the desired file number 3 Press the Enter key The screen will now show File will be saved Note File name is optional 4 Optional naming a file If you do not wish to name it press the Enter key to return to the Single Main Menu The file will be saved as the number selected in Step 2 above To begin the run proceed to Section 5 C Performing a Single phase run To name the file proceed to Step 5 below 5 A file name may contain up to five characters The characters may be alphabetical or numeric or a combination To name a file use the ALPHA key in combination with the 12 keys on the right side of the unit a Note the three small red lamps above the DEF key When all of the lamps are off the key pad is in numeric mode Any lit lamp indicates that the key pad is in the alphabetical mode and the active letter position is the same as the active lamp position for positions 0 9 b If you wish to type alphabetical characters and no lamps are lit press the ALPHA key The lamp above the D farthest left will light In this position the letter or symbol to
8. the chromosome of Staphylococcus aureus J Gen Micro 136 2231 2239 1990 El Adhami W L Roberts A Vickery B Inglis A Gibbs and P R Stewart Epidemiological analysis of a methicillin resistant Staphylococcus aureus outbreak using restriction fragment length polymorphisms of genomic DNA J Gen Micro 137 2713 2720 1991 Su C J S F Dallo H Alderman and J B Baseman Distinctions in DNA and protein profiles among clinical isolates of Mycoplasma pneumoniae J Gen Micro 137 2727 2732 1991 Genomic Analysis Van Daelen R A J and P Zabel Preparation of high molecular weight plant DNA and analysis by pulsed field gel electrophoresis Plant Molec Biol A15 1 25 1991 Van Daelen R A J J J Jonkers and P Zabel Preparation of megabase sized tomato DNA and separation of large restriction fragments by field inversion gel electrophoresis FIGE Plant Molec Biol 12 341 352 1989 El Adhami W L Roberts A Vickery B Inglis A Gibbs and P R Stewart Epidemiological analysis of a methicillin resistant Staphylococcus aureus outbreak using restriction fragment length polymorphisms of genomic DNA J Gen Micro 137 2713 2720 1991 YAC s Carle G F M Frank and M V Olson Electrophoretic separations of large DNA molecules by periodic inversion of the electric field Science 232 65 68 1986 Long Range Physical Mapping Kamiura S C M Nolan and D Meruelo Long range physical m
9. 3 0 1 3 0 1 Note If the sample DNA is larger than 50 kb it must be in agarose plugs Single phase program for DNA from 50 to 1000 kb including lambda ladders and S cerevisiae chromosomes Voltage 200 V 6 9 V cm Run in Time 0 Run Time 28 hrs Pulse Time 1to50s Mode Reverse REV F R Ratio 3 0 1 Note The sample must be in agarose plugs Amersham Biosciences PC 500 SwitchBack Pulse Controller User Manual 19 14 Care and Maintenance Cleaning Do not immerse any part in water Wipe the unit components with a soft damp cloth after each run Do not use any strong detergents or acids or bases Replacing fuses Important Fuses protect equipment by disconnecting loads too large for the circuit design so it is imperative that replacement fuses conform to the fuse rating specified on the label Figure 3 shows the back panel and Figure 4 illustrates how to replace input fuses in the mains power module 1 Caution Turn the mains power supply switch off and detach the power cord before replacing input fuse s 2 Open the fuse compartment by inserting a small flat blade screwdriver into the slot at the end of the power module opposite the power cord receptacle Twist the screwdriver 1 8 turn pulling the hinged compartment cover open Note The 115 V model has one fuse cassette located on the right The 230 V model has two fuse cassettes 3 Insert the screwdriver above the arrow on one fuse cassette catch the cassette
10. 43 71 MultiTemp III Thermostatic Circulator 115 V 1 18 1102 77 230 V 1 18 1102 78 DNA PFGE markers 27 4530 01 Yeast DNA PFGE markers 27 4520 01 Amersham Biosciences PC 500 SwitchBack Pulse Controller User Manual 1 10 11 12 13 14 15 16 PULSE CONTROLLER FUNCTION AND DESCRIPTION A PC 500 PULSE CONTROLLER B APPLICATIONS C SPECIFICATIONS IMPORTANT INFORMATION USER INTERFACE OPERATING INSTRUCTIONS SINGLE PHASE RUNS A PROGRAMMING A SINGLE PHASE RUN B SAVING A SINGLE PHASE PROGRAM C PERFORMING A SINGLE PHASE RUN MULTI PHASE RUNS A PROGRAMMING A MULTI PHASE RUN B SAVING A MULTI PHASE PROGRAM C PERFORMING A MULTI PHASE RUN LOADING A PROGRAM EDITING AND SAVING A PROGRAM DELETING A PROGRAM FORMATTING THE DIRECTORY VIEWING THE DIRECTORY PRINTING THE DIRECTORY RUNNING PARAMETER GUIDELINES CARE AND MAINTENANCE BIBLIOGRAPHY CUSTOMER SERVICE INFORMATION Amersham Biosciences oo po 12 13 14 15 16 16 17 17 17 18 19 21 21 PC 500 SwitchBack Pulse Controller User Manual Amersham a UY Biosciences 24598 Rev C 4 97
