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UNICORN® OS User Manual - GE Healthcare Life Sciences

1. Figure 5 6 The Method editor in Text instruction mode showing the block window top Text instruction window centre and instruction dialogue box bottom Up to four windows can be displayed together with the instruction dialogue box Click on the Windows toolbar button or choose the View Windows menu command to open a dialogue box for choosing which windows to display Windows Block Graph _ Flowscheme W InstructionBox Figure 5 7 The Windows dialogue box for selecting which windows to display in the Method editor M ethod editing operations which can be performed in the various windows are summarized in the table below UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 5 7 5 Creating and editing methods Widow opaan seson Block window Select block 3 Text window Display and hide block instructions 5 4 Select current instruction M ove instructions within a breakpoint Graph window Display block durations throughout the method N ot recommended for use with UNICORN OS Flow scheme For information only 5 4 5 window Instruction box Specify breakpoints instructions 5 4 2 5 4 4 parameters and variables Insert change and delete instructions 5 3 Method blocks M ethod blocks represent modules in the structure of the method M ost commonly each block handles one functional unit within the method e g column washing amid
2. All users should be given access to the FAILED directory on each local station in a network installation This will ensure that users can access results saved in the FAILED directory in the event of a network communication error 6 Select the system s to which the user will have access from the System s list and the access level of the user from the Access level list Y ou can continue to define new users as long as the User setup dialogue box is open Click on OK to close the dialogue box If you close the box by clicking on Cancel all changes made since you opened the dialogue box will be lost 12 3 2 Changing user passwords Every user can change his or her own password with the User Password command in the M ain menu Enter the old password and the new password twice in the appropriate fields Passwords are not displayed explicitly in this dialogue box The password will not be changed if either the old password is incorrect or the two copies of the new password differ from each other In addition a user with User setup Levels access can change the password for any user To change a password for another user open 12 12 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Administration 7 2 Administration User setup select the user and enter a new password In the Password field see Section 12 3 1 Change passwords regularly and avoid obvious passwords like secret and open sesame
3. Choose Any in the Column pull down list Click on OK once you have made your selections The method template will now be opened as an untitled method 4 2 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Creating methods from method templates 4 4 2 Saving and running a test sequence method All newly created templates already contain a partially built method for a pre defined 20 base sequence which is 5 ATA CCG ATT AAG CGA AGT TT 3 This sequence can be viewed in the sequence editor in the Sequence page of Run setup or the blocks for the sequence as blocks in the T ext instruction panel This partial method has two main uses eeU sed specifically with the fixrec method template you can save the method and directly perform a test run of UNICORN OS to synthesize the sequence see below eeUsed with any method template you can replace the supplied sequence with a sequence of your own choice and then generate a ready to run method for that sequence see Section 4 3 To use the 20 base sequence for a test run of the instrument E 1 Createa new method according to Section 4 1 Run setup 2 Click on the Run set up button on the toolbar or select View Run setup 3 Select the Sequence page to display the pre defined sequence in the sequence editor field IE gi a GEEF Fh aji 5 Daa Pope ATA DOG ATT Akh CGA AGT TI me fe i aani T Pye 25 an W Page ioe oer ee Paral ipa lan a foe ee
4. Curveop FRET_SHIFT C Integr SMOOTH_AR illeaa SMOOTH_MA SMOOTH_MEDIAN J Export Es SUB Description CI Chom TOM of the JOther instruction Curve subtraction Figure 8 19 Instructions field in the Procedure editor dialogue box In the Parameters field the specific parameters contained within the instruction are displayed and clearly identified Some instructions have many such parameters that can be seen by scrolling up and down using the vertical scroll bar O ther instructions have no parameters at all To edit the parameters of a specific instruction either type in the new value or choose from the sub list for a given parameter if such exists When you have made the desired changes click on the Replace command button Y ou will now see that the selected instruction in the evaluation procedure is updated in accordance with the new parameters assigned to it To Insert a new Instruction line select the procedure instruction after the position where you want the new instruction to be inserted Choose an instruction from the Instructions field and then enter parameter values Click on the Insert command button The new instruction will now be added to the evaluation procedure To remove an instruction from the evaluation procedure select it in the procedure listing and click on the Delete command button Select File Save or File Save as from the menu bar in the dialogue box Give the procedure a name
5. Results filtered igf To display all files choose Filter and click on View All UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Login and file handling 3 3 2 4 Finding files To find a file 1 Choose Find from the pull down panel menu or from the File menu Enter a file name specification in the Search for field Find Search for Type W Search all directories 90 01 01 95 06 15 Start directory E Found item c defaulttimethoditest20 c defaulthmethod testo cc defaulthmethodtestsu Figure 3 6 The Find dialogue box Y ou can use standard O S 2 wildcard characters in the file name specification see above 2 You can restrict the search further if required eeC hoose file type from the pull down menu for Type All Directory Method or Sequence for the methods panel All Directory Result or Sequence Scouting for the results panel eeC lick on Date Range for files last written between specified dates eeC heck Search all directories to search through all the directories to which you have access If Search all directories is not checked the search will be restricted to the current directory and sub directories below 3 Click on Search when you have entered all parameters The result of the search is shown in the Found item box UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 3 7 3 Login and file handling 4 Doubleclick ona file in thi
6. 7 7 Installation 11 8 11 12 13 14 Choose to Install additional systems until you have installed all systems physically connected to the computer The number of systems is not related to the number of System control windows installed in step 9 If you installed systems with different drivers make a note of which driver was selected for each system number When you later define systems in M ain menu Administration System the control unit number for each system must correspond to the correct driver If you have chosen to install Strategy and or Method Templates insert the Strategy templates disk when prompted to do so Enter a suitable name for the strategy Unicorn OS 1 10 Installation O Enter name for strategy OligCorn Templates are installed automatically together with the strategy Choose to Install additional strategies if you have more than one configuration of synthesis system N ote Strategies are installed independently of systems A strategy iS assigned to a system when the system Is defined see Section 12 1 After installation is complete you must remove the diskette and restart the computer for the installed software to be properly configured For computers in a network make the required changes to STARTUP CM D see Section 10 7 before restarting the computer If 3 or 4 systems are connected to a computer either physically or through the network the line TH READS 256 In the CONFI
7. Choose File Exit from the menu bar in the dialogue box T he Procedures Edit command on the M ain menu bar can be used to delete or rename created procedures 8 3 3 Points to watch In recording and editing evaluation procedures for automatic evaluation beware of the following potential pitfalls eeM ake sure that the procedure addresses the right curves Curves are identified by storage position alone thus the instruction ADD 01 02 03 will try to add curve 01 to curve 02 and store the result in 03 regardless of the contents of 01 and 02 If 03 contains a curve 8 18 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Evaluating results 8 which is not a raw data curve the existing curve in 03 will be overwritten If 03 contains a raw data curve the procedure will stop with an error message The raw data curves will always occupy the same positions for a given strategy e g UV in position 01 If the Operation is not valid when the procedure is run the procedure will stop at the instruction with an error message Any subsequent instructions in the procedure will not be executed eeln calculating a baseline UNICORN OS suggests default values for the four control parameters see Section 10 1 2 based on the appearance of the curve To instruct UNICORN OS to use default values appropriate for the curve every time the procedure Is run choose the Default setting in the appropriate fields for the parameters
8. Important If the Questions box is not checked questions will not be displayed even if they are defined as mandatory Since the answers to questions can form an important part of the run documentation you are recommended always to check the Questions box N otes If this box is checked the notes page will be displayed at the start of the run Y ou can enter notes in the Start notes field but not in any of the other fields Y ou can use the scroll bar if necessary to read notes in the Method notes field The start of the run is the only occasion when you can enter start notes If the Notes box is not checked the notes will not be displayed and you cannot enter start notes for the run Evaluation procedures If this box is checked the evaluation procedures set to execute at the end of the method will be displayed at the start of the run Y ou can change the choice of procedures to execute but you cannot add or remove procedures Procedures are stored as part of the method file which cannot be changed at the start of the run If the Procedures box is not checked the procedure settings saved in the method will apply at the end of the method System settings If this box is checked the system settings including alarms monitors and curve configuration will be displayed for information at the start of the run UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 5 37 5 Creating and editing methods
9. To change system settings use the System Settings command in the System control menu before starting the run see Chapter 13 Result name changeable If this box is checked you can change the result name when the run is started Click on the Set button to change the result directory If the Result name changeable box is not checked the result name will still be displayed but neither the name nor the directory can be changed Sequence This option should be selected to display the sequence that the method has been created for This is for information only 5 7 Saving the method 5 7 1 Saving as a method When you click on the Create button in the Sequence editor page UNICORN OS generates the method updates the method variables and automatically opens the Save As dialogue box The method must be saved before it can be run Figure 5 38 Dialogue box for saving a method The Methods panel will be shown in icon or detail view according to the current setting in the Main menu You can change the display mode from the pull down menu activated by clicking with the right mouse button in the panel see Section 3 2 3 1 Enter a name for the method M ethod names may be up to 8 characters long and may contain letters A Z digits 0 9 and underscore characters T he case of letters is not significant The method name must be unique for the chosen system within the directory see steps 2 and 3 below 5 38 UNICORN for Oligo Synthesi
10. eeQ S 2 version 3 0 or later eeEtherlink III network card with 3C5x9 SY S driver version dated 10th M arch 1995 or later eeN ovell N eW are version 4 02 or later eeA valid network connection 1 Hardware recommendations apply in February 1997 Contact your local sales office for details of the latest hardware recommendations 2 OS 2 is a registered trademark of IBM Inc 3 Novell and N amp W are are registered trademarks of N ovell Inc UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA A 1 A Technical specifications A 2 Control capacity A 2 1 Stand alone installations Simultaneous control of up to four synthesis systems Each module is separately configured in a system strategy supplied by Amersham Biosciences A 2 2 Network installations Up to four synthesis systems may be connected to each local station in the network Each station local or remote may control up to four systems simultaneously Up to 20 computers with UNICORN OS installed may be logged in to the network at the same time Each local station supports up to 8 simultaneous connections throughout the network if more than one chromatography system is connected to a local station the total number of connections to the systems installed on that system may not exceed 8 regardless of which station the connections are made from A 3 Data sampling Data from synthesis system monitors is stored temporarily in data buffers in the loc
11. A Z digits 0 9 and the underscore character Use underscore characters instead of spaces if you want to separate words in aname eg Flow_rate Names must be unique within the method The case of letters is retained but is not significant The names Flow _Rateand FLOW_RATE are treated as identical UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 5 23 5 Creating and editing methods When you define a variable the value in the parameter field applies as the default value for the variable Note Only one variable which affects block length may be defined within each block Any number of other parameters may however be defined as variables within a block Default variable values can be changed either by editing the instruction in the instruction box or by changing the value in the Variables page of Run setup Changes made in the Text instruction are automatically made in the Variables page and vice versa Figure 5 25 Emma Ca aa i HAE Flier s r biia voue yng i 4 STS ram i a Vigh of appr we or r HE a aTi pper Dresi EET ea PEE EN iSol_Chore lem jion WEI We Figure 5 25 Relationship between variables in Text instructions and in the Variables page of Run setup Important If a breakpoint is defined as a variable changing the variable value when the method is started will shift other instruction breakpoints accordingly This functionality is equivalent to using Change to alter a
12. If you cannot remember your password you cannot log in to UNICORN OS Ask your system administrator or other user with sufficient authorization to give you a new password Press the Cancel button to abandon the log in attempt If you press Cancel during initial start up of UNICORN OS the system returns to the OS 2 desktop N etwork installations In a network installation you must log in to the network before starting UNICORN OS If you want to control a system from the local computer without logging in to the network press Enter when the startup cmd window appears Do not just minimize or close the startup cmd window Y ou can log into UNICORN OSon any computer in the network provided that UNICORN OS software is installed on that computer Y ou can log in with the same username and password on any number UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 3 1 3 Login and file handling of computers simultaneously All computers can connect to and display a running system but a given system can only be actively controlled from one computer M ultiple log ins with the same username are treated internally as separate users for the purpose of System control Note Do not confuse network log in with UNICORN OS log in Y ou log in to the network to gain access to network resources shared drives printers and other networked equipment Y ou login to UNICORN OS to gain access to the oligonucleotide synthesis
13. PMT HLD dhe ice of bp SST peN nT eT chee Dice oi ipie SST EEN a eee ii TT Aches Circe i Spe SPST EMT mT TEN cei Circe an ipiam SST CH Ta ed Se ced Core m ipie SYSTEM Pml EA aed Er m Speen SYSTEM TTEA TL a See ch Sa TE ee Eee m ipiam SST ER Figure 12 11 System audit trails Run End Manual Displays times for run start and completion and for manual operation Alarms Warnings Displays alarms and warnings for the system Errors Displays system errors Calibration Displays monitor calibration operations Viewing older audit trails When you choose Administration Audit trail the audit trail window displays the current audit trail To view an older audit trail click on Open in the audit trail window and choose the file to open The drive is automatically selected according to the type of audit trail file Files are named by date and serial number Choose Current log file to return to the current audit trail after viewing older files 12 16 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Administration 7 2 Open log file Drive Logdive E Log files Figure 12 12 Dialogue box for opening old audit trail files Printing audit trails Click on Print in the audit trail window menu prints the audit trail file as currently displayed in the window 12 4 2 Renewing audit trail files The audit trail file is renewed at regular intervals between 1 and 30 days To set the interval c
14. Unicorn OS 1 10 Installation Select items to install Program W System W Strategy Method Templates Cancel UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Installation 7 7 Stand alone installation eeC heck all components N etwork installation eeProgram must be checked on all stations both local and remote eeSystem must be checked on all local stations but should not be checked on remote stations eeUsers should be checked for the first installation only If Users is checked during later installation on other computers in the network any users already defined will be deleted and only the default user will be available eeStrategy and Method Templates should be checked for each new strategy to be installed usually when a new system is installed on a local computer Each strategy need only be installed once Method Templates should not be checked for BioProcess System installation 6 Enter the disk where the program is to be installed and click on OK This should be a physical disk usually C in the computer where you are Installing UNICORN OS not a network disk Unicorn OS 1 10 Installation select OK to accept the suggested target drive or enter another drive Cancel 7 Choose whether the installation is stand alone or network Fora stand alone installation the Network options settings are ignored For a network installation choose the server disk from the
15. XIV UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Index R record off procedures 8 15 record on procedures 8 15 recovering deleted blocks 5 13 deleted instructions 5 19 recovering deleted files 3 9 red instructions 5 16 reference curves displaying in system control 6 6 remote station 2 2 2 12 10 3 removing blocks 5 13 method instructions 5 19 questions 5 30 variables 5 24 rename block 5 14 renaming blocks 5 14 evaluation procedures 5 33 files 3 8 variables 5 25 replacing instructions 5 20 report 7 28 reports batch runs 8 20 printing 7 28 saving a format 7 31 reports in methods 8 20 restore mode for calls 5 10 result file contents 7 1 opening 7 1 result files 2 6 changing names at method start 5 36 directory 5 35 FAILED directory 9 2 naming 5 35 opening 3 4 serial number 5 35 result name in start protocol 5 38 result page in run setup 5 35 Run button system control 6 9 run data 6 5 Run data contents 6 5 run data layout 6 5 run hours 12 18 run method UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA XV Index XVI access 12 6 run notes 6 10 run sequences access 12 7 run setup 4 3 4 4 5 25 evaluation procedures page 5 31 info page 5 34 method notes page 5 30 result file page 5 35 result page 5 35 sequence page 4 3 4 4 5 34 start protocol page 5 36 variables page 5