11. PC 500 SwitchBack Pulse Controller User Manual 1 Pulse Controller Function and Description 1 A PC 500 Pulse Controller The PC 500 SwitchBack Pulse Controller is designed for performing field inversion gel electrophoresis FIGE of double stranded DNA molecules The SwitchBack is used in conjunction with a power supply to run either a horizontal or vertical electrophoresis unit When used in the reverse field mode for FIGE the SwitchBack periodically reverses the polarity of the electrodes As a result molecules in the gel migrate in a forward direction and then when the polarity is reversed reorient themselves and travel backward Because the forward pulse is always longer than the reverse this results in a net forward migration By selecting the appropriate pulse times FIGE enables you to maximize resolution of DNA fragments in desired regions from the kilobase to megabase size range The SwitchBack also provides an alternating cross field mode for OFAGE and TAFE The SwitchBack can be programmed for single or multi phase runs A single phase run is controlled by one program A multi phase run is controlled by several single programs concatenated together A single phase run can have a constant pulse time throughout or a ramped pulse time that increases linearly during the run For a ramped separation all you need to enter is the total run time and the initial and final pulse times the SwitchBack automatically compute
12. all outlet See Figure 3 3 Connect the leads from your electrophoresis unit to one of the two sets of OUTPUT receptacles on the front of the Pulse Controller 4 Connect the INPUT leads from the back of the SwitchBack to an approved power supply Plug the power supply into a grounded wall outlet Figure 2 illustrates instrument hook up Do not turn the power supply on yet 5 Optional If you intend to print screens or programs connect the printer cable to the parallel printer port at the back of the Pulse Controller 6 For ease in viewing the screen a retractable metal stand is attached to the underside near the front feet of the unit To use pull the stand forward and allow it to snap securely into place 7 Turn the Pulse Controller on by setting the mains power switch to the on position 8 To program the Pulse Controller see either Section 5 Programmng and Performing a Single phase run or Section 6 Programming and Performing a Multi phase run 9 When programming is complete prepare and load your samples Amersham Biosciences PC 500 SwitchBack Pulse Controller User Manual 10 11 Turn on the power supply We recommend using constant voltage Maximum voltage is 500 V maximum current is 500 mA See Section 13 Running Parameter Guidelines Perform the run according to the instructions in Section 5 C Performing a Single phase run or Section 6 C Performing a Multi phase run Note In the event o
13. and numbers in a particular directory go to either the Single Main Menu or the Multi Main Menu and press 3 FILER MENU On the Filer Menu Screen press 6 PRT DIR The screen will display Printing directory Press any key to abort When the printout is complete the Filer Menu screen will display Press the EXIT key to return to the Single or Multi Main Menu Amersham Biosciences PC 500 SwitchBack Pulse Controller User Manual 18 13 Running Parameter Guidelines For each set of conditions these factors were the same 1 GTG agarose gels were run on the HE 100 SuperSub Horizontal Electrophoresis Unit with buffer circulation The 0 5X TBE buffer was cooled to 10 C The power supply was operated in constant voltage mode Single phase program for DNA in the size range of 0 5 25 Kb Voltage 230 V 7 9 V cm Run in Time 10 min Run Time 16 hrs Pulse Time 50 msec no ramp Mode Reverse REV F R Ratio 2 5 1 Single phase program for DNA in the size range of 8 3 to 48 kb Voltage 150 V 5 2 V cm OR 150 V 5 2 V cm Run in Time 10 min 10 min Run Time 32 hrs 28 hrs Pulse Time 0 7 to 2 3 s 0 6 to 2 0 s Mode Reverse REV Reverse REV F R Ratio 2 0 1 3 0 1 Multi phase program for DNA in the size range of 5 to 250 kb RUN 1 RUN 2 Voltage 200 V 6 9 V cm 200 V 6 9 V cm Run in Time 10 min 0 Run Time 8 hrs 8 hrs Pulse Time 1to20s 0 8to1 5s Mode Reverse REV Reverse REV F R Ratio