16. breakpoint see Figure 5 18 for an illustration of how Change affects instructions 5 5 3 Removing a variable To convert a variable to a fixed value 1 IntheT ext instruction window select the instruction where you want to remove the variable The parameters for the instruction will be shown in the Instruction dialogue box 2 Locate the required parameter in the instruction box Press the VAR button 3 Click on Clear to delete the variable name and click on OK 5 24 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 5 6 Run setup Run setup Creating and editing methods 5 5 5 4 Renaming a variable To change the name of an existing variable 1 IntheT ext instruction window select the instruction where you want to rename the variable The parameters for the instruction will be shown in the Instruction box 2 Locate the required parameter in the instruction box Press the VAR button 3 Enter anew variable name in the dialogue box and click on OK The Run setup is a series of pages for setting the behaviour of the method when it is started To access the Run setup pages press the Run setup button at the top of the M ethod editor toolbar To access a given page in the Run setup click on the respective tab at the right of the Run setup screen Alternatively press the small arrow buttons at the bottom right of each page to scroll forwards and backwards through the pages Note Some o
17. conditions 5 44 WATCH instructions 5 10 5 43 in imported blocks 5 15 WATCH_OFF instruction 5 10 view mode connections 2 11 window 2 4 windows 3 2 in text instruction editor 5 7 volume base 2 7 L Ziegler N ichols tuning method D 2 zoom function evaluation 7 13 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA
18. log in passwords locked systems can normally only be unlocked using the locking password see Section 6 5 3 We recommend that this authorization is restricted to a few users in an installation The user who locks a system does not require this authorization to unlock the same system eeRun methods Required for starting methods Section 6 1 eeManual interaction Required for issuing manual commands in System control Section 6 3 eePause Required for pausing a running process with the PAUSE button in System control Section 6 3 1 The PAUSE instruction in methods does not require explicit authorization eeCalibrate tune Required for using the Calibrate and Tune commands in System control Section 6 6 eeSystem settings Required for changing system settings with the Settings command In System control Chapter 13 Any user may view the system settings but this authorization is required to make changes to the settings 12 6 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Administration 7 2 eeEdit sequences R equired for using the sequence editor eeRun sequences R equired for running sequences eeEdit global lists Required for saving a method as a method template an evaluation procedure or a report format as globally available Also required for deleting method templates global procedures or global report formats We recommend that this authorization is restricted to only one user in an
19. or deletes the character to the right of the cursor if no text is marked lt Backspace gt Clears the marked text without saving it in the clipboard or deletes the character to the left of the cursor if no text is marked In the default method templates supplied with UNICORN OS the method notes describe the system setup required by the method We recommend that you use method notes for this purpose in your own methods to provide documentation of the method requirements Bear in mind that method notes are saved with the method and apply to all runs made with the method Use the start or run notes for run specific information The date and time when the method was created and last edited are saved automatically in the method information and need not be entered in the method notes 5 6 4 Evaluation procedures Evaluation procedures can be called automatically at the end of a method to evaluate and or print the results User defined procedures are created in the Evaluation module and may be saved in method files see Section 7 6 Procedures saved with one method file can be imported to another Note A procedurein a method will not be updated when a procedure with the same name is changed in Evaluation The same applies to report formats saved in a procedure Note If you usean evaluation procedure to print results automatically from a run controlled from a remote station in a network installation the results will be printed on the
20. see Section 3 2 This operation does not require special authorization UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 12 13 7 2 Administration 12 3 6 Deleting home directories To delete home directories click on Delete in the Access panel of the User setup dialogue box Select a directory to delete in the Delete directory dialogue box that appears and click on OK Important It does not matter which directories are marked in the Directories list in the User setup dialogue box when you click on Delete This list shows the directories to which the currently marked user has access not the directories that will be deleted All methods result files and subdirectories in a directory will be deleted when the directory is deleted Delete directory Directories fed Delete directory C francis Figure 12 9 Dialogue box for deleting home directories Y ou cannot delete eea home directory to which a user is assigned To delete such a directory you must first either delete the user or change the home directory assignment for this user Select the user in the User setup dialogue box and assign a different home directory eea directory to which several users share access To delete such a directory remove the access rights from each user first 12 14 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Administration 1 2 12 4 Audit trails The audit trail
21. stacking and stretching 7 24 style and colour 7 9 X axis units 7 11 Y axis units 7 6 curves settings 13 5 custom strategies 4 11 cut curve 7 14 D D_Baseline setting 5 46 data sampling A 2 data security 2 14 9 2 data storage A 2 date and time incorrect F 5 decompressing files 11 3 default password 12 12 settings 13 1 user 9 1 12 10 default user installation 11 9 default variable values 5 22 defining access levels 12 5 evaluation procedures 5 32 home directories 12 11 password 12 11 run data layout 6 5 systems 12 2 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA V Index vi users 12 11 variables 5 23 delete blocks 5 13 delete template 5 39 deleting blocks 5 13 evaluation procedures 5 33 files 3 9 home directories 12 14 method instructions 5 19 questions 5 30 run data layout 6 6 system defintions 12 4 templates 5 39 users 12 13 variables 5 24 deleting files 3 9 access 12 6 desktop 2 3 details view 2 6 3 4 detecting air in eluent line 5 45 differentiate 8 12 directories for result files 5 35 directories for methods 4 10 disconnect system 6 15 6 16 disconnecting a system 6 15 6 16 displaying block instructions 5 17 curves 6 6 reference curves in system control 6 6 run data 6 5 documentation 7 2 printing 7 28 E edit baseline data point deletion 8 7 data point insertion 8 6 drawing the baseline 8 7 Inte
22. 1 Showing part of a curve This section deals with the selection of just part of a curve for purposes of closer examination of details and for presentation This can be done in three different ways eemagnification using the zoom function eefixing the axes eecutting the curves The zoom function In the active chromatogram window it is possible to zoom in on a designated area of the chromatogram This is the easiest and quickest way to magnify different parts of a curve 1 Place the mouse pointer in any corner of the area to be magnified 2 Press and hold the left mouse button A magnifying glass icon will replace the mouse pointer arrow on the screen 3 Drag a box from the point of origin to cover the area to be magnified R elease the mouse button The selected region will now be displayed in the entire chromatogram window together with appropriate scales for the y and X axes GRA Ped SS04 L431 Cond Figure 7 13 Illustration of the chromatogram zoom function The zoom process can be repeated many times within an already magnified region Y ou can also use the lt PAGE UP gt and lt PAGE DOWN gt kKeys to zoom out and zoom in respectively on the whole chromatogram Y ou can also use the cursor arrow keys on the keyboard to move around in the chromatogram at the current magnification scale UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 7 13 Presenting results 4 Displa
23. 18 1118 52 Edition AA Evaluating results 8 Export Documentation Export Documentation Parts W Method notes i Start protocol W Start notes Into H Run notes i System settings W Evaluation notes i Scouting WI Questions i Variables W Evaluation procedures H Calibration i Result name W Reference curves i Column parameters Export format Export to file ASCII EXPDOC Directory Figure 8 22 Export Documentation dialogue box Curves are exported as two series of numerical co ordinates referring to time volume and signal respectively Only one curve can be exported in each file When exporting a curve you may choose to export only a portion of the curve by inserting the limiting retention values directly into the boxes in the Cut curve field or by visually selecting the part of the curve using the CUT GRAPHICALLY command button option Y ou may also reduce the number of data points that are exported For example reducing the number of data points by a factor of five will export every fifth point to the file Cf Se Pe TEHN F Peat i EHH C Wi EHIH IF eil Figure 8 23 Export Curve dialogue box The file name for the exported data is suggested according to the item being exported eg EX PDOC for Documentation or EX PCRV for Curve You may change the file name if desired The extension of the exported file will be asc or wks depending on the file format By default files are stored on the
24. 3 8 sorting files 3 5 view mode 3 4 parameters for method instructions 5 18 password 3 1 changing 12 12 defining 12 11 minimum length 11 6 path for result files 5 35 pause access 12 6 Pause button system control 6 9 pausing a method 5 43 6 9 peak asymmetry 8 11 Peak asymmetry Chromatogram Layout box 8 11 peak integration 8 1 accept negative peaks 8 5 baseline calculation 8 1 HETP 8 11 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA xiii Index peak window 8 4 reject peaks 8 4 peak integration 8 2 peak table adjusting peak boundaries 8 7 filtering peaks 8 3 peak labels 8 3 peak table 8 2 peak tables Chromatogram Layout box 8 3 Peak max condition 5 44 PeakH eight setting 5 45 PID tuning D 1 pipe server name 10 5 in network installations 12 4 in stand alone installations 12 3 Pl regulator D 1 P regulator D 1 previewing questions 5 29 print 7 32 printer setup 7 32 printing audit trails 12 17 chromatograms 7 32 reports 7 28 process monitoring 6 4 process operators 12 9 process supervisors 12 8 program settings O S 2 F 1 pull down menus 3 3 y questions 5 27 authorized 5 27 6 3 deleting 5 30 editing 5 30 in start protocol 5 37 input field 5 28 inserting 5 29 mandatory 5 27 multiple choice 5 28 no answer 5 28 preview 5 29 value 5 29 quit program 3 10 quitting UNICORN access 12 7 quitting UNICORN OS 3 10R
25. 5 groups according to function the list shows available instructions In the currently selected group For short help on the purpose of each instruction click on the instruction and press F1 c Enter values for instruction parameters in the Parameters panel If a scroll bar appears on the right hand side of the Parameters panel additional parameters are required The scroll bar blinks to alert you until you have accessed the additional parameters Hrany iuie ai aires Pic ara igre pil Dele ma _ a Frem ee oe mA R Pog int E Figure 5 17 The Method editor instruction box 4 Click on Insert The new instruction will be inserted in the block either a atthe position of the breakpoint of the new instruction if there are no other instructions at that breakpoint b immediately after the currently highlighted instruction if the highlight is at the same breakpoint as the new instruction c asthe last instruction at the breakpoint if there are several instructions at the same breakpoint as the new instruction and none of these is highlighted UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Creating and editing methods 5 Note Instructions which are placed at the same breakpoint are executed simultaneously with the exception of i CALL instructions which are executed in the sequence in which they are written and ii pump instructions whereby the first pump instruction is
26. 7 templates 11 8 instruction box displaying 5 17 variable identification 5 22 instructions 2 8 see method instructions or evaluation procedure instructions editing 4 12 in evaluation procedures C 10 in methods B 1 instructions at the same breakpoint 5 40 Int_Status condition 5 44 invalid method instructions 5 16 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA IX Index L language for OS 2 displays F 5 layout Chromatogram Layout box 7 12 layout Chromatogram Layout box 7 7 leaving systems 6 16 9 2 Less than condition 5 44 Less than_or_ valley condition 5 44 limits for alarms and warnings 6 12 linear flow rate 5 43 local station 2 2 2 12 10 2 local station failure 9 3 locked processes 12 6 locked system 6 10 9 1 locking systems 6 16 logbook 6 8 logging in 3 1 logging out 3 10 M macros evaluation See evaluation procedures main menu panels 2 6 3 3 mandatory questions 5 27 manual control 6 9 manual instruction box 6 11 manual instructions 6 11 during a method run 6 11 manual interaction access 12 6 MCU 11 2 installation 11 2 messages 5 42 method creating 4 6 new 4 2 5 3 5 48 method blocks see blocks method developers 12 8 method editor access 12 5 block window 5 8 graph window 4 9 run setup 5 25 text instruction window 5 16 method files 2 6 naming 5 38 Opening 3 4 UNICORN for Oligo Synthesis version 1 10 User M
27. AT a As aT TT I I I I I I I I I I I I OI UI U UIBR BRB PW WWNONYONNFP eS FE vii Contents viii UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Introductory material Methods and Runs Evaluation System management Appendices This page is deliberately left blank Introduction 7 1 Introduction UNICORN OSis an adapted version of the UNICORN control system which allows real time control of oligonucleotide synthesis systems from a personal computer The package operates together with OligoPilot II and OligoProcess from Amersham Biosciences UNICORN OS runs under the operating system O S 2 Warp version 3 0 or higher from IBM Functional features of UNICORN OS include e OnePC may control up to 4 synthesis and or chromatographic separation systems directly e Network support allows any number of systems to be run from one PC e Method templates providing method frameworks for most common applications eliminate the need to program methods from scratch e Modular methods are defined in the method templates reflecting the separate steps in a synthesis process e g detritylation coupling oxidation thiolation capping and wash purge steps e Dynamic graphical overview of active runs e User definable alarm and warning limits for monitor signals e Batch operation and process documentation in accordance with the requirements of Good M anufacturing Practice GM P and Good Labora
28. For example CALCULATE BASELINE 01 06 XXX XXX XXX XXX can be changed to CALCULATE BASELINE 01 06 DEFAULT DEFAULT DEFAULT DEFAULT 8 3 4 Running evaluation procedures To run a procedure for a specific chromatogram first make sure that the desired chromatogram is active Click on Procedures Run and choose the desired evaluation procedure Click on OK and the procedure runs at once Y ou can also open the Procedure editor dialogue box and choose Control Run or click on the Play button 8 3 5 Batch runs It is possible to apply an evaluation procedure to a designated batch of result files even if they are not open on the Evaluation workspace It Is especially useful for example to perform integration with the same parameter settings on many results or to print a number of results with the same settings The batch run is done in the background of the Evaluation module and thus the results of the run are not seen Y ou will of course receive any print outs or report documentation if this was one of the steps in the run procedure see Section 7 6 1 Select Procedures Batch run The Batch run dialogue box is displayed UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 8 19 8 Evaluating results 96047707 gaid 1717 9604170 goid i707 MTE FEN Figure 8 20 Batch run dialogue box 2 Select File Open related Curves Select the search criteria for the directory result chromatogram and
29. I wo AH Lal ae ne al HH i me HLH B E PEE LEL on Ae MHI Fem Ti AA Figure 5 21 The Method editor flow scheme window UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 5 21 5 Creating and editing methods 5 5 Method variables Variables can be assigned to any instruction parameter including breakpoints V ariable values can be changed immediately before the start of a run without using the M ethod editor allowing one method to be used for runs under a variety of conditions see Chapter 4 Variables are defined with names which can be explicit descriptions of the variable function e g Column_volume Load_of support Suitable choice of variable names can make the method easier to read and understand and also help the operator in setting variable values at the start of a run Each parameter defined as a variable is also assigned a default value which is used if no changes are made to variable values at the start of a run Up to 64 variables can be included in any one block although only a total of 64 variables can be used in the whole method 5 5 1 Identifying variables Parameters defined as variables are identified in two ways eein the Text instruction window the default value for the parameter is expressed by variable value V ariable type variable units Detrit_wash 0 00 Base SameAsMain 0 00 FFlow_ACN 2 50 bart 0 00 FFLow_ACN 2 50 0etritwash_Pressure bar O0
30. M anual 18 1118 52 Edition AA Contents 4 5 M ethod notes 4 6 Saving the method 4 7 Starting a run 4 8 Editing text instructions 5 Creating and editing methods 5 1 T he sequence editor 5 1 1 M odifying cross references in the sequence editor 5 2 T ext instructions editor 5 3 M ethod blocks 5 3 1 Viewing blocks 5 3 2 Calling blocks 5 3 3 Adding blocks 5 3 4 Deleting blocks 5 3 5 Renaming blocks 5 3 6 Importing and copying blocks 5 4 M ethod instructions 5 4 1 Viewing instructions 5 4 2 Adding instructions 5 4 3 Deleting instructions 5 4 4 Changing instructions 5 4 5 The flow scheme window 5 5 M ethod variables 5 5 1 Identifying variables 5 5 2 Defining variables 5 5 3 Removing a variable 5 5 4 Renaming a variable 5 6 Run setup 5 6 1 Variables 5 6 2 Questions 5 6 3 M ethod notes 5 6 4 Evaluation procedures 5 6 5 Info 5 6 6 Sequence 5 6 7 Result 5 6 8 Start protocol 5 7 Saving the method 5 7 1 Saving as a method 5 7 2 Saving as a template 5 7 3 Deleting a template 5 8 H ow to use selected method instructions 5 8 1 BASE and METHODBASE instructions 5 8 2 Instructions at the same breakpoint 5 8 3 Controlling block and method length 5 8 4 M essages 5 8 5 Pausing a method 5 8 6 Linear flow rates 5 8 7 Conditional instructions 5 9 Creating methods from scratch 5 25 5 34 5 35 5 36 5 38 5 38 5 39 5 39 5 40 5 40 5 40 5 41 5 42 5 43 5 43 5 43 5 47 UNICORN for Oligo Synthesis version 1 10 User M anua
31. Run or Edit to run or edit the method respectively To change the size of a panel drag the panel border Click on the Maximize button at the right hand end of the panel title bar to toggle panel size between normal and maximized M aximizing a panel will hide but not close the other panel 3 2 3 Presenting files The way files are presented in the panel can be set from the File menu or from the pull down menu that appears when you click with the right mouse button in the panel Presentation options are eeview mode icons or detail display eesorting order eefilter for displaying only a chosen set of objects e g methods for one system View Y ou can display the contents of the panels either as graphical icons View Icons or as a details list View Details The details list includes a small icon identifying the type of object file size last written date and time and creation date and time To change the view mode choose View from the pull down panel menu or from the File menu and choose the appropriate mode from the sub menu UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Login and file handling 3 Srl i Lasi armad CS a Nae Oe dana Creaa eres ody coal IEE Ou oe Drieie MI E bode ca ig Cece crt i 04 21 86 ord at eth BG Poe aly igi 1926 ia 3 20 56 bone Figure 3 4 Icon and detail display modes illustrated for the methods panel Sort Files can be sorted in the panel accord
32. See ae r i mf a E 0 nil l Mi Nu a I LG in io E Pesk name Pet Peak start Faak end ml ml m j 3 4 41 85 2 L3 Les 3 01 57 lz J F l 24 ag a m r b Feskrame Deiis pask Spitpent tombe _ Jierigt Ok Gaon Hep Figure 8 8 Edit Peak Table chromatogram window Deleting a peak in the peak table To delete a peak from the table click on the peak in the chromatogram or in the peak table and click on the Delete Peak command button N ote that the remaining peaks will be renumbered after the deletion Splitting a peak A peak is defined within two limiting drop lines one to the left and right of the peak It is possible to split the peak to create two new peaks by inserting a new drop line A new drop line is always positioned at the middle point between the two existing drop lines Thus the area of each new peak is dependent on the symmetry of the original peak To make a split select the desired peak in the list or mark it in the curve Press the Split peak command button Note The peaks will be renumbered according to the split Refer to the description below about adjustment of the drop lines UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Evaluating results 8 Joining a peak It is possible to join the areas of adjacent peaks if separated by a drop line 1 Select a peak either on the chromatogra
33. UNICORN OS installed automatically Y ou must always log in to the network before installing or setting up UNICORN OS software See Chapter 10 for full details of network setup and administration 11 3 Hardware installation In most cases an installation of your system will be performed by Amersham Biosciences authorized personnel If your system is not pre installed then follow the steps below to install the main control unit M CU expansion card in your PC This procedure Is only required in stand alone installations i e systems not connected to a network and on local computers in network installations 1 Turn off the power to your PC and unplug the mains cable 2 Openthe PC cover Refer to your PC documentation if you are not sure how to do this 11 2 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Installation 7 7 3 Locate an empty full length expansion slot 4 TaketheMCU card out of the anti static bag H andle the card by its edges and avoid touching the electronic components as far as possible Discharges of static electricity can permanently damage electronic components on the card If you are working in a room where static electricity tends to build up discharge any electricity from your body by touching an earthed metal surface e g a water tap or radiator before handling the card Install the M CU card into the expansion slot Y our PC documentation should describe how to
34. UNICORN OS include eeA ccess security Useof UNICORN OS is restricted to authorized users Each user is assigned an access level which defines the functions that the user is permitted to use eeC onnection security Running systems may only be controlled from one connection Systems may be locked with a password to prevent other users from changing run parameters eeD ata security Result files can be saved automatically at pre set intervals during a run to minimize data loss in the event of system failure In a network installation results are saved on the local station if network communication fails Security features are discussed in more detail in Chapter 9 N etwork and administrative aspects are discussed in Chapters 10 and 11 respectively 2 14 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Login and file handling 3 3 Login and file handling 3 1 Logging in To start UNICORN OS double click on the UNICORN OS icon on dl bs the O S 2 desktop The program takes a little while to start aP UNICORN OS If UNICORN OS is already started and the previous user has logged Desktop icon out click on the Login button at the bottom of the UNICORN OS desktop LOGIN MENU OF Cancel Help Figure 3 1 The Login Menu dialogue box Click on your username in the list or type the name directly in User field Type your password in the Password field and then click on the OK button to log in
35. a colour and line style dependent on their position in the curve list To reassign the colour and or line style of a specific curve do the following 1 In the Chromatogram Layout box click on the Style Colour button A panel will appear which shows the colour and line style options 2 Tochangethe colour and or line style of a curve select the curve of interest from the list 3 Select the desired colour and or style UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 7 9 Presenting results Line Style And Colour 02 96041 704 1_PressDet 03 96041 704 1_PressAChH 04 96041 704 1_CondP Figure 7 10 Line Style and Colour dialogue box 4 Click on OK to return to the Chromatogram Layout box 7 2 7 Changing and fixing the axes By default the y axes are automatically scaled for each curve to show the whole curve The x axis is automatically scaled to show the whole run ml or minutes for OligoPilot and litres or minutes for O ligoProcess It is possible to fix the minimum and maximum values for the axes of any curve and thereby select a specific part of the curve to be displayed Figure 7 11 Axis dialogue box 7 10 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Presenting results y axis 1 Click on the Axis command button in the Chromatogram Layout box 2 Select the appropriate curve from the list for which you want to fix the sca
36. ain the block will inherit the main base in the new method regardless of the base in the source method UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 5 15 5 Creating and editing methods 5 4 Method instructions Use the instruction dialogue box in Text instruction mode to enter edit and delete instructions 5 4 1 Viewing instructions Instructions are displayed in the T ext instruction window J piii Pria a om Mee Fi pe oe aP b TE ber hbr a ry men Te aT bo Part E E g OE Heas Fren CORT INE a ee ee m0 Panga iE oy Bea cl _ il im Call Foaral STAAT parmin piahi pesse Cis Cy E se hire litle FE BTS ote Call aru pT Coe Cake od 3 Oi ll Erd iki oo Hi Call Pera Pem E si Perra Pep C si Piega TT Ti kiij Eii Wrk p im Purp ry oy is bred aed wv Veh oll ert dri Erd rahi 1 Eki wil ee ee baat by Boo fia aml TE ee ey Me ee 3 pr aiaa We 51AT smki Hd wE TS LEF J I Figure 5 14 The Text instruction window top with the instruction dialogue box bottom Instructions are colour coded in the Text instruction window as follows Blue Black Red 5 16 Valid call instructions i e CALL and WATCH instructions to other blocks in the method Valid instructions other than CALL and WATCH Instructions with invalid syntax These may be a calls to blocks which are not defined in the method or b in
37. and Replace have different functions if the breakpoint is changed eeChange shifts all subsequent instructions in the block according to the change in the breakpoint Change does not affect the relative order of instructions in the method Y ou cannot change the breakpoint of an instruction to earlier than the nearest previous breakpoint in block Furge_cappingAB 0 00 0 00 0 00 3 00 6 00 4 00 4 00 Change Base Volume 0 00 Flow _Reag 10 00 4ml min 5 00 solvent Cap_aA vaste gt 500 solvent Cap_B vaste gt 6 00 Solvent ACM 3 1 Waste 17 00 Flow_Reag O 00 ml min m 14 00 End_block gt 14 00 Furge_cappingAB Base Volume Flow_Reag 10 00 ml min solvent Cap_A vaste solvent Cap_B vaste Solvent ACM 3 1 Waste Flow_Reag 0 00 ml min End block Figure 5 18 Change moves the selected instruction and all subsequent instructions eeReplace moves the selected instruction but does not change the breakpoint of any other instruction Replace can change the relative order of instructions in the method Furge_c 0 00 0 00 0 00 3 00 6 00 4 00 4 00 Replace appingAB Base Volume Flow_Reag 10 00 ml min 0 00 Solvent Cap_A Waste ee Fai solvent Cap_bB vaste 3 00 Solvent ACM 3 1 vaste 6 00 Flow _Reag 0 00 ml min 4 00 End block 9 00 Furge_cappingAB 0 00 Base Volume oolvent Cap A vaste Flow _Reag 10 00 ml min solvent Cap_B vaste Solvent ACM 3 1 W
38. anual 18 1118 52 Edition AA Index method information in start protocol 5 36 method instructions 5 16 adding 5 17 at the same breakpoint 5 19 colour codes 5 16 current instruction 5 17 deleting 5 19 groups 5 18 in start protocol 5 37 insertion point for new instructions 5 17 invalid syntax 5 16 moving within a breakpoint 5 21 parameters 5 18 recovering after deletion 5 19 method text instructions 5 6 method notes 4 9 5 30 editing functions 5 31 in templates 5 31 method templates 4 2 test sequence 4 3 methods 2 6 adding to the system control file menu 6 3 advanced editing 5 1 blocks see blocks creating 4 2 5 3 creating new 4 4 directory 4 10 file names 4 10 for system 4 2 5 3 5 48 for technique 5 3 for template 5 3 graphical overview 4 9 hold 5 43 overall length 5 42 pause 5 43 saving as templates 5 39 saving for different systems 4 10 5 39 saving from result files 7 2 starting 6 1 structure 2 7 submethod calls 5 9 techniques 5 40 variables see variables minimizing UNICORN desktop 3 3 11 6 windows 3 3 minimum password length 11 6 M inPeak 5 45 M inPeak setting 5 45 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA xi Index XII mirror images of curves 7 27 modules 2 2 monitoring a run 6 4 monitors calibration 6 18 settings 13 4 moving files 3 8 access 12 6 moving instructions within a breakpoint 5 21 moving method
39. be used The File name parameter may include a path from the current users root directory C 4 4 Export EXPORT _ Exports the curve in Curve source to the file defined in Curve source CURVE_ ASCII Export to File in ASCII format In the part of Curve Left limit source limited by Left limit and Right limit Every lt n gt Right limit samples are exported Every lt n gt sample Export to file EXPORT _ Exports the curve in Curve source to the file defined in Curve source CURVE_WKS Export to File in WKS format In the part of Curve Left limit source limited by Left limit and Right limit Every lt n gt Right limit samples are exported Every lt n gt sample Export to file UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA C 13 C Evaluation functions and instructions EXPORT _ Exports the documentation in the current result file in ONOFF Variables DOC_ ASCII ASCII format to the file defined in Export to file If all ONOFF Scouting parameters to this function is OFF then no ONOFF Start Protocol documentation is exported If at least one of them is ONOFF Questions ON then the documentation will be exported and the ONOFF RefCurves corresponding parts will be included in the exported ONOFF EvalProc file ONOFF Method Info ONOFF Method Notes ONOFF StartNotes ONOFF RunNotes ONOFF EvalNotes ONOFF Sys Settings ONOFF Calibration ONOFF LogBook ONOFF Result Name ONOFF Column Parameters NAME Export to file EXP
40. choosing panels to display in the System control window 6 2 1 General panel techniques Panels in the System control window are always displayed at the full width of the System control window H orizontal yellow lines separate multiple panels in the same window Y ou can drag these lines with the mouse to change the vertical size of the panels The Hide panel button in the top left hand corner of each panel closes the panel The Maximize Restore button toggles the panel size between adjustable and full window A panel which is maximized will hide any other panels The Contents buttons in the run data and curves panels open the Run data contents and Curve contents dialogue boxes respectively see below UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Performing a run 6 6 2 2 Run data The run data panel displays the current values for selected run parameters V alues are updated at least every 5 seconds the actual interval is defined in the system strategy SN DATA Ey Figure 6 4 The run data panel Click on the button or choose View Run data contents to open a dialogue box for selecting which parameters to display The list of available parameters depends on the system strategy 31A Tun one Saaleched ims a depkyad n Be run dete wane Aun date bn sguts ALP OAT A Figure 6 5 The View run data contents dialogue box Different run data layouts each consisting of a subset of th
41. curve name using the respective Browse command buttons Alternatively click on the respective All button to include all result files chromatograms and or curves Wildcard characters and can also be used to specify the search parameters see Section 7 4 1 under Alternative A 3 Deselect those chromatograms that you do not want to be included in the batch run Click on OK to perform the batch run Any created curves and peak tables will be saved in each result file automatically 4 To view the results of the batch run on a given result chromatogram open this on the Evaluation workspace Note If oneof the result files on which you perform a batch run is the currently opened result file the created curves will not be shown in the open result file H owever the curves are stored in the original result file with the same name Therefore do not select Save on the open result file if you do not want to overwrite the curves created during the batch run It Is recommended that you do not perform a batch run on the open result file 8 3 6 Evaluation procedures and reports The creation of evaluation procedures combined with batch runs is a very useful tool to produce printed documentation simultaneously for 8 20 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Evaluating results 8 many result files e g for a number of scouting runs This removes the necessity to open import result files onto the Ev
42. different system Warnings and alarms are also recorded in the logbook for the run AlarmAvarming 1 Downtime 0 00 30 Figure 6 13 Alarm warning display box In a network installation warnings and alarms are only displayed on computers that are connected to the system An alarm can be acknowledged only from the computer connected in control mode alarms are displayed but cannot be acknowledged on computers connected in view mode Note For this reason we discourage passive operation of a system i e with no controlling connection 6 12 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Performing a run 6 6 4 If communication fails This section summarizes the consequences of system failure during a run See Section 11 3 in UNICORN 2 10 User M anual for more details 6 4 1 Network failure If network communication fails during a run that has been started from a remote station the run will continue and the results will be saved in the FAILED directory on the local station A control mode connection can be established on the local station to control the running system 6 4 2 Local station failure Intermediate results up to the time of the failure are saved if the Autosave interval is set to a suitable value in the system definition see Section 12 1 6 5 Managing system connections UNICORN OS installed on a given computer may have up to four System control windows the actual number i
43. epic Figure 4 2 Sequence page in Run setup containing the 20 base sequence pre defined in the method template UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 4 3 4 Creating methods from method templates Click on the Create button see Section 4 3 which fully generates the method and inserts default variable values The Save As dialogue is then displayed Save the method with the name test20 The method must be saved before you can make a run The test20 method will be displayed in the Methods box of the M ain menu To run the test20 method follow the instructions detailed in Section 4 7 and Chapter 6 You can change the method variables prior to the commencement of the run 4 3 Creating a sequence and method As described in Section 4 2 all of the method templates contain a partially built method for a pre defined 20 base sequence By replacing the pre defined sequence with a sequence of your choice you can quickly and easily create a ready to run method L Select a method template in the New Method dialogue box as described in Section 4 1 Click on the Run set up button on the toolbar or select View Run setup Select the Sequence page to display the pre defined sequence Select the pre defined sequence in the sequence editor and delete it using the lt Delete gt key on the keyboard Enter a new sequence up to a maximum of 200 bases in the sequence editor in the 5 3 direction Re
44. fit the card if you are uncertain contact your computer supplier 4 Close the cover on your PC 5 Connect the M CU card to the liquid handling module CU connector using the communication cable provided Additional liquid handling modules up to a total of four may be connected in series Note The connection to the M CU card is made with a 15 pin connector for ISA bus computers Compaq and a 9 pin connector for MCA bus computers IBM PS2 11 4 Software installation UNICORN OSwill normally be installed by your Amersham Biosciences representative Follow the instructions below if you need to install the program yourself The software must be installed on all stations in a network 11 4 1 Installing UNICORN OS for the first time The installation procedure described below assumes that the operating system O S 2 is correctly installed on your computer Refer to the Operating system documentation for details For network installations the network must be correctly set up as described in Chapter 10 Note System fonts must be installed in O S 2 for correct functioning of UNICORN OS UNICORN OSis supplied on 3 5 high density diskettes Files on the distribution diskettes are compressed and cannot simply be copied to the hard disk The installation procedure described below creates the required directory structure on the hard disk and decompresses the distribution files Do not attempt to decompress the distribution files using any ot
45. for maximum security If you forget the password for the only user with User setup Levels access you must re install the default user as described in Section 11 4 2 12 3 3 Viewing and changing user definitions To view the setup for any user click on the user name in the User setup dialogue box To change the user definitions make changes in the dialogue box fields as appropriate and click on OK 12 3 4 Deleting users To delete a user select the user from the username list and click on Delete User Y ou may delete all users except the last user with User setup Levels access This ensures that at least one user has the right to perform administration functions Deleting a user does not affect the user s home directory or method and result files 12 3 5 Defining new home directories To define an existing directory as a home directory for a user select the directory in the Home directory field in the User setup dialogue box Any directory may be used as a home directory As a recommendation in network installations place the home directory on a drive which is addressed by the same drive letter from all computers in the network To create a new home directory at the same time as you create a user Check the Create home directory box in the New user dialogue box and enter the directory name and drive in the appropriate fields To create new directories under a home directory use the New directory command in the M ain menu
46. if the corresponding report contents option is not selected Information will only be included in the report if the appropriate option under Report Contents is selected 4 When you have decided on the report contents you can preview the report by clicking on Preview and or print the report by clicking on Print Two of the report options warrant more detailed description namely Synthesis data and Chromatogram Synthesis data This option can be used to print out all of relevant information concerning the actual synthesis including the sequence detritylation table and cross reference list The detritylation table contains information about the efficiency of the coupling reaction for the UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 7 29 Presenting results addition of each base to the oligonucleotide Efficiency is automatically calculated by measuring the conductivity in the cell during the cleavage of DM Tr at the detritylation step The level of conductivity is determined by the amount of cleavage and is directly correlated to the area under the relevant peak in a coupling cycle The area is determined by peak Integration which is automatically performed if one of the supplied method templates has been used to create a method Click on the appropriate radio button and select the options to be included C hromatogram Selection of the radio button associated with the report content Chromatogram al
47. installed from other stations in a network installation will be deleted but method and result files will not be erased from the disk Y ou can regain access to these files by redefining users with appropriate directory access see Section 12 3 for further details of defining users For example assume that the system has a user called ARN OLD with access to the home directory ARN OLD To regain access to Arnold s files after installing the Users option simply define a new user with ARNOLD asthe home directory Users are installed on the network server in a network installation UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 11 9 7 7 Installation Program Before reinstalling the program you must delete STARTUP CM D from the root directory on drive C and reboot the computer Check the Program button to reinstall UNICORN OS This will not affect any existing method or result files in the system The program is always installed locally even in network installations In network installations remember to make the required changes to STARTUP CM D after reinstalling the program see Section 10 7 System Check the System button if you are installing a new or additional system on a stand alone computer or a local station in a network Systems are only installed on local stations in a network installation Strategy Check the Strategy button to re install system strategies or to install additional stra