14. ap of the Ly 6 complex mapping the Ly 6 multigene family by field inversion and two dimensional gel electrophoresis Genomics 12 89 105 1992 Fingerprinting Grothues D and B Tuemmler New approaches in genome analysis by pulsed field gel electrophoresis application to the analysis of Pseudomonas species Molec Microbiol 5 11 2763 2776 1991 General Pulsed Field Gel Electrophoresis Lai E B W Birren S M Clark M I Simon and L Hood Pulsed field gel electrophoresis BioTechniques 7 1 34 42 1989 16 Customer Service Information Technical service and repair Amersham Biosciences offers complete technical support for all our products If you have any questions about how to use this product or would like to arrange to repair it please call or fax your local Amersham Biosciences representative Important Request a copy of the Amersham Biosciences Health and Safety Declaration Form before returning the item No items can be accepted for servicing or return unless this form is properly completed Ordering information Qty Code No PC 500 SwitchBack Pulse Controller 0 500 V 500 mA 115 V 1 80 6073 92 230 V 1 80 6074 11 Fuses 115 V input T 0 25A 250V 5x20 mm 5 80 6106 98 output F 0 5A 250V 5x20 mm 5 80 6335 74 230 V input F 0 16A 250V 5x20 mm 5 80 6270 76 output F 0 5A 250V 5x20 mm 5 80 6335 74 Companion Products HE 100 SuperSub Submarine Electrophoresis Unit complete 1 80 60
15. aving a Program 1 2 To edit a single or multi phase program first load it into memory See Section 7 Loading a Program Once the program is loaded press the EXIT key to return to the Single or Multi Filer Menu Press 1 Edit Use the FWD key to step the cursor to the desired position Use typeover or the DEL key to change values To cancel the edit press the EXIT key This will return to the Main Menu without altering either the program copy loaded into memory or the original program stored in the directory To load the changes into memory press the Enter key This will return you to the Main Menu If desired you may test the edited program by pressing 2 Run To save the edited program to the Directory press 3 Filer Menu from the Main Menu Then press 1 Save You can either replace the original program see a below or save it as a new program see b below a To accept these defaults press the Enter key This replaces the original program in the Directory with the edited version b To change the default name and location use typeover or the DEL key to specify a new name and location Then press the Enter key This saves the edited version to the Directory without replacing the original program 9 Deleting a Program 1 2 To delete a single phase file go to the Single Filer Menu and press 2 DEL To delete a multi phase run file go to the Multi Filer Menu and press 2 DEL The sc
16. ding a separation from the smaller DNA Many types of PFGE instrumentation are available The simplest equipment is designed for FIGE which periodically inverts the polarity of the electrodes during electrophoresis subjecting DNA to a 180 reorientation Only an electrical field switching module is needed and any standard vertical or horizontal gel box with temperature control can be used Although extremely useful for separating relatively small DNA 4 1000 kb FIGE s reorientation angle of 180 results in a separation range limited to under 2000 kb Like other PFGE techniques FIGE may exhibit mobility inversions during electrophoresis in which larger DNA can move ahead of smaller DNA Pulse length ramping in which the orientation pulse length is constantly increased during separation will minimize inversions and this capability is included in the SwitchBack Ideally the DNA should migrate in straight lanes to simplify lane to lane comparisons The original pulsed field systems used inhomogeneous electric fields that did not produce straight lanes making interpretation of gels difficult The simplest way to achieve straight lanes is to periodically reverse the electric field with a Pulse Controller A standard gel box with parallel electrodes produces a homogeneous electric field Amersham Biosciences PC 500 SwitchBack Pulse Controller User Manual 1 C Specifications Power Requirements Fuse values Programmable po