48. instructions 5 20 multiple choice questions 5 28 multiple connections 6 14 multiply 7 26 N named pipes 10 5 names Chromatogram Layout box 7 8 naming blocks 5 11 result files 5 35 variables 5 23 network administration 10 2 failure 6 13 9 2 overview 2 12 network administration 12 1 network installation 2 2 alarms and warnings display 6 12 communication failure 9 2 connections 6 17 logging into UNICORN OS 3 1 network environment 10 1 organization 10 2 requirements 10 4 security 10 2 system definition 10 8 user profile 10 8 network installations control A 2 network login 3 2 network requirements A 1 new method 5 3 4 2 5 48 new block 5 10 new chromatogram 7 21 new method 4 2 new procedure 8 16 no answer questions 5 28 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Index no template creating anew method 5 48 normal mode for calls 5 10 normalize 7 24 N oscreen parameter for messages 5 42 notes in start protocol 5 37 N ovell N etware 10 3 number of system control windows 11 7 YO on line help 2 5 open related curves 7 21 open chromatogram 7 19 open curves 7 23 Open result 7 1 Opening files 3 4 O S 2 language version F 5 OS 2 requirement 11 2 overall method length 5 42 P pages in run setup 5 25 panel presentation 3 3 panels copying files 3 8 file filter 3 5 finding files 3 7 moving files 3 8 renaming files
49. left unlocked with no control mode connection has no identified responsible user Systems may be locked even when they are idle to allow users to reserve a system for later use 9 3 Data security 9 3 1 Network communication failure If the network communication fails while a synthesis is running any control mode connection on a remote station will lose control of the system and results destined for network drives cannot be saved in their correct directories In this event the run continues under control from the local station Results are saved on the local station with the original result file name in the directory UNICORN OS LOCAL FIL FAILED from which they can be retrieved after the run is complete To retrieve results from the FAILED directory 1 Start UNICORN OS if it is not already started on the local station where the synthesis process was run 2 Login asa user with access to the FAILED directory 3 You can now open the result file in the Evaluation module and move it to a suitable location on the network server when network communication is reestablished so that it is accessible from remote stations Note Result files are saved directly under the FAILED directory and are identified by result file name Files with the same result file name base are distinguished by an incremental serial number in the same way as result files in any other directory Access to the FAILED directory The system administrator may choos
50. name field Available strategies are determined when UNICORN OS is installed see Section 11 4 If you have several strategies installed make sure that the selected strategy is appropriate for the chromatographic system being defined Click on Information in the Strategy dialogue box to display information about the selected strategy 3 Set the system title bar colour if desired by clicking on Colour Y ou can choose any combination of bar colour and text colour from the palettes in the dialogue box that appears T he selected colours will be used in the title bar for System control windows for the particular system and can provide a quick identification of which systems are connected to the different System control windows 4a Stand alone installations For stand alone installations the Pipe server name field is a dummy field with default value LOCAL Leave this value unchanged UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 12 3 7 2 Administration 4b Network installations In the Pipe server name field enter the pipe server name of the computer to which the system being defined is physically connected See Section 10 6 for information concerning pipe server names The pipe server name can be found if necessary in the CONFIG SYS file on the line RUN C NETWARE NPDAEMON EXE lt pipe server name gt 5 Select the control unit number 1 4 in Control unit number This is the physical connection nu
51. not available on a remote station in a network installation the user list in the login dialogue box is empty make sure that the computer is logged in to the network before starting UNICORN OS A remote station accesses the user list directly from the network server If the user list on a local station in a network installation is not up to date make sure that the computer is logged in to the network before starting UNICORN OS The user list is stored locally on a local station and is updated automatically from the network server if the computer iS logged in to the network F 2 2 Error message Strategy file error If you receive the error message Strategy file error Can t load strategy in a stand alone installation the strategy file is probably corrupt Reinstall the strategy as described in Section 11 4 2 In a network installation the error may appear if you try to create a method for a system not physically connected to the computer M ake sure that the computer is logged in to the network before UNICORN OS is started so that the strategy file on the server disk is accessible F 2 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Troubleshooting F 3 UNICORN OS access problems F 3 1 Unable to access certain UNICORN OS functions UNICORN OS functions to which you do not have access appear grey in the menu and cannot be used Y our user profile is determined by the system administrator from Ad
52. numbers close to zero At those points where Curve source 1 has amplitude less than Threshold1 or Curve source 2 has amplitude less than Threshold2 the result of the division is defined to be 1 0 The y axis of the curve will be the same as that of the first Curve source Curve source Resulting curve Curve source Resulting curve First fraction to pool Last fraction to pool Curve source Resulting curve Multiplication factor Curve source Resulting curve Shift factor Curve source Resulting curve Filter Curve source Resulting curve Filter width Curve source Resulting curve Filter width First curve source Second curve source Resulting curve First curve source Second curve source Resulting curve Threshold1 Threshold2 C Evaluation functions and instructions C 4 2 Integration CALCULATE _ Calculates a baseline from the curve in Curve source Curve source BASELINE The baseline is stored in the Resulting curve Resulting curve DEFAULT can be selected in the Baseline parameters Noise Window which will than calculate default baseline parameters Shortest baseline for each new curve segment Slope limit Max baseline level PEAKTABLE working memory of the computer TABLE Resulting peak table Resulting peak table NEGATIVE _ Controls the baseline behaviour in subsequent PEAKS baseline calculations If OnOff is ON then the baseline may be drawn above the curve and negative peaks may be detected by PEAK_INTEG
53. on the screen V arious report layout changes can be made in the Chromatogram Layout dialogue box For example ee CUFVES e axes ee header information to be included on the same page as the chromatogram Select Header in the Chromatogram Layout box Select Variables and or Questions H eader information for imported chromatograms can not be printed 4 Click on OK when all changes in the Chromatogram Layout box have been performed 7 Click on Print or Preview 7 6 1 Saving a report format you can save a customized report format for later use A saved report format also includes any selections you made in the Chromatogram Layout box with respect to the report Note Theformat cannot be saved with Current layout The layout saved is always the one shown in Define All saved formats can be globally saved i e made available for any user if you have Edit global lists authorization or can be specific for the current user only 1 Select all the relevant information for your report format 2 Click on the Save as command button and type in a name for your format in the box that appears Click on OK to save the format Note A format saved with thename DEFAULT in upper case letters will be used as the default format for all result files when they are opened for the first time UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 7 31 Presenting results 7 6 2 Selecting a format The newly sav
54. open File Print allows you to print any components in the documentation 7 7 Exiting Evaluation If you want to quit from the Evaluation module of UNICORN OS select File Exit You will then be asked if you want to save the results of the evaluation session that you have performed If you answer Yes the previous version of that result file will be unconditionally overwritten This may be undesirable if you have included the current result file within an evaluation procedure batch run see Section 8 3 5 7 32 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Evaluating results 8 8 Evaluating results This chapter will mainly describe how to eeintegrate peaks eeautomate evaluation operations eeex port data and curves 8 1 Integrating peaks UNICORN OS allows you to identify and measure on a curve such values as peak areas retention times and peak widths This uses peak integration and should be used if you are not satisfied with the automatic integration performed during a run or if the automatic integration function was not used 8 1 1 Baseline calculation for integration Integrating peaks is divided into two steps calculating the baseline and calculating peak areas As a correct baseline is crucial for accurate calculation of peak areas several ways of calculating the baseline are availablein UNICORN OS eoU sing the Calculate baseline instruction for automatic calculation of the baseline whic
55. pull down list in the Server disk field and enter the pipe server name of the station see Section 10 6 in the Pipe server name field The pipe server name can be found if necessary in the CON FIG SYS file on the line RUN C NETWARE NPDAEMON EXE lt pipe server name gt For installation on a local station make sure that Remote only is not checked For installation on a remote station i e a computer to which no synthesis systems are physically connected check Remote only UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 11 5 7 7 Installation ul iiralealion eT ipt ol ination J Stand alone Hetero Server disk G T Pips serem meme fim 412 i Bemata ondy W hen you perform a network installation the necessary parts of UNICORN OS software will be copied automatically to the network server disk Note Ifyou perform a stand alone installation and later want to connect the system to a network you must re install UNICORN OS software with the appropriate settings 8 Choose the minimum number of characters required for passwords this number applies to both login and locking passwords Alternatively if you do not require password protection check No password required W ith this setting users can be defined with or without passwords N ote In a network installation make sure that you enter the same password settings on each station in the network Check Allow minimizing program if
56. results 8 14 Click on OK to return to the Create curve box and again on OK when you have made your selections there W ith Point mode selected you can use the left button to insert new curve points on the chromatogram T he co ordinates of each new point are automatically entered into the Point List It is possible to precisely define the retention value of the point by activating the XY box and selecting the appropriate coloured scale within the box Using the right mouse button the cursor bar can be dragged along the x axis and retention values be read in the active XY box By eye you can insert a point on the chromatogram at the desired amplitude at the position of the active bar The right mouse button can also be used to magnify a region of the chromatogram Selecting Zoom mode switches over the mouse button functions i e the left mouse button can be used to drag the cursor bar or perform zoom functions and the right mouse button inserts points onto the chromatogram SRS nd TREO us I7B087 494 en00 196 454 494 G00 LO14 821 407 101 on 02S 003 494 600 L 4T 37i 48s j L0G SE DF 07E Faa 48 ft wn LUET aZ3 482 0 L1L40 598 SMF oo Libs L95 1 oo i Lb 924 4 7000 rH 4 A 3 467 L 700 m 3 FE ed nn uk Ti 25 89 100 a E i j or 7o P a k 4 il pet lh a UON T l eee aT 26 SiG 1000 me ines i Lari hiii L ight TAS SLA On il f L si b H
57. run the method after you have provided information about the sequence you want to synthesize see Section 4 3 M ore advanced usage of UNICORN OS requires that you understand the principles and concepts of methods Blocks Methods in UNICORN OS are usually divided into blocks Blocks typically contain the subroutines that control the complete synthesis procedure A synthesis cycle is generally based on the following order of subroutines eeD etritylation eeD etrit wash eeC oupling eeQ xidation thiolation eeC apping M ethod templates supplied with UNICORN OS contain all the blocks that are likely to be used in a specific method W hen the desired sequence is created the blocks needed to build up the method to synthesize the sequence are automatically copied in from the method template The methods derived from the method templates can be directly used to process the run Additionally the methods are convenient starting points for developing customized methods Fully adequate customized methods for many applications can be created simply by adjusting the values of method variables see below N ew blocks can also be created in the Text instructions or in the cross reference list of the Sequence page in Run setup M ethod base M ethod blocks are written in one of three method bases which defines the unit for the breakpoints in the block eetime min eevolume ml or according to the strategy eecolumn volume set by the use
58. server disk and directory where UNICORN OS network components are to be installed 13 Install UNICORN OS see Chapter 11 10 6 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Network setup 7 0 10 7 Startup files UNICORN OS network stations require some background processes to be started to enable communication over the network These processes are suitably started from the STARTUP CM D file in the root directory of the local boot drive The STARTUP CM D file is created during UNICORN OS installation Each station must also be logged in to the network with the correct logical drive mapping before UNICORN OS is started A routine should be included in STARTUP CM D to prompt for username and password to ensure this login The example below shows an extract of STARTUP CM D for a local station with one connected system The OCI multinet drivers and open connection interface are not required on remote stations W hen STARTUP CM D is first created the lines marked with an arrow are commented out Remove the REM text on these lines and edit the M AP line to address the correct server disk and directory before starting UNICORN OS REM Example of STARTUP CMD for a l1 system local station REM Prepare Novell Netware for automatic login CD NETWARE login REM Edit this line to map the server disk gt MAP Q SERVER PROCESS lt dirname gt gt gt REM Storage process for local hard disks Ge CD UNIC
59. templates supplied with UNICORN OS are defined with a set of questions for the synthesis scale i e column volume support load weight of the support and delay volume To define questions which will be shown when the method is started open the Questions page in Run setup Mintheed elite bet fr a Fi feign 1 Pa ob rr l Fraja arankan air i Pomp km Tar Lisle se i Coste chr gil E EFTTI i k i wi T A i j fate a at mimi l i PETH E 3 Ei is draw PET Piip i tal Figure 5 28 The Questions page in Run setup Questions may have the following status eeMandatory these questions must be answered before a method is started eeAuthorized answers to these questions must be acknowledged by a user with Confirm Unlock authorization see Section 12 2 The user s password must be given to acknowledge the answers eeChromatogram these questions will be printed with the answers on the same page as the chromatogram if Diagram Header is chosen in an evaluation report Note These options are non exclusive i e a question can be mandatory require authorization and or be printed on the chromatogram header UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 5 27 5 Creating and editing methods Questions may be defined to accept four types of answers eelnput field accepts any alphanumerical input as the answer Input field questions may ha
60. the first listed call to a block will be completed before proceeding onto the next instruction or call to block To ensure that instructions are executed in a defined sequence where this is important separate the instruction breakpoints by 0 1 base units The revised formulation for the first example above Is 0 00 Base SameAsMain 0 10 WasteOut Waste_ACN 0 20 PFlow_ACN 2 00 bar 10 00 CV_Column_wash End_block 5 8 3 Controlling block and method length The length of a block is determined by the breakpoint of the last instruction in the block A block in which all breakpoints are at 0 will take no time or volume during a run e g START parameters 0 00 Base SameAsMlain 0 00 Scale 1 754Weight_oft_support g si Loading of supportumol g 0 00 ColDiam O 00 Col Diarn mm 0 00 Delayol 1 3 Delay_volumel ml 0 00 End_block To extend the length of a block without performing any other operation set the breakpoint of the END BLOCK instruction appropriately eg 0 00 Base SameAshlain 400 End block UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 5 41 5 Creating and editing methods During arun the overall time or volume is determined by the sum of the block lengths N ote in particular that the length of the main block does not indicate the overall length of the method the main block often consists only of calls to other blocks and has zero length The accumulated time and volume are displayed i
61. the main Export to file method is included and if Blocks is ON then all blocks are included in the exported file EXPORT _ Exports the peak table in Peak table source to the file Peak table source PEAKTABLE __ defined in Export to file in ASCII format Export to file ASCII EXPORT_ Exports the peak table in Peak table source to the file Peak table source PEAKTABLE_ defined in Export to file in WKS format Export to file WKS C 4 5 Chromatogram functions Instruction Description Parameters COPY _ Creates a copy of the specified chromatogram If is From chromatogram CHROM used as source then the current default name chromatogram is used If is used as destination To chromatogram name then a default name will be created for the copy CREATE _ Creates a new chromatogram with the given name If Name NEW_CHROM is used for the chromatogram name a default name will be generated and used DELETE _ Deletes the named chromatogram If is used as the Chromatogram name CHROM name then the current default chromatogram is deleted Opens the specified chromatogram from the specified CHROM file Chromatogram name RENAME Renames the specified chromatogram If is used From chromatogram CHROM as From then the current default chromatogram is name used To chromatogram name RESTORE _ Resets the destination for the subsequent curve and DESTINATION peak table operations to the default chromatogram _CHROM Used in pai
62. the synthesis process The run status can be displayed as eenumerical display of run data from selected monitors eegraphical display of curves from monitors eea flow scheme showing the current flow path in the system eea logbook recording the control events in the run Systems can be controlled either manually with interactive commands or through pre programmed methods Sa paleomnls lt Matha hich rd ISDE Fl View Manual System F1 Help Fire Hd Pama Car _Eaa ot E ee RUH D T a E wj towered ES a OO O ee eee re D a Cored HHT rkr abel 1 Figure 2 5 The System control window with run data and curves displayed 2 10 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 2 5 Evaluation UNICORN OS concepts 2 2 4 2 System connections To control a synthesis process the operator establishes a connection between the computer and the oligonucleotide synthesis system in one of the System control windows on UNICORN OS desktop Two kind of connections may be established eeC ontrol mode connections which permit full control of the connected system ee V iew mode connections from which the progress of the synthesis can be monitored but the system cannot be controlled Each oligonucleotide synthesis system may have only one control mode connection but may have several view mode connections In a network installation the same or different users may establish simultaneous view