17. er Manual 8 5 Enter the values for each parameter a Key in the desired value The cursor itself does not move but the numbers entered scroll from right to left Press the DEL key to delete the right most character b Press the FWD key c Enter in the next value and press the FWD key Run in Run in time is an optional setting that designates a conventional electrophoresis period before the pulse program begins Run in time is only programmable in the reverse field mode REV Run in time can be set from 0 to 99 min Note It is not included as part of the run time Run Run time can be set from 1 to 299 hrs 59 min Pulse Time When entering the pulse time PT the initial and final pulse times must have the same time units i e s min or hr To switch between time units press the UNITS key Initial and final pulse times can be set from 0 01 to 99 99 s 0 01 to 99 99 min or 0 01 to 99 99 hr The intermediate pulses used during the run are automatically determined by the program Mode The default mode is reverse field REV for FIGE Alternating mode ALT for OFAGE or TAFE can be selected by pressing the MODE key Forward Reverse Ratio F R When you select reverse field REV F R indicates the forward reverse ratio of each pulse It can be set from 2 0 1 to 4 0 1 The default setting is 3 0 1 d To change a value press the DEL key The value will be deleted from right to left Enter the correct value e If you enter
18. f a power interruption the SwitchBack automatically continues the programmed run once power is restored SwitchBack Mains In Figure 2 Instrument hook up Amersham Biosciences PC 500 SwitchBack Pulse Controller User Manual 5 Single phase runs 5 A Programming a Single phase run 1 When the mains power switch is set to on three long beeps sound and the screen displays SwitchBack Pulse Controller Rev X XX Scientific Press any key The screen will now display SINGLE MAIN MENU lt l gt Edit lt 2 gt Run lt 3 gt Filer Menu lt 4 gt Print File Press 1 EDIT to begin programming a single phase run The screen will now display Run in m blank S Run Chr min PT 39 07 to 03 73 Sec Mode REV F R 3 1 blank S is the default name and location of an as yet unspecified file The default mode of operation is reverse field REV for FIGE To switch to alternating mode for OFAGE and TAFE press the MODE key The edit screen for alternating mode will appear blank S 99 Run 9 99 hr min PT 93 09 to 00 09 Sec MODE ALT The programming for the alternating mode is similar to that for the reverse field mode except there is no run in time or forward reverse pulse time ratio to be set Saving loading creating multi phase runs and all other functions are identical to those for the reverse field mode Amersham Biosciences PC 500 SwitchBack Pulse Controller Us
19. he power supply Disconnect the power leads Disconnect the leads to the electrophoresis unit 7 Loading a Program 1 To load a single phase program go to the Single Filer Menu Screen To load a multi phase run go to the Multi File Menu screen Press 3 LOAD The screen will now show either the directory of single files or multiple files If you had two single phase programs saved and named the Single File Directory would look like this S 1NAME1 S 2NAME2 S 3empty SM4empty S 5empty SMbempty LOAD PROGRAM AT SSI If you are loading a multi phase program the screen will look the same as above except that the file numbers will begin with M The default setting is SO1 for single phase run files M 1 for multi phase run files To load a different program use the DEL key to delete the 1 and then enter the desired file number e g O2 3 Press the Enter key The screen will display Filer Message File is loaded The screen will return to the Single or Multiple Filer Menu Press the EXIT key The Single or Multi Main Menu screen will appear To run the program press 2 RUN The screen will momentarily display Starting a Single or Multi File Run For a complete description of run progress see either Section 5 C Performing a Single phase run or Section 6 C Performing a Multi phase run Amersham Biosciences PC 500 SwitchBack Pulse Controller User Manual 16 8 Editing and S
20. me as for a single phase program For instructions see Step 5 in Section 5 B Saving a Single phase Program If you do not wish to name the file press the Enter key The file will be saved as the number you selected in Step 3 above 6 After entering the file name press the Enter key The screen will momentarily display Filer Message File is saved Then the Multi Filer Menu screen will appear 7 Togo back to the Multi Main Menu press the EXIT key If you are ready to start the multi phase run proceed to Section 6 C below Amersham Biosciences PC 500 SwitchBack Pulse Controller User Manual 14 6 C Performing a Multi phase run 1 Make sure the leads from an approved power supply are securely connected into the receptacle marked INPUT at the back of the Pulse Controller Make sure the leads from your electrophoresis unit are securely connected to the receptacles marked OUTPUT on the front of the Pulse Controller Load your samples turn on the power supply and set the voltage We recommend using the constant voltage setting Maximum voltage is 500 V maximum current 500 mA For suggested running conditions See Section 13 2 To start the run go to the Multi Main Menu and press 2 RUN The screen will display Starting a Multi File Run Then Starting a Single File run from a Multi File 3 The run screen will then appear If a run in time was specified the screen will be similar to the following
21. n will show Multi Main Menu lt 1 gt Edit lt 2 gt Run lt 3 gt Filer Menu lt 4 gt Print File 2 To program a multi phase run from the Multi Main Menu press 1 EDIT The screen will show the file numbers and names of the first four single phase run files as well as a line of S characters which you will use to select the single file numbers to be concatenated into your multiple multi run S 1 TEST1 S 2 TEST2 S 3 TEST3 S 4 empty Multi Run blank M S D S D S D S D S D To display other single phase programs on the screen press the NEXT or PREV keys Two files are displayed at a time The entire directory of 30 files can be scrolled through by pressing these keys Note For a printed directory of the single files refer to Section 12 Printing the Directory 3 Key in up to five single phase file numbers in the order you wish them to run Press the FWD key to advance the cursor from one file number to the next S is considered the end marker for multi phase runs if there are fewer than five single phase programs Note When a single program is used to create a multiple program the run in time is still executed If this is not desired edit and save a version of the single program without a run in time See Section 9 Editing and Saving a Program 4 After the last file number is entered press the Enter key to return to the Multiple Main Menu screen 5 To save the pr
22. nd the twelve alpha numeric keys are at the right side of the control panel See Figure 1 Function keys Hoefer SwitchBack Pulse Controller PC 500 x 7 ABC DEF GHI 1 2 3 JKL MNO PQR OUTPUT 4 5 6 MODE UNITS ALPHA PRINT STU VWX WE 7 8 9 NEXT PREV FWD EXIT DEL Fa Enter 0 Figure 1 Switchback front panel MODE When editing a single phase program the FWD Movesthe ursor to the next item MODE key changes the operation from reverse field REV to alternating ALT The default EXIT Exits the current screen mode for FIGE is reverse field Pressing the MODE key toggles between the Single Main Menu and the Multi Main Menu UNITS When editing a single phase program pressing the UNITS key cycles the Pulse Time PT setting through seconds minutes and hours ALPHA Determines the selection of either the number or one of the three characters above the alpha numeric keys indicated by a lit lamp above the 2 key The ALPHA key only operates while naming a file see Step 5 in Section 5 B Saving a Single phase Program PRINT Prints set up screens and directories NEXT Scrolls down in the file directory PREV Scrolls up in the file directory When a run is in progress pressing EXIT pauses the run Pressing EXIT again cancels the run Alpha numeric keys The twelve keys to the right of the control keys are similar
23. ogram press 3 FILER MENU and proceed to Section 6 B Saving a Multi phase program We recommend always saving the multi phase program because it will be lost when the Pulse Controller is turned off at the end of a run or when the program is cancelled 6 If you are ready to start the run proceed to Section 6 C Performing a Multi phase run Amersham Biosciences PC 500 SwitchBack Pulse Controller User Manual 13 6 B Saving a Multi phase Program 1 After you have entered the single file numbers you wish to concatenate into a multi phase file and pressed Enter the Multi Main Menu Screen will appear 2 From the Multi Main Menu Press 3 FILER MENU The Multi Filer Menu screen will appear Multi Filer Menu lt 1 gt Save lt 2 gt Del lt 3 gt Load lt 4 gt Format lt 5 gt Dir lt 6 gt Prt Dir 3 Press 1 SAVE The screen will change to the Multi phase Directory MO1 empty M 2 empty M 3 empty M 4 empty M 5 empty M 6 empty Store program at M 1 The program will automatically point to the first empty file for storage If you wish to store the program in a different file use the DEL key to delete the file number and then enter the desired file number using the numeric keys 4 Press the ENTER key The screen will display File will be saved ase oes Note File name is optional 5 Optional Naming a Multi phase file The procedure for naming a multi phase program is the sa