63. the system The current status of a run is indicated by the colour of the status indicator and by the available buttons buttons which are not available are grey ET ET Figure 6 11 The control bar at the top of the System control window Run Starts a run when the system is idle and a method is loaded Hold Suspends execution of a method but continues to pump liquid at the current flow rate and valve setting Accumulated time and volume continue to be incremented Any method instructions which are set to the time volume when Hold is pressed are executed Later method instructions are not executed until Cont is pressed Pause Suspends execution of a method and stops all pumps so that the system comes to a standstill In OligoPilot and O ligoProcess the valves remain in the position they were in before the pause Accumulated time is not incremented during Pause Any method instructions which are set to the time volume when Pause is pressed are executed Later method instructions are not executed until Cont is pressed Cont Resumes execution of a paused or held method End Terminates method execution and puts the system in the idle state The colour of the status indicator and the available buttons indicate the control status of the connection buttons which are not available are grey Status Indicator Available buttons Idle W hite Run Running Green H old Pause End M anual Green Run Pause End H old Y ellow Pause
64. time monitoring and subsequent evaluation of the synthesis process Strategies Part of UNICORN OS software referred to as the strategy is system specific The strategy defines what is available in method and manual instructions system settings run data curves and method templates M ost of this manual describes the user interface in UNICORN OS independent of the strategy Strategy dependent instructions are listed in Appendix B UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 2 1 2 UNICORN OS concepts 2 1 2 Stand alone and network installations UNICORN OS may be installed either on a stand alone computer or in a network ee n a stand alone installation up to four oligonucleotide synthesis and or chromatographic separation systems may be physically connected to and controlled from the computer where UNICORN OS is installed ee n a network installation each oligonucleotide synthesis system Is physically connected to one computer in the network but may be controlled from any computer in the network on which the software is installed A computer to which oligonucleotide synthesis systems are physically connected is referred to as a local station O ther computers in a network installation are called remote stations Note Itisnot necessary to install UNICORN OS for network control if you only want to be able to save method and result files on a server disk J ust define a home directory on the serve
65. to control the synthesis systems directly connected to the PC without logging in to the network The station must however have been logged in to the network once previously so that the necessary files are copied from the network server to the local computer This copying is performed automatically M ethod and result files stored on network drives will of course not be accessible For runs performed in this stand alone mode where the result file is directed to a network drive the results will be saved in the FAILED directory on the local station see Section 9 3 10 4 Network requirements The following are minimum network requirements for running UNICORN OSin a network installation eeQ S 2 Warp version 3 0 or later eeEtherlink Ill network card with 3C5x9 SY S driver version dated 10th M arch 1995 or later eeN ovell N etware version 4 02 or later eeA valid network connection 10 4 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Network setup 7 0 10 5 Shared network resources Installation of UNICORN OS in a network requires access to a shared disk which is accessed through the same logical drive letter from all local and remote stations in the network On the N etwork a directory should be created by the network administrator in which all UNICORN OS users must be given the minimum N etwork access rights of Read Write Create Erase and Filescan N ote that this could be an existing common dir
66. to create methods If however you intend to save the created method as a template for future use with the sequence editor then you must manually create all of the relevant cross references between the bases and options used in the method to the created blocks To create your own method from scratch 1 Click on the New Method toolbar button or choose the File New Method menu command in the M ethod editor or choose File New Method or New Method in the M ain menu These alternatives are equivalent When you choose the command from the M ain menu the M ethod editor is opened automatically To rio Tear Po m Figure 5 42 The New method dialogue box 2 Choose the system for which the method is intended The instructions available for a given system are determined by the system strategy A method developed for one system may not be valid on another 3 Placea check mark in the No Template box UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Creating and editing methods 5 4 Click on OK once you have made your selections An untitled empty template containing only a M ain block with a Base instruction will be opened 5 Select View Text instructions or click on the Text instructions button 6 Click on View Windows and select Text and Instruction Box from the list Click on OK 7 Create a method using the information described in Sections 5 2 to 5 6 Information about the specif
67. to the oligonucleotide Trityl ON synthesis will not display the last 5 base in the Trityl table since the last base is not detriylated Thus in a synthesis of a 25 mer trityl ON there will only be 24 Detrit values in the synthesis data Synthesis data can also be obtained by printing a report see Section 7 6 Daniana Figure 7 4 Log Book page Evaluation log This lists all of the evaluation operations performed for the current result file for all sessions including procedures executed at the end of the method Click on the Refresh button to update the list PFRI Tha Wr Figure 7 5 Evaluation Log page 7 4 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA ooo oo y Presenting results 7 1 3 Temporary icon The Temporary icon is specific to the Evaluation module and is essentially an empty chromatogram Thus curves can be copied into Temporary and comparisons and or evaluations can be performed This iS particularly useful if you do not want to clutter up your original chromatograms with a large number of curves Information contained within the Temporary chromatogram is automatically saved from one evaluation session to the next but is not saved within the result files Double click on the icon or select Windows Temporary to open it The contents of the temporary chromatogram can be removed by selecting Edit Clear temporary chromatogram 7 2 Basic presentation of chromatograms This sec
68. values between 1 and 10 C 1 3 Median For each data point in the source curve the processed target curve Is calculated as the median of the data points within a window centred on the source data point The width of the window is determined by the parameter value expressed as number of data points W hen the source point is less than half the window size from the beginning or the end of the curve the median is calculated symmetrically round the source point over as many data points as possible Increasing the window width increases the smoothing effect To completely remove a noise spike the window width should in principle be slightly more than twice the width of the spike The filter algorithm only accepts odd integer parameter values between l and 51 If an even number has been given it is incremented by one C 2 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Evaluation functions and instructions C C 2 Baseline calculation theory The baseline calculation can schematically be described in three steps 1 Find baseline segments 2 Select baseline points 3 Draw the baseline C 2 1 Finding baseline segments In the first step four baseline parameters are used to find the baseline segments The parameters can be seen in the Integrate Calculate baseline function or by pressing the Baseline settings command button in the Integrate Peak integrate function T he default values for the parameters are
69. you want to be able to minimize the UNICORN OS window on the OS 2 desktop Password and minimizing options Password C Ho password required Minimizing _ Allow minimizing program 11 6 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Installation 7 7 9 Choose the number of System control windows that should be available in the installation maximum 4 Unicorn OS 1 10 Installation Enter number of system control windows that should be available Cancel The installation will now start If global procedures and report formats exist already e g in a network you will be asked if you want to replace the corresponding files Normally you should answer No If you answer Yes all existing global procedures and report formats will be deleted and replaced by the defaults Change to diskette no 2 and 3 when prompted to do so 10 If you have chosen to install Systems choose the system to install and the driver For OligoPilot Il select Oligo W hen the software is installed for the first time the choice is made automatically Y ou can choose the system to install if you have more than one system already present and you wish to reinstall for one of the existing systems x system installation select system System 1 fusien 2 finie 3 RENI 4 Select driver CJ BioPilot Oligo UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 11 7
70. zi pe rie on on Si i F i T ra IT M gt g e gt a Figure 8 13 Measurement of the Slope limit after differentiation 8 12 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Evaluating results 8 4 Usethe zoom function to magnify the curve over an appropriate area Place the vertical bar at the beginning of a peak where you want the W atch conditions to be fulfilled i e where the slope becomes higher Read the actual slope value in the active XY box 5 Inthe M ethod editor enter the slope value as a parameter for the W atch instruction 8 2 2 Creating a curve It is possible to create a curve based on any external measurements 1 Select Operations Create Curve In the box that is displayed select a help curve 2 Select the minimum and maximum values of the y axis Also choose the appropriate units from the list that is displayed when you click on the drop down arrow The help curve determines the min and max values for the x axis S504 12 Pree OY ASIN 0401 Fra H Di Sei iI Conde W in ET Condi iBA et Figure 8 14 Create Curve dialogue box 3 Ifyou want to create new unit click on the N ew unit button and enter the new unit name and number of decimal places the values will receive Create new unit Number of decimals Figure 8 15 Create new unit dialogue box UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 8 13 8 Evaluating
71. 0 User M anual 18 1118 52 Edition AA 6 17 6 Performing a run 6 6 Calibrating monitors Certain system monitors need to be calibrated regularly for correct results According to the routines established in the laboratory or process department monitors may be calibrated at pre set intervals by the system technician or calibrated as required by the user before each run This section describes the calibration procedure for the ACN and Detrit solvent pumps in OligoPilot Il For other synthesis systems users are recommended to read the relevant system manual for calibration procedures To calibrate the solvent pumps for OligoPilot II do the following 1 Select System Calibrate to display the Calibration box 2 Select each of the pumps in turn and perform the procedures described below 3 Click on Exit once the calibrations have been made D etrit solvent pump a i ET Cee piah Fae LL A ee pe bet 1 Aabmarca valm nooo Te R 1 Reame yoke 2 iii et Baie th Fd Figure 6 16 Dialogue box for pressure calibration of the detrit pump Select PressDet in the Monitor pull down list to calibrate the pressure reading of the Detrit P 6000 solvent pump The calibration is based on the maximum set pressure of the pump 1 Thevaluein the Reference value 1 box should be zero 0 0000 bar If not enter 0 0000 Click on the Read value 1 button 2 Enter the desired maximum pressure value lt 20 0 bar in the Reference value 2 b
72. 118 52 Edition AA File organisation E E File organisation This appendix documents the file structurein a UNICORN OS installation E 1 Stand alone installations In a stand alone installation the entire directory structure resides on the local hard disk System and user definition files and system strategies are duplicated in the 1ocal and server directories E 2 Network installations E 2 1 Local and remote computers The bin dll and local directory structures are installed on each computer E 2 2 Network server The server directory structure is installed on the network server The local fil directory structure is also installed if home directories are created on the server disk System and user definition files and system strategies are copied automatically from the network server to each station so that local UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA E 1 E File organisation E 2 stations can be used as stand alone systems in the event of a network communication failure UNICORN The main UNICORN OS directory Executable modules Device drivers and dynamic linked library dll modules local H olds a copy of the global system definitions system tab N ot available on remote computers fil H olds a copy of the files containing user definitions user20 mpm N ot available on remote computers lt nome gt H ome directories for users in the system There may be any
73. 25 Ge SameAsM ain base 5 11 save evaluation 7 28 method editor 4 10 Save AS evaluation 7 28 main menu 3 8 method editor 4 11 5 39 Save as template 5 39 save templates access 12 7 saved audit trails 12 16 saving manual run results 6 11 methods as templates 5 39 saving a sequence 4 5 saving methods from result files 7 2 saving results evaluation 7 28 Screen parameter for messages 5 42 search 3 7 security 1 1 2 14 features 9 1 file backup 3 9 in network installations 10 2 sequence editor 4 3 creating a new method 5 2 modifying the cross reference list 5 3 new sequence 4 4 sequence grouping 4 5 sequence page 4 3 4 4 5 34 new sequence 4 4 saving a sequence 4 5 serial number in result file names 5 35 settings UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Index for watch conditions 5 45 in start protocol 5 37 System menu system control 13 1 Shift 7 26 simultaneous connections 6 14 Slope greater than condition 5 44 Slope less than condition 5 44 smoothing algorithms C 1 autoregressive C 1 median C 2 moving average C 1 software installation 11 3 software requirements 11 2 A 1 software upgrades 11 10 sorting files 3 5 Stable baseline condition 5 44 stand alone installation 2 2 stand alone installations control A 2 start protocol at method start 4 12 6 3 evaluation procedures 5 37 method information 5 36 method instruct
74. 5 40 r calculate baseline 8 1 calibrating pump 6 18 calibrating monitors 6 18 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Index calibration access 12 6 CALL instruction 5 10 inserting when block is created 5 12 CALL instructions in imported blocks 5 15 calling blocks 5 9 see calls calls conditional 5 10 normal and restore modes 5 10 unconditional 5 10 cancelling WATCH instructions 5 10 cascade 7 6 7 20 changing base 5 40 breakpoints 5 20 method instructions 5 19 passwords 12 12 strategy for system definition 12 4 variable default values 5 24 Chromatogram Layout dialogue box 7 7 axes 7 10 base 7 11 curves 7 7 filter peaks 8 3 header 7 11 HETP 8 11 layout 7 7 7 12 names 7 8 peak asymmetry 8 11 peak tables 8 3 style colour 7 9 tag 8 3 chromatograms Chromatogram Layout dialogue box 7 7 contents 7 2 7 5 presentation 7 5 zoom function 7 13 Clear temporary chromatogram 7 5 colour codes for method instructions 5 16 column volume base 2 7 communication failure 6 13 communication problems F 3 comparing chromatograms 7 17 curves 7 20 runs 7 17 concepts 2 1 conditional calls 5 10 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA iii Index conditional instructions 5 43 actions 5 47 conditions 5 44 system settings 5 45 conditions for watch instructions 5 44 CONFIG SYS F 1 confirm access 12 6 Co
75. 6 valve6pos1 to column 1 5 to column 1 7 to column 1 6 to column 2 1 to column 2 2 to column 2 4 to column 2 3 to column 2 4 to column 2 5 ACN_T U to column 2 7 T U to column 2 6 ACN X to column 1 1 X to column 1 8 ACN_Y to column 2 1 Y to column 2 8 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA B 3 B Strategy for Oligo Synthesis Solvent Beaucage 3 7 1 0 5 2 6 1 ACN_ 3 1 to column 3 1 p1 0 Cap A to column 3 2 p1 0 Cap B to column 3 3 p1 0 ACN_ 3 4 to column 3 4 p1 0 OX to column 3 5 p1 0 Extra_3 6 to column 3 6 p1 0 Tetrazole to column 3 8 p1 0 Every amidite and solvent setting can also be set to waste This means setting valve 6 from position 1 to position 2 WasteOut Waste_ACN Waste_Detrit Waste_A Waste C Waste G Waste_T Waste_X Waste_Y ACN Pump Detrit Pump Recycle valve 6 1 valve 6 2 valve 6 3 valve 6 4 valve 6 5 valve 6 6 valve 6 7 valve 6 8 to waste to column to waste to column flowrate LFlow 0 1000 cm h flowrate LFlow 0 1000 cm h B 4 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Strategy for Oligo Synthesis B B 1 3 Alarms amp Monitors Tetrazole Percent tetrazole of the column 1 100 volume Base_lId Identification of the base that gives the trityl peak used because Last eff only considers bases with the same ID ColDiam Sets the column diameter in mm 1 100 mm use
76. 8 21 8 Evaluating results Procedures Record on Edit gt Run Batch run Menu added to menu Global print evaluation procedures peakintegrate Figure 8 21 Procedures menu To take away a procedure from the menu deselect it from the Procedures Menu list 8 4 Exporting data or curves Data and curves can be exported to other file formats Select File Export and then the appropriate sub menu choice Alternatively the Edit Copy to clipboard function can be used which is the quickest and easiest way to copy a chromatogram into for example M icrosoft Word 8 4 1 Exporting results Data can be exported to ASCII text and Lotus 1 2 3 WKS spreadsheet formats The choice of format depends on the subsequent use of the data If the data is to be inserted into a word processing program e g M icrosoft W ord it is preferable to export the data in ASCII format Calculations or diagrams are most easily produced if the data is exported in Lotus 1 2 3 format which can be easily read by most charting and spreadsheet programs e g M icrosoft Excel Documentation and Evaluation log are exported as text strings regardless of file format Peak tables and Curves however are exported as text strings in the ASCII format but as numerical values in the Lotus 1 2 3 format When exporting peak tables all possible columns in the table are exported 8 22 UNICORN for Oligo Synthesis version 1 10 User M anual
77. Cont End M ethod pause Red H old Cont End M anual pause Red Run Cont End UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 6 9 6 Performing a run 6 10 Other buttons on the control bar are O pens a dialogue box for choosing which window panels to display Clicking on this button is equivalent to choosing the menu command View Window Opens the run set up pages Run notes may be entered in the Notes page and scouting settings may be changed for scouting run that have not yet started Other pages are displayed for information only As The connection mode button has three states which indicate and change the connection mode as described in the table below System control window Click to connect to a system disconnected Control modeconnection Click to leave system View mode connection Click to establish a control mode connection if possible The control bar also displays a text message indicating the connection status of the window Controlled by lt user gt The indicated user has a control mode connection to the system Other users may establish a view mode connection Locked by lt user gt The indicated user has left the system in s a locked state Users who can supply the required password may unlock the system and establish a connection System is available Any user may establish a connection System connections are described in more detail in Section 6 5 UNICORN for