24. reen will display the first six files in the directory along with the message Delete file at SG1 for the Single File Directory or MOI for the Multiple File Directory To see other files in the directory press the NEXT key To review previous files press the PREV key Note The default setting is SG1 or M 1 To delete a different file press the delete key DEL to delete the number 1 and then enter the correct file number Press the Enter key The screen will momentarily display the message that the file has been deleted and the Single or Multi Filer Menu will appear Amersham Biosciences PC 500 SwitchBack Pulse Controller User Manual 17 10 Formatting the Directory To reformat and clear the entire directory of single phase and multi phase files bring up the Single or Multi Filer Menu and press 4 FORMAT The screen will display the message Are you sure Press for no or 1 for yes If you want to completely clear all stored files press 1 The screen will momentarily display Format complete and then return to the Filer Menu 11 Viewing the Directory To view the directory go to the Single or Multiple Filer Menu and press 5 DIR The first eight files will appear To view more files press the NEXT key To review previous files press the PREV key In this way you can page through the entire directory To return to the Filer Menu press the EXIT key 12 Printing the Directory For a printout of the file names
25. s and executes the intermediate pulse times A multi phase run is a concatenation of up to five single phase runs As many as 30 single phase and 30 multi phase programs can be stored in the SwitchBack memory 1 B Applications and Theory Applications of Pulsed Field Gel Electrophoresis PFGE are numerous and diverse These include preparing large DNA fragments for cloning in yeast artificial chromosomes YAC s and P1 vectors identifying restriction fragment length polymorphisms RFLP s and construction of physical maps detecting in vivo chromosome breakage and degradation and determining the number and size of chromosomes electrophoretic karyotype from yeasts fungi and parasites such as Leishmania Plasmodium and Trypanosoma Although the theory of pulsed field electrophoresis is still a matter of research some qualitative statements can be made about the movement of DNA in agarose gels during PFGE Briefly during continuous field electrophoresis DNA above 30 50 kb migrates with a mobility independent of size This is seen on a gel as a single large diffuse band If however the DNA is forced to change directions during electrophoresis different sized fragments within this diffuse band begin to separate from each other With each reorientation of the electric field relative to the gel smaller sized DNA will begin moving in the new direction more quickly than the larger DNA Thus the larger DNA lags behind provi
26. secs Connections de haut voltage Toutes les prises de courant haut voltage utilis doivent tre intactes et non effiloch es Toutes les prises ont une manche protectrice fixes en plastique qui les prot gent quand elles sont branch es au g n rateur de courant ou le Pulse Controller Des prises expos es non prot g es sont un danger d lectrocution et doivent tre remplac es par des prises avec protection L utilisation de multi prises qui permettent de brancher plus d un gel sur une sortie lectrique du g n rateur ne sont pas recommand es et devraient tre remplac es par des prises prot g es Cuves lectrophor tiques elles devraient tre couvertes durant leur utilisation Assurez vous qu il n y aie aucune ouverture suffisement grande pour permettre des doigts ou autre objets d occasionner des contacts avec le tampon ou les lectrodes Si l instrument n est pas utilis en conformit avec les recommandations du fabriquant les protections de s curit qui quipent cet appareil peuvent tre rendues in fficaces Seulement les accessoires et pi ces d tach es approuv s ou fornis par Amersham Biosciences sont recommand s pour l utilisation l entretien et r paration de cet appareil Amersham Biosciences PC 500 SwitchBack Pulse Controller User Manual 3 User Interface The user interface includes two sets of keys the eight function keys are below the liquid crystal display LCD a