78. G SY S file must be changed to THREADS 512 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Installation 7 7 Unicorn OS 1 10 Installation The installation process is now complete Please remove any floppy disks and restart the system To restart the computer click with the right mouse button on the O S 2 desktop and choose Shutdown from the desktop menu W hen the shutdown procedure is complete press Ctrl Alt Del or turn the power off and on again to restart the computer 11 4 2 Installing selected software components after the initial installation If your UNICORN OS installation should be damaged due to accidental file deletion or hard disk failure or if you want to install additional systems strategies or templates you can use the installation program to re install selected parts of the software The installation program detects the presence of existing UNICORN OS files and suggests the existing disk for installation Check some or all of Users Program System Strategy and Method templates according to which part s you wish to Install The respective parts will be copied into the existing directory structure Note For network installations remember to log in to the network before installing any UNICORN OS software components Users Check the Users button to re install the default user if the existing user definitions are damaged Any other users defined in the system including users
79. Installation eeQuit program Required for endinga UNICORN OS session with the Quit command in the M ain menu 12 2 2 Access level examples Below are some examples illustrating the way access levels might be used in a multi user installation System administrator The administrator has special responsibility for maintaining user system and audit information and for file management in the PC directory structure backup routines etc The administrator may not edit or run methods or sequences issue manual instructions calibrate monitors or change the system configuration Important W e recommend that only one user in an installation or network Is assigned system administrator rights If several users can change user definitions and system connections confusion can rapidly follow UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 12 7 7 2 Administration Define access levels Level name Administrator Figure 12 3 Suggested authorization profile for the system administrator D evelopment staff Developers need to be able to edit and run methods and sequences issue manual instructions configure system parameters calibrate monitors and evaluate data They may also copy move or delete files Detine access levels Level name Development ltem Figure 12 4 Suggested authorization profile for development staff Process supervisors Supervisors may pause a method and issue man
80. Lois Alii Mii ELL ui 8 ni nm ai D Owai Tma phie Spine Thagh Ci uer mireg iri io red o E Fou i ITER F al ral I aij Te Ti Le Conca Peg va Figure 8 16 Create Curve chromatogram window To delete a point from the Point List double click on the appropriate choice in the list Alternatively select the co ordinates in the list and click on the Delete button To delete all of the points in the list click on the Delete All button To draw the curve click on either Draw Spline or Spline Through Draw Spline creates a smooth curve from the data but does not necessarily pass through every point that you have entered By UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Evaluating results 8 contrast Spline Through creates a curve that passes through all of the data points In cases where you have created a curve using Draw Spline you may want the curve to pass through a selection of those points currently lying away from the curve Y ou may force a straight line between two points by selecting the first of the two points in the Point List and then clicking on the Draw straight to next point command button This may have to be repeated for several consecutive points to achieve the desired curve Click on OK and save the curve Y ou can change the curve name from the default CreatedCurve and also the curve destination 8 3 Automated evaluation procedures An evaluation procedure is a rec
81. OFA T 000 CallHomal Add OFA C 0 00 Cali Mommal Add Dh E 000 Calima Add OHA amp 000 Call Mormal Add OHE G UO all Monel Miral driny 000 Ered_methed Figure 2 3 Relationship between blocks and instructions The method left is written as a series of calls to blocks each of which consists of instructions for performing one or more specified tasks right Breakpoints Each instruction in a method block is issued at a specified breakpoint according to the method base The first instruction in a block is always at breakpoint 0 and all other breakpoints are counted from this point For example in the following instructions from a block 0 00 Base Time OPRL Flow_Reag 5 00 ml min 9 00 Flow_Reag 0 00 ml min 2 8 UNICORN for O ligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA UNICORN OS concepts 2 At breakpoint 0 00 the reagent flow rate is set to 5 00 ml min After nine minutes have elapsed at the next breakpoint the flow rate of the reagent will be set at 0 00 ml min i e no flow at all M ethod variables Breakpoint values and instruction parameters may be defined as variables This is a powerful facility for constructing a method which contains default parameter values T hese default values may then easily be changed either to create variants of the same method or to adjust the parameter values at the start of a run see Section 4 4 Using variables makes it easy to adapt a method to
82. ORN OS BIN detach storage gt nul REM OCI multinet drivers REM One for each connected system detach pmnt_drv 0 gt nul REM detach pmnt_drv 1 gt nul REM detach pmnt_drv 2 gt nul REM detach pmnt_drv 3 gt nul REM start open connected interface REM One for each connected system detach OCI 0 gt nul REM detach OCI 1 gt nul REM detach OCI 2 gt nul REM detach OCI 3 gt nul When you make changes to STARTUP CM D you must reboot the computer log in to the network and restart UNICORN OS for the changes to take effect UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 10 7 1 0 Network setup 10 8 System and user definitions 10 8 Synthesis systems accessible from UNICORN OS network stations are defined by the system administrator for UNICORN OS with the Administration System command in the M ain menu see Section 12 1 Parameters that must be defined for each system in a network include eethe system name to help in identifying the system in the network e g M ichaels OligoPilot or O ligoPilot 2 eethe strategy associated with the system eethe local station name i e the pipe server name on the PC to which the system is physically connected eethe control unit number on the local station each local station can handle up to 4 systems Each UNICORN OS user has a user profile which defines access rights data storage locations etc These parameters are defined by the s
83. ORT _ Exports the documentation in the current result file in ONOFF Variables DOC_WKS WKS format to the file defined in Export to file If all ONOFF Scouting parameters to this function is OFF then no ONOFF Start Protocol documentation is exported If at least one of them is ONOFF Questions ON then the documentation will be exported and the ONOFF RefCurves corresponding parts will be included in the exported ONOFF EvalProc file ONOFF Method Info ONOFF Method Notes ONOFF StartNotes ONOFF RunNotes ONOFF EvalNotes ONOFF Sys Settings ONOFF Calibra tion ONOFF LogBook ONOFF Result Name ONOFF Column Parameters NAME Export to file EXPORT _ Exports an evaluation log in ASCII format to the file Export to file EVAL LOG _ defined in Export to file ASCIl EXPORT _ Exports an evaluation log in WKS format to the file Export to file EVAL LOG _ defined in Export to file WKS Exports a method to the file defined in Export to file in Main ASCII format If all parameters are OFF then no Blocks method is exported If Main is ON then the main Export to file method is included and if Blocks is ON then all blocks are included in the exported file C 14 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Evaluation functions and instructions C EXPORT _ Exports a method to the file defined in Export to file in Main METHOD _ WKS format If all parameters are OFF then no Blocks WKS method is exported If Main is ON then
84. Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Performing a run 6 6 3 2 Manual instructions The oligonucleotide synthesis system can be controlled with manual instructions see Appendix B issued from the Manual menu To save the results of a manual run issue the instruction RECORD ON in the Other instruction group at the beginning of therun UNICORN OS will prompt for a result file name at the beginning of the run Manual Pump Flowpath Alarms Mon Other The Manual menu opens a dialogue box similar to the text Instruction box in the M ethod editor see Section 5 4 Figure 6 12 The manual instruction box The number in parentheses in the title bar of the dialogue box identifies the System control window to which the box applies Available instructions are determined by the strategy for the connected system Instructions for OligoPilot and OligoProcess strategies are listed in Appendix B M anual instructions are entered in the same way as method instructions from the dialogue box in the M ethod editor The Insert button places the current instruction in the list at the bottom left of the dialogue box Clicking on Execute executes all instructions in the list at the same time or executes the currently marked instruction if the list is empty N ote that although all instructions are executed simultaneously some may take some time to complete in the liquid handling module T he Dele
85. R equired for using the M ethod editor for creating and editing methods Chapters 4 and 5 eeEvaluation R equired for using the Evaluation module for processing result data Chapters 7 and 8 eeUser setup Levels R equired for defining and changing access levels and user IMPORTANT Werecommend that only one user in an installation or network is assigned this access eeAudit trail System setup R equired for examining the audit trail and for defining connected systems Section 12 4 IMPORTANT Werecommend that only one user in an installation or network Is assigned this access UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 12 5 7 2 Administration eeDelete move Home only Required for deleting and moving files and directories within the user s home directory Sections 3 2 5 and 3 2 6 Does not authorize these operations on other directories eeDelete move Required for deleting and moving files and directories outside the user s home directory Sections 3 2 5 and 3 2 6 Also authorizes these operations within the home directory eeCopy files R equired for copying files Section 3 2 5 The user must have access to both the source and target directories for moving or copying between directories eeConfirm R equired for authorized confirmation of answers to start protocol questions Section 5 6 2 eeUnlock locked system Permits a user to unlock locked systems by providing the user s own
86. RATE If OnOff is OFF then the baseline is never drawn above the curve PEAK _ Performs a peak integration on the curve in Curve Curve source INTEGRATE source and stores the resulting peak table in Resulting peak table Resulting peak table It is assumed that the baseline is subtracted PEAK _ Specifies which part of the curve in Curve source that Curve source WINDOW will be integrated Peaks between retention Left limit Left limit and Right limit will be detected if the OnOff parameter Right limit is set to On If OnOff is set to Off the whole curve will OnOff be used for integration REJECT _ Specifies which peaks that are interesting to include in Height less than PEAKS a peak table when peak integrating If Area Less Than Width less than has a numerical value not OFF then only peaks Width more than larger than this area will be included in the peak table Area less than If Height Less Than is specified then no peaks lower Peak must be one of xx than this height are included Similarly if the Width largest Less Than parameter is not OFF then only peaks wider than this value are accepted and if Width More Than is not OFF peaks narrower than this value is accepted Finally if Peak Must be One of xx Largest is specified then at most so many peaks as specified will be included in the peak table and if more than number of peaks are detected the ones with the largest area are selected Any combination of conditions is
87. UNICORN QS version 1 10 User M anual Amersham Edition AA o K e Biosciences Important user information Reading this entire manual is Amersham Biosciences shall in no event be liable for incidental or consequential damages includi recommended for full understanding without limitation lost profits loss of income of the use of this product of business opportunities loss of use and other related exposures however caused arising from the faulty and incorrect use of the product Should you have any comments on this manual we will be pleased to receive them at Trade marks UNICORN OligoPilot I1 OligoProcess and Prii Support are the exclusive trade marks of Amersham Biosciences AB In view of the risk of mark degeneration it is respectfully suggested tl authors wishing to use these designations refer t their trade mark status at least once in each arti Amersham Biosciences AB M arketing Department SE 75182 Uppsala Sweden Amersham Biosciences reserves the right to make changes in the specifications without prior notice x Copyright 1997 Amersham Biosciences Warranty and Liability All rights reserved No part of this publication r Amersham Biosciences AB guarantees that the product be reproduced stored in a retrieval system ar delivered has been thoroughly tested to ensure that transmitted in any form or by any means withot it meets its published specifications The warranty in written permission fro
88. _Capping_wash CV Sets the number of column washes after Capping contact flow CV_Column_wash CV Number of column volumes used in column wash block CV_Coupling_wash CV Number of column volumes of wash after coupling CV_detrit_wash CV Number of column volumes of detrit wash CV_tetrazole DNA Sets the of the column volume for tetrazole in DNA cycles CV_tetrazole RNA Sets the of the column volume for tetrazole in RNA cycles CV_Thio CV Sets the number of column volume after of Beaucage reagent cm h The linear flow used in detritylation block Detrit_Pressure_Flow Maximum flow pressure of detrit Efficiency_threshold 9 Minimum acceptable threshold efficiency Earann oa ner oranan ofan min Recycle Time Sets the time of recycling of amidite and tetrazole Weight_of_support Fo To calculate the Scale B 12 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Evaluation functions and instructions C C Evaluation functions and Instructions This appendix describes the functions implemented in the Evaluation module There are four sections in the appendix C 1 describes how the smoothing functions are calculated C 2 gives an basic introduction into baseline calculation theory which is an essential part of peak integration C 3 describes the peak table column components C 4 the Procedure Editor instruction types are described which are used to build up an evaluation procedure C 1 Smoothi
89. a particular oligonucleotide synthesis run For example in the block below the values of the start parameters variables can be seen START parameters 0 00 Base SameAsMain 0 00 Scale 2 Weight_of_support g 90 Loading_of_support umol g 0 00 DelayVol 2 00 ml 0 00 ColDiam 20 Col_Diam mm 4 00 End block The variables are expressed variable value V ariable_type variable units In the block above it is possible to see that the variable values have been set at 2 g weight of support 90 umol g loading of support and a 20 mm column diameter By using variables a method may be displayed either in detail as text instructions or in a condensed form as variable values in Run setup mode This is illustrated in Figure 2 4 The Run setup mode is displayed when the method is run allowing variable values to be set at the beginning of the run TE Mi i Cinn Wola lk oft 010 An z PENATI pe E a Wigh a reco nt O TON F Eia LI a ee Le oi suppor a ee Op PH alee Ce el e Pere Tol_Ceare Jew O08 1 Figure 2 4 Relationship between variables in text instructions and in the Variables page of run set up UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 2 9 2 UNICORN OS concepts 2 4 System control 2 4 1 Control facilities The System control module allows independent control of up to four oligonucleotide synthesis systems from one computer with continuous real time monitoring of
90. accessed under Administration Audit trail in the M ain menu provides a full record of UNICORN OS usage and system activity for the system administrator The audit trail may be viewed in global mode all systems in the installation or system mode one chosen system 12 4 1 Examining audit trails In the audit trail window choose whether you wish to view the global or system trail G lobal audit trails Check the items you want to display in the Global panel All items are recorded in the audit trail the check boxes in the Global panel only control which items are displayed Global audit trail files are saved on the server disk in a network installation and a network connection is required to examine global audit trails Audit trail 95 06 23 05 32 42 Loain default Global C System 95 0623 05 33 00 Connection change default at LOCAL connected to Sr Global 35 06123 05 41 16 New system BioPilot 2 900623 08 41 47 New system BioPilot 1 W User 95 06123 06 24 43 Connection change default at LOCAL disconnected fror 35 06123 06 25 56 Connection change default at LOCAL connected to Sr W Backup 99 06123 06 38 19 Connection change default at LOCAL disconnected fror W Log in i out 95 06 23 06 38 39 Connection change default at LOCAL connected to Sr 95 0623 06 43 39 Connection change default at LOCAL connected to 5 r W Define system 35 06123 06 47 16 Login default 95 06123 08 47 43 Connection change default at LOCAL connec
91. ain block in the method The corresponding BASE Instruction will be inserted in the block at breakpoint 0 If you choose Column volume enter a value for the column volume In general use SameAsMain wherever possible for the block base Y ou can then change the base of the whole method simply by changing the base of the M ain block UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 5 11 5 Creating and editing methods Length Y ou can enter a length for the block if required An END BLOCK instruction will be inserted in the block at the corresponding breakpoint This field may not be left blank Call Y ou can create a CALL instruction to call the new block from an existing block e g the M ain block Choose the block in which the CALL instruction is to be inserted in the From field and enter the breakpoint at which the CALL is to be made in the At field If you do not want to create a CALL instruction e g when the block being created is to be activated by a WATCH instruction choose an empty line in the From field Blocks which are created without a CALL instruction are placed last in the method and the Instructions are grey Notes If the CALL instruction is placed at the same breakpoint as the END BLOCK instruction the CALL instruction will be placed immediately beforeEND BLOCK Do not call a block from within itself Y ou will generate a potentially infinite loop which exceeds the maximum number
92. al system controller Data is transferred from the buffers to disk storage by UNICORN OS whenever a chromatogram Is closed Data is also saved to disk at preset intervals during a run minimizing data loss in the event of communication failure The capacity of the data buffers is 16000 points for all The initial sampling frequency for each monitor is set in the system strategy and can be viewed and changed in the Curves group of System Settings A 2 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Technical specifications A At an initial sampling frequency of 10 sample s 10 H z the following resolutions apply for the curves Duration Sampling frequency N o of points Resolution s point 0 27 min 10 Hz 0 16000 0 1 27 53 min 5Hz 8000 16000 0 2 53 107 min 2 5 Hz 8000 16000 0 4 1 25 Hz 8000 16000 0 8 107 203 min To ensure maximum resolution for part of a run issue a NEW _CHROMATOGRAM instruction at the beginning of the part This empties the data buffers and resets the sampling frequency to that specified in the system strategy UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA A 3 A Technical specifications A 4 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Strategy for Oligo Synthesis B B General strategy for Oligo synthesis This appendix alphabetically lists the instructions for methods manual control system settings and va
93. allowed If all parameters are OFF then every detected peak are included in the peak table C 12 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Evaluation functions and instructions C SET _ Sets the column height for the peak integration Column Height COLUMN _ calculation of the HETP value The Column height HEIGHT parameter is the height of the column in centimetres If Column height is OFF then the HETP value is not calculated for the following integrations C 4 3 File Operations CURVE_ OPEN Opens the curve specified in the Result file defined in File name File name and stores it in Resulting curve If is Curve name entered as File name the current result file will be Resulting curve used The File name parameter may include a path from the users root directory IMPORT _ Imports a curve to the current chromatogram from Chromatogram name CURVE another chromatogram in the current file and stores Curve source it in the Resulting curve Resulting curve IMPORT _ Imports a peak table to the current chromatogram Chromatogram name PEAKTABLE from another chromatogram in the current file and Peak table source stores it in the Resulting curve Resulting peak table PEAKTABLE __ Opens the specified Peak table in the Result file File name OPEN defined in File name and stores it in the Resulting Peak table name peak table If is entered as File name the current Resulting peak table Result file will
94. aluation workspace By importing a procedure to a method printouts can be automatically generated at the end of arun 1 Begin recording a new procedure by clicking on Procedures Record on 2 Click on File Report and choose a report format see Section 7 6 3 Select Print in the Generate Report dialogue box as the final instruction Stop the record function by selecting Procedures Record Off 4 Save the procedure 5 Now perform a batch run see Section 8 3 5 on all the desired result files to get the printed reports The procedure can also be imported into a method to get automatic printouts at the end of a run Section 5 6 5 Note If the selected report format is changed in File Report the new format will be applied when the procedure is run If the format is subsequently deleted the procedure cannot be run 8 3 7 Placing a procedure on the menu and running It is possible to place a maximum of 15 created evaluation procedures onto the Procedures menu Select Procedures Menu and select the evaluation procedure to be added to the menu Click on OK Activate a chromatogram and select the Procedures menu Y ou will see the procedure that you added to the menu Select this procedure and it is automatically run for the active chromatogram The menu addition is remembered in UNICORN OS even if the results of the current evaluation session are not saved UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA
95. am from the M ain menu Y ou will be prompted to save any unsaved work in the M ethod editor or Evaluation module Y ou can only quit the program from the M ain menu Note You can quit UNICORN OS during a run which does not use scouting or a sequence without affecting the run Do not however shut down OS 2 or turn off the computer while the run is in progress ICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Introductory material Methods and Runs Evaluation System management Appendices This page is deliberately left blank Creating methods from method templates 4 4 Creating methods from method templates UNICORN OS is supplied with a set of method templates which are used as the starting point for creating customized methods T hese method templates are defined with variables for critical parameters in the synthesis so that customized methods can be created for most purposes simply by setting appropriate values for the method variables This chapter describes how to create and edit methods at this level See Chapter 5 for a description of advanced method editing facilities Briefly the steps in creating a method by editing method variables are as follows 1 Choose New Method in the M ain menu or the M ethod editor and select a system and template Run setup 2 Choose View Run setup or press the Run setup button UIJ O pen the Sequence page and enter the desired sequence and cr
96. ange icons 7 2 audit trails 12 15 access 12 5 backup 12 18 printing 12 17 saved files 12 16 starting new files 12 17 authorized confirmation 12 6 authorized questions 5 27 6 3 automated evaluation and operations See evaluation procedures autominimize 3 3 autosave 6 13 9 3 12 4 auto scaling in system control display 6 7 axes Chromatogram Layout box 7 10 axis scales system control 6 7 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Index B backup audit trails 12 18 base 2 7 5 40 changing for the whole method 4 8 Chromatogram Layout box 7 11 for blocks 5 11 for system control curves 6 7 baseline calculation edit baseline 8 5 types of baseline 8 1 baseline calculation 8 1 baseline calculation evaluation estimating baseline parameters C 5 theory C 3 basic concepts 2 1 batch run 8 19 block window 5 8 blocks 2 7 5 8 base 2 7 5 11 calls 5 9 copying within a method 5 15 creating 5 10 deleting 5 13 determining length 5 12 displaying and hiding instructions 5 17 execution control 5 9 importing from other methods 5 15 in block window 5 8 in text instruction window 5 9 length 5 41 names 5 11 recovering after deletion 5 13 renaming 5 14 variable length 5 24 breakpoints simultaneous instructions 5 19 breakpoints 2 8 for END BLOCK 5 41 moving instructions within a breakpoint 5 21 moving method instructions 5 20 simultaneous instructions
97. are descriptive comments that form part of the method documentation T here are four separate notes fields for method editing start up run and evaluation respectively Only the method notes can be edited from the M ethod editor the other notes are accessible at the respective stages in a run To view the method notes open the Notes page in the Run setup mj M ethod templates are supplied with notes describing the system requirements for running the method Read through these notes carefully before using a method Click on the Maximize notes button to expand a notes field to fill the notes page Click on the same button again to restore the default display with all four notes fields visible Maximize notes Method editor teste Figure 5 33 The Notes page in Run setup 5 30 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Creating and editing methods 5 The notes are entered as free text and may be edited using standard O S 2 editing functions to edit the notes Words wrap automatically at the end of the field lt nsert gt Toggles between insert and type over mode In type over mode the cursor marks the current character lt Shift D el gt Cuts the marked text saving it on the clipboard lt Ctri Hnsert gt Copies the marked text to the clipboard lt ShiftHnsert gt Pastes the contents of the clipboard at the cursor lt D elete gt Clears the marked text without saving it in the clipboard
98. area or ee type the desired left and right limit values in the boxes marked Left limit and Right limit Note Theareas outside of the Left limit and Right limit will not be saved in the newly created cut curve Thus the x axis of the new saved curved will not begin at zero unless designated as one of the limits The original curve is not changed by this Operation 4 Click on OK A new panel will appear Select whether to save the new cut curve in the Source chromatogram curve list or in a New chromatogram If you select the latter option you can change the name of the new chromatogram Click on OK UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 7 15 Presenting results Figure 7 16 Save Cut Curve dialogue box The original source chromatogram will be displayed If the destination of the cut curve was the source chromatogram open the Chromatogram Layout box and select the new cut curve from the curve list If the destination of the cut curve was a new chromatogram this will be represented as an icon Double click on this icon to open it ll a Figure 7 17 Cut curve displayed in the chromatogram The y axis has been fixed to best display the curve see Section 7 2 7 7 3 2 Entering text in the chromatogram Up to five basic annotations can be added to the chromatogram L 7 16 Select Edit Text Add The mouse pointer is replaced with an ABC pointer Position the pointer
99. arger values have greater effect and a value of 0 has no effect M ost often a simple Pl regulator is preferable for control of flow rate and BioProcess System is configured by default with the D parameter set to zero setpoint stationary error response P regulator Pl regulator Figure D 1 A simple P regulator left gives a stationary error A Pl regulator right eliminates the stationary error but introduces stable oscillations in the response UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA D 1 D Feedback tuning Tuning a feedback control system in practice is largely a matter of trial and error The following recommendations summarize the Ziegler N ichols method for finding suitable PID values Small empirical adjustments in the suggested values may be required for optimal feedback control D 1 Flow rate tuning 1 Select System Tune in System control Select Flow for Feedback Control O pen the valves and start the pump with the column in line 2 Set P 0 05 9999 and D Set the flow rate in Setpoint Press New parameters and the new values will apply 3 Notethe response Increase the value of P until the actual flow rate oscillates with a constant period and amplitude N ote When changing to new PID values set the new values and flow rate and press New parameters The flow rate must be changed in Setpoint every time the PID values are changed When the o
100. aring different runs 7 17 7 4 1 Comparing chromatograms from different runs 7 17 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA iii Contents 7 4 2 O verlaying curves 7 4 3 Stacking and stretching curves 7 4 4 Mirror images of curves 7 5 Saving results 7 6 Printing chromatogram and documentation reports 7 6 1 Saving a report format 7 6 2 Selecting a format 7 6 3 Printer set up 7 6 4 Printing active chromatograms 7 7 Exiting Evaluation 8 Evaluating results 8 1 Integrating peaks 8 1 1 Baseline calculation for integration 8 1 2 Performing a basic integration 8 1 3 Optimizing peak integration 8 1 4 M anually editing a baseline 8 1 5 Adjusting the peak boundaries 8 1 6 Measuring HETP 8 1 7 Measuring peak asymmetry 8 2 Other evaluations 8 2 1 Finding the slope values for W atch instructions 8 2 2 Creating a curve 8 3 Automated evaluation procedures 8 3 1 Recording a procedure 8 3 2 Editing an existing procedure 8 3 3 Points to watch 8 3 4 Running evaluation procedures 8 3 5 Batch runs 8 3 6 Evaluation procedures and reports 8 3 7 Placing a procedure on the menu and running 8 4 Exporting data or curves 8 4 1 Exporting results 8 4 2 Copying results to the clipboard System management 9 Security features 9 1 Access security 9 2 Connection security 9 3 Data security 9 3 1 Network communication failure 9 4 Security recommendations for control stations 10 Network setup 10 1 Summary of network s
101. ase which most closely suits the purpose of the block Be careful when changing the base for an existing method Changing between time and volume base can affect the relative duration of steps in the method if different steps use different flow rates Note For method blocks which use a volume or column volume base make sure that the flow rate is not zero Volume breakpoints are calculated from the flow rate of the pump and the method will not progress if the flow rate Is zero The parameters for the BASE instruction differ slightly according to whether a named column definition is used 5 8 2 Instructions at the same breakpoint Instructions placed at the same breakpoint in a block are executed simultaneously with the exception of successive CALL instructions which are executed in the sequence in which they are written This can have important consequences in some situations For example the instruction sequence 5 40 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Creating and editing methods 5 0 00 Base SameAsMain 0 00 WasteOut Waste ACN 0 00 PFlow_ACN 2 00 bar 10 00 CV_ Column wash End block will set the waste valve to Waste ACN at the same time as 10 column volumes of ACN are pumped through at a pressure of 2 00 bar Conversely in the instruction fragment O00 Call Normal Detritylation 0 00 Call Normal Detrit_wash 000 Call Normal Coupling_recycle_DNA_T the instructions contained in
102. aste Flow_Reag 0 00 ml min End block Figure 5 19 Replace moves only the selected instruction 5 20 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Creating and editing methods 5 M oving instructions within a breakpoint To change the order of instructions within the same breakpoint in a block mark the instruction to move with the right mouse button and drag the instruction to its new location holding the right mouse button down Y ou can only move instructions in this way within a group of instructions at the same breakpoint Move Purge_cappingAB Purge_cappingAB 0 00 Base Volume 0 00 Base Volume 0 00 Flow _Reag 10 00 ml min 0 00 ookvent Cap A Waste 0 00 solvent Cap A vaste ie Vii Flow _Reag 10 00 mlsrmin 3 00 solvent Cap_B vaste 3 00 solvent Cap_B vaste 6 00 solvent ACM 3 1 Waste 6 00 solvent ACM 3 1 Waste 4 00 Flow _Reag 0 00 ml min 9 00 Flow _Reag 0 00 ml min 9 00 End block 9 00 End block Figure 5 20 Instructions can be moved within the same breakpoint by dragging with the right mouse button 5 4 5 The flow scheme window The flow scheme window displays the configuration of system components This window is for information only and can be useful e g in identifying valves for flow path instructions I E ie fet a iu H he 7 p 4 5 HHH akti FP Castor Cee e C C E Eh amp F D amp E l ee i g 7 Ta Eee ye A z b Boo z o B p n E Ei Fi J
103. ave As dialogue so that the method can be saved before performing a run Enter a name for the method select the destination and click on OK see Section 4 6 for more details The method is saved with default values for the method variables These can later be changed before you start a run see Section 4 7 and Chapter 6 or you can change the variables and save the method under a new name Note Users with the appropriate access authorization have global access to methods created by all users In such circumstances an already saved method can be used as the basis for generating a new method with a different sequence This is particularly useful if for example a method was saved with specially modified blocks see Section 5 2 or cross reference lists see Section 5 1 1 4 4 Editing method variables H aving created a method you may want to change the default values for the method variables The method templates contain all the blocks that are likely to be needed for a typical synthesis run By defining a sequence and then creating a method the program automatically builds up a method to synthesize the specific sequence by copying in all of the relevant blocks from the method template Each block has a set of method variables displayed on the Variables page in Run setup Y ou set default values for the variables in the M ethod editor and can change these values for a particular run when the run is started 1 After you have
104. ay UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 8 11 8 Evaluating results rely on the slope of the curve as a parameter by which to initiate a specific change in the run It is therefore important to use accurate slope values for the specific W atch instruction parameter Slope Figure 8 12 The slope of a curve To determine slope values you must first make a run with the sample you intend to purify Then use this result to find slope values in the Evaluation module 1 Ensure that you have selected Time as the x axis scale for retention in the Chromatogram Layout box 2 Select Operations Differentiate Select the desired Cond1 curve check that a First order calculation is selected and click on OK The differentiated curve will appear in the active chromatogram 3 Measure the y axis values on the differentiated curve by clicking on the XY icon choosing the y axis differentiate scale and reading the curve co ordinates in the active XY box It may be necessary to smooth the differentiated curve The units for the differentiated curve is uS min Co ordinates are based upon the position of the vertical cursor line on the chromatogram in relation to where it bisects the curve Any y axis value for the differentiated curve is the UV curve slope at the selected retention point mom Maleae g ry n eee Ge Parad T 1c T F if fi i ad ot E i sT fi f m o a pi L mam
105. b 1 Gb 3 5 1 44 Mb Etherlink III network card with 3C5x9 SYS driver version dated 10 M arch 1995 or later N ovell N etware registered trademarks of N ovell Inc version 4 02 or later A valid network connection As supported by OS 2 Warp operating system M icrosoft mouse or compatible Colour SVGA Compaq Q Vision 1 Hardware recommendations apply in February 1997 Contact your local sales office for details of the latest hardware recommendations UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 11 1 7 7 Installation The M CU for communication between UNICORN OS and the synthesis system s is supplied as part of the system s and is not included with UNICORN OS TheM CU is available for E ISA and M CA bus computers 11 2 2 Software requirements IBM Operating System O S 2 Warp version 3 0 or later N ovell N etW are version 4 02 or later for network installations N etwork installation UNICORN OScan be installed in network configurations using N ovell N etW are version 4 02 or higher Computers to which synthesis systems are physically connected are termed local computers Computers from which systems are to be controlled over the network are termed remote computers Systems can also be controlled remotely from a local computer The table below summarizes the installation requirements for UNICORN OS on computers in the network Local UNICORN OS Remote UNICORN 0s N etwork server
106. boundaries are now represented by a drop line above and below the curve respectively and the peak areas are automatically recalculated Drop lines on preceding or following peaks can be similarly adjusted M ovement of these drop lines can be up to but never beyond any other drop line A drop line may also not be moved beyond a point where the peak meets the baseline Identification names for peaks Double click on the peak of interest in the list A dialogue box will appear with the peak number its retention value and a text field in which you can enter a specific tag for that peak Click on OK Y ou will see the peak names in the chromatogram only if you have earlier accessed the Chromatogram Layout box pressed the Peak Label command button and chosen Peak names for labelling the peaks 8 1 6 Measuring HETP HETP height equivalent to a theoretical plate calculations allow you to check how well the column has been packed l 8 10 Perform a run with injection of a non interactive substance for example a small volume of acetone N ote that the injection must be at zero time UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Evaluating results 8 2 In the Integrate dialogue box type in the column height cm in the appropriate field area Perform the peak Integration according to your other selected parameters 3 View the results of the integration and select highlight Plate height HETP in
107. cally each time the method is run eethe first 6 characters in the method name plus a 2 digit serial number eethe date of the run in a 6 digit format determined by the country setting in OS 2 plus a 2 digit serial number eea freely specified name of up to 6 characters within the file naming restrictions in the operating system plus a 2 digit serial number If the result file directory already contains files with the same file name base the serial number is incremented automatically By default result files are stored in the home directory of the user who starts the run To change the directory where the result file will be stored press the Set path button double click on the required directory icon and press Exit UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 5 35 5 Creating and editing methods Note Theresult name may be specified as changeable in the start protocol see Section 5 6 8 In that case the specification in the Result page serves to generate the suggested result name which may be changed at the start of the run The Scouting Directory box is not relevant for UNICORN OS 5 6 8 Start protocol The start protocol determines which items of the Run setup are displayed and may in appropriate cases be altered at the start of a run O pen the StartProt page and check the items that are to be displayed O Meje eee Te CP wiew Adiser Fi jeip ka a Wf Maasi there _ Eaniuet
108. can be controlled from either remote or local stations A local station in a network can also be used asa remote station to control other systems Note Ifa run is being controlled from a remote station and a network communication error occurs the run will continue under the control of the local station Results will be saved in the FAILED directory on the local station see Section 9 3 A control mode connection can be established on the local station to control the run For a synthesis system to be accessible in the network the local station must be switched on and logged in to the network The user interface for UNICORN OS does not however need to be started on the local station System control from a remote station is managed through network level routines which are started in STARTUP CM D on the local computer UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 10 3 7 0 Network setup Local station Remote station SPE hh i ES E N afl F Liha CGR hd Leer Infertece madube i Lusi w east w Hehask cae cos weston fon fonnetion inicio ae Chromatography systems Figure 10 2 The connection interface module in a local station is started automatically and runs separately from the user interface module This allows systems connected to the local station to be controlled from a remote station without running the user interface module on the local station A local station can be used