27. the left will be entered when a key 0 9 is pressed If you press ALPHA again the center lamp above the E will light In this position the letter or symbol in the center will be entered when a key 0 9 is pressed Amersham Biosciences PC 500 SwitchBack Pulse Controller User Manual 10 If you press ALPHA a third time the lamp on the right above the F will light In this position the letter or symbol at the right will be entered when a key 0 9 is pressed If you press ALPHA a fourth time no lamps will be lit indicating the keyboard is in numeric mode c To change a character press the DEL delete key and enter a new character 6 After entering the file name press the Enter key to save the program The screen will momentarily display before returning to the Single Filer Menu screen Filer Message File is saved 7 Press the EXIT key to return to the Single Main Menu At this point you may print the file see Steps 8 and 9 below or run the program see Section 5 C below 8 To print a file after it has been entered and saved press 4 PRINT FILE The screen will display the first six files and the message PRINT PROGRAM AT S _1 If you wish to print a different file use the DEL key to delete the default number Use the numerical keys to enter the desired file number Press the Enter key The screen will display File now printing Press any key to abort 9 After the printing is complete the
28. to the keys on a touch tone telephone in that each key represents either the number on the key or one of the three letters or characters above the key When naming files pressing the ALPHA key sequentially lights the lamps above the 2 also D E F key The position of the lit lamp indicates which of the three characters is activated press the ALPHA key until the correct position is selected When all three lamps are off the numeric keys are active The DEL key deletes an entry The Enter key completes a program file entry Amersham Biosciences PC 500 SwitchBack Pulse Controller User Manual 5 4 Operating Instructions General Operating Tips Use longer pulse times to separate larger DNA fragments A longer ramp the difference between the initial and the final pulse time will separate a wider range of DNA sizes Band sharpness can be improved by using a higher concentration and or a higher quality agarose sample contains DNA fragments larger than 50 kb the sample must be in agarose plugs for sample oading If the sample contains DNA fragments larger than 200 kb the gel should be cooled to 10 14 C Operating Procedure 1 Cast the gels and assemble your horizontal or vertical electrophoresis unit If the unit has cooling capability we recommend cooling to 10 C during FIGE 2 Connect the power cord to the MAINS IN socket on the back panel of the Pulse Controller and plug into a properly grounded w
29. wer parameters Timer parameters Memory storage Operating environment Relative humidity Altitude Installation category Pollution Degree Dimensions wxdxh Weight Product Certifications 115 V 60 Hz 5 W 230 V 50 Hz 5 W 115 V input T 0 25A 250V 5x20 mm output F 0 5A 250V 5x20 mm 230 V input 2 F 0 16A 250V 5x20 mm output F 0 5A 250V 5x20 mm Switched Voltage 0 500 V Switched Current 500 mA max Minimum 1 min Maximum 299 hrs 59 min Run in Time 0 99 min Pulse Time Range 0 01 s 99 99 hrs Pulse Increments 0 01 s 0 01 min or 0 01 hr Forward and Reverse Pulse Ratio 2 0 1 to 4 0 1 in increments of 0 1 30 single and 30 multi phase programs An internal battery maintains memory when power is shut off or interrupted Indoor use 15 40 C ambient lt 80 for 15 31 C decreasing linearly to 50 for 31 40 C lt 2000 m Il 2 22x23x13 cm 8 6x9x5 in 1 6 kg 3 54 lbs UL3101 1 CSA C22 2 1010 1 CE This declaration of conformity is only valid for the instrument when it is used in laboratory locations gt used as delivered from Amersham Biosciences except for alterations described in the User Manual and connected to other CE labeled instruments or products recommended or approved by Amersham Biosciences Amersham Biosciences PC 500 SwitchBack Pulse Controller User Manual 2 Important Information Electrical Safety The following items should be
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