109. ch result files within sub directories of the directory you have chosen Y ou must instead select the specific sub directory to be searched 7 18 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Presenting results 3 You may instead want to search for result files and or chromatograms with a specific name type e g all result files with the name 9604 and all chromatograms contained therein In this example by typing 9604 in the Results field and clicking on All for the chromatograms the chromatograms for all result files with the same first four characters will be located in the search within the selected directory Y ou can use standard OS 2 wildcard characters in the filename specification represents a string of alohanumeric characters and for any single character For example 9604 will display only files named 9604 9604 will display all files with names beginning 9604 9604 will display all files with names ending 9604 29604 will display only 5 character names ending in 9604 4 Click on the Search command button and a list of chromatograms will be displayed based on the designated search criteria A new search can be performed with new search criteria without erasing the first found chromatograms from the list 5 If you do not want to include a chromatogram in your list deselect it Click on OK and all selected chromatograms are transferred to the Evaluation workspace and displayed as icon
110. created and saved during an evaluation session 7 1 2 Documentation icon The full documentation of a run is stored within the result file The important documentation pages for UNICORN OS are Variables M ethod Questions N otes Evaluation procedure Sequence Result Log Book Start Protocol and Evaluation Log A few of these are described below To open the Documentation either double click on the icon or select Windows Documentation To print the contents in any of these select File Print or File Report The contents can be saved as anew method by selecting the page Method and clicking on Save as 7 2 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Presenting results Variables This displays the parameters that were used during the run et Figure 7 2 Variables page N otes This displays notes that you have made at various points during the run You are also able to enter new comments in the Evaluation Notes field ROWAN Ah Figure 7 3 Method Notes page UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 7 3 Presenting results Log book This displays exactly what happened during a run including information concerning alarms the method manual changes errors the system and the oligonucleotide sequence Selecting checking the Synthesis Data option provides information about the coupling efficiency of each base addition
111. created your method see Section 4 3 select the Variables page in Run setup W ork through the variable list adjusting the values to suit your synthesis To change a variable value simply type the required value in the field Remember that the values you enter here will be default values suggested each time the method Is run 4 6 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Creating methods from method templates 4 Figure 4 5 The Variables page in Run setup If the whole variable list does not fit on one screen a scroll bar see Figure 4 5 will be shown to the right of the list Click on the arrows at the ends of the bar to scroll one variable at a time or on the bar itself to scroll one screen at a time Y ou can also drag the slider button to scroll but this is not recommended since you can easily miss variables by scrolling too far 4G ane varrable UO ae SCreen Slider button AT OG SCrEEH Ane Ge ANAE Figure 4 6 Scroll bar functions Typical variables are illustrated with the list below taken from a method created for OligoPilot II with the fixree method template The list is organized according to the order of the blocks in the method Other method templates may vary in their structure and variables UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 4 7 4 Creating methods from method templates eeStart_parameters These variables toge
112. current setting in the Main menu You can change the display mode from the pull down menu activated by clicking with the right mouse button in the panel see Section 3 2 3 1 Enter a name for the method M ethod names may be up to 8 characters long and may contain letters A Z digits 0 9 and underscore characters T he case of letters is not significant The method name must be unique for the chosen system within the directory see steps 2 and 3 below 2 By default the method will be saved in your home directory To change the directory double click on the appropriate directory icon in the Methods panel 3 If you have more than one system connected to the PC choose the system for which the method Is intended The method can only be run on the system for which it is saved Remember that different systems may have different configurations and control capabilities 4 Choose the technique for which the method was written 5 Click on OK 4 10 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Creating methods from method templates 4 Note Themethod templates are written for standard strategies If you receive a syntax error message when the method Is saved one or more instructions in the method are Invalid These may be calls to blocks which are not defined or instructions which are invalid in your customized strategy this can also arise if a method is written for one system then saved on another I
113. d Noise window The rectangular box is allowed to be tilted with a maximum slope corresponding to the Slope limit see Figure C 2 The box is not allowed to move up above the Max baseline level Max baseline level Slope limit Figure C 2 Slope limit and Max baseline level When looking for baseline segments the box is virtually moved along the source curve in steps of 1 3 of the Shortest baseline segment A baseline segment is found whenever the currently examined part of the source curve fits completely within the box The found baseline segments are joined by connecting adjacent segments provided that the slope of the joining lines does not exceed the Slope limit C 4 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Evaluation functions and instructions C C 2 2 Selecting baseline points In the second step the baseline segments are replaced by a large number of baseline points A baseline point is placed at the start and end of each segment The line between these will also be filled with points The baseline points are shown as pale blue crosses in the Integrate Edit baseline function C 2 3 Drawing the baseline The baseline points are used to create the baseline curve using a spline interpolation The spline function ensures that the baseline curve is guided by the baseline points but the curve does not nec
114. d as either the baseline intersection or drop line to the baseline Peak endpoint Amplitude above the baseline at left A in Figure C 4 heights and right peak limits E G in Figure C 4 Fraction tube id Fraction number at peak start peak maximum and peak end Baseline height Baseline amplitude at peak start peak maximum and peak end A F and G in Figure C 4 Sigma Standard deviation for a Gaussian shaped peak For definition see below Resolution Peak resolution For definition see below Plate height HETP Height equivalent to theoretical plate and plates metre The column height must be entered in the Integrate dialogue box for this parameter to be calculated For definition see belowr Asymmetry Peak asymmetry nacatar of column packing For definition see below Sigma L Y ina Sigma o peak where Y isthe amplitude at retention sample number i on the peak Imax IS the sample number of the peak maximum A peak IS the peak area The peak width for a Gaussian peak is 4 x Sigma R esolution Vko Vg xX1 177 Wai Wn R S where Vp 1 elution volume for peak 1 Vpz2 elution volume for peak 2 Wh1 peak width at half height for peak 1 Wh2 peak width at half height for peak 2 C 8 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Evaluation functions and instructions C Asymmetry if Asymmetry width B width A where A and B are
115. d in Lflow functions Cond1Alarm Sets the alarm and warning limits for the signal from the monitor CV Sets the column volume for 1 200 calculation of CT flows and volume of Capping and Beaucage CV_Cap Column volume of capping 1 2 0 1 10 CV CapA and 12 CapB CV_Thiolat Sets the column volume for 0 1 1 0 CV thiolation Cycle_Start Marks the start point for an integration and thus where from retention is calculated DelayVol The dead volume from port1 tothe 0 1 10 ml column inlet Equivalents of amidites dependent 1 10 eq on the scale Eq OX Equivalents of oxidation dependent 1 10 eq on the scale pAlarm_Det Sets the alarm and warning limits pAlarm_ACN for the pressure on the Det and ACN pumps An alarm will set the system in Pause A warning will issue a warning message with the system in Run UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA B 5 B Strategy for Oligo Synthesis B 6 CycleStart Indicates the synthesis cycle start in order to calculate retention and used by Pause_at_Cycle_End to Pause a synthesis PeakCond1 Sets the minimum peak MinPeak Not to be regarded as a disturbance when using the instruction Watch and the limits D_ Baseline used by the instruction Watch Stable_baseline Enables disables the alarms from the pumps wit Loading mol g weight g 10 250 umol g and 0 1 100 g respectively Integrate_Start Indicates when the method can start looking fo
116. ddle button on a three button mouse to open the icon menu Click on the arrow to the right of Open and choose Settings The following settings are required Path and filename drive UNICORN BIN M INIT EXE Working directory drive UNICORN BIN Both the CONFIG SYS and UNICORN OS icon settings are set when UNICORN OSis installed Y ou should not normally need to change these settings If the settings appear to be correct and UNICORN OS still fails to start try reinstalling UNICORN OS UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA F 1 Troubleshooting F 1 3 OS 2 cannot create swap file Depending on the amount of memory in your computer UNICORN OS may require several megabytes free disk space to start If O S 2 reports that the swap file cannot be created delete unwanted files from the hard disk to create more free space About 30 M b free space Is required for the swap file UNICORN OS will issue a warning at start up if the amount of free disk space Is limited F 2 Login problems F 2 1 Unable to log into UNICORN OS Choose your username from the list and enter your password If you have forgotten your password ask the system administrator for a new one If you cannot log In using your correct username and password the USERS20 M PM filein the UNICORN SERVER FIL directory may be corrupt Restore the file from the latest back up copy or reinstall the default user see Section 11 4 2 If users are
117. defined in the method instructions organized by block Values for variables can be entered or changed here for the current run Questions are data entry fields which are filled in by the operator when the run is started Some questions may be mandatory and some may require authorized confirmation M ethod notes are displayed and start notes can be entered Information about the method being run The sequence of the oligonucleotide to be synthesized The name of the result file is displayed here This page Is always displayed and the name may be changed if this is permitted in the start protocol The last page of the start protocol has a Start button instead of Next Click on this button to start the run M ethods for most purposes can be created by adjusting the method variable values as described above The method is however actually programmed as a series of instructions which use these variables as parameters To see and or change the instructions click on the Text instruction button on the M ethod editor toolbar or choose View Text instructions from the menu To see an overview of block the structure and buffer concentration in the method choose View Windows Block and View Windows Graph respectively in the text instruction window see Section 5 2 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Creating methods from method templates 4 W ith the instruction editor you have complete facil
118. determined from the source curve The baseline segments are found by searching for all parts of the source curve which 1 arelonger than the Shortest baseline segment This parameter determines the minimum length for a part of the source curve to be considered a possible baseline segment 2 haveno point outside the Noise window The noise window is defined as a rectangular corridor parallel to the slope of the curve and centred on the first and last points within the currently inspected segment 3 slope less than the Slope limit This limits the maximum slope of the baseline to differentiate baseline segments from peaks 4 arelower than the Max baseline level Determines the highest acceptable signal level for the baseline This parameter is by default set to have no influence on the baseline calculation and is seldom necessary to adjust The parameters can be illustrated as a rectangular box in which the source curve has to fit to be identified as a baseline segment see Figure C 1 The length of the box corresponds to the Shortest baseline segment and the height of the box corresponds to the maximum level of noise on the baseline segments and is referred to as the Noise window UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA C 3 C Evaluation functions and instructions Shortest baseline segment Noise Window Figure C 1 Baseline box with Shortest baseline segment an
119. directory on the local station see Section 6 4 A control mode connection can be established on the local station to control the run For an oligonucleotide synthesis system to be accessible in the network the local station must be switched on and logged in to the network UNICORN OS user interface does not however need to be started on the local station System control from a remote station is managed through network level routines which are started in startup cmd on the local computer see Figure 2 7 Local station Remote station WOOF Uer miera Tiida Leer intertace Teeth ested s Figure 2 7 The connection interface module in a local station is started automatically and runs separately from the user interface module This allows systems connected to the local station to be controlled from a remote station without running the user interface module on the local station A local station can be used to control the oligonucleotide synthesis systems directly connected to the PC without logging in to the network M ethod and result files stored on network drives will of course not be accessible For runs performed in this stand alone mode where the result file is directed to a network drive the results will be saved in the FAILED directory on the local station see Section 6 4 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 2 13 2 UNICORN OS concepts 2 7 Security Security features in
120. down list and select the method template block that you want assigned to the selected base T he base in the Block field will now be associated with the block name that you chose Figure 5 3 In the above example the base A has been cross referenced to the block Column_wash UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Creating and editing methods 5 Y ou can select any block contained in the method template even including those that you have modified or created see Section 5 3 Repeat this process for other bases as appropriate 7 You can also reassign the cross references for the optional method steps selected in the Sequence page The blocks for column wash and final detritylation can be assigned using the pull down lists for Column wash block and Final Detriylation respectively Column wash black Column wash Final detritylatian black Final_cetritylatian a Figure 5 4 Fields for cross referencing the column wash and final detritylation blocks in a method Additionally you can click on the Purge solvent list and Purge amidite list radio buttons respectively to display the options in the Block field These options can be cross referenced to different blocks in exactly the same way as described for bases in step 5 Cima raira bet i sees TTT A Coping bee i i Pane sokan bgi JI A Pra pe makis ki e aa a Bode iin E i Cohr aat bik o hiisi E Pep a hio Ceskbyt
121. ds 2 6 UNICORN OSM ain menu interface divides user files into two categories for methods and results see Figure 2 1 Only directories to which the current user has access are shown In the M ain menu panels Files may be displayed in icon view or details view in the M ain menu panel see Chapter 3 for more details 2 2 1 Method files M ethod files contain instructions for controlling a run and are shown in the Methods panel of the M ain menu 2 2 2 Result files Result files are created by UNICORN OS when a synthesis process is run and contain ee Settings for the synthesis run method variables and questions ee Filename sequence and description for the sequence ee Efficiency values for each coupling stage Oligonucleotide synthesis runs are programmed as methods in UNICORN OS UNICORN OS is supplied with method templates which enable most synthesis processes to be performed The user needs only to insert the desired sequence into the sequence editor and then click on the associated Create button to automatically produce a ready to run method see Chapter 4 For more advanced usage it is possible to edit and create methods for specific synthesis processing needs see Chapter 5 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA UNICORN OS concepts 2 2 3 1 Method structure This section introduces the main components of a method and how they are structured In most cases it is sufficient to just
122. e Calculate baseline option with the default settings for the parameters displayed if the Baseline settings command button is pressed is the most common choice 4 Click on OK to perform the peak integration when you are satisfied with your selection Following integration the peaks in the chromatogram will be automatically labelled with their respective retention The start and end point of each peak will be marked by drop lines The peak table will be displayed underneath the active chromatogram 8 2 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Evaluating results 8 Ei oO ThE BESI i T TT s 1h Ta Ty ma Pant ALM 194 11 al i is i iir 1af Pi 257 AT 6 550 77 Asing JEEN piai s Se i i ah et Le a E AIE us Bn a S08 3 mA m Figure 8 2 The results after peak integration In addition to peak areas several other peak characteristics such as retention time and peak width are automatically calculated The characteristics displayed in the peak table may be selected in the Chromatogram Layout box see Appendix C 3 All characteristics are automatically calculated for each integrated peak when the Peak Integrate function is used although only the selected items will be displayed in the peak table C hanging peak labels Asan alternative to using retention as the peak label the peaks can be sequentially numbered or be marked with specific identif
123. e UNICORN OS Calibration dialogue that an internal Measured value is assigned Click on the Read value 2 button and then release the SET button on the pump Click on Save to save the calibration Refer to the P 6000 Pump Instruction M anual for more detailed instructions Note A value for the actual calibrated pump pressure can be obtained by multiplying the values obtained in the Measured value and Measured 1 fields UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 6 19 6 Performing a run 6 20 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Introductory material Methods and Runs Evaluation System management Appendices This page is deliberately left blank Presenting results 7 Presenting results A result file holds a complete record of a run including method system settings curve data run log and Trityl data The Evaluation module offers extensive facilities for presenting and evaluating run data All of the functions and nomenclature in the Evaluation module of UNICORN OS are the same as that used for chromatographic separations in UNICORN 2 10 Only those functions relevant to the presentation of synthesis run results is described in this chapter This chapter describes how to eeview the results from a run eepresent the chromatograms and curves of your result file eecOmpare chromatograms and curves eeprint reports 7 1 Opening a result fi
124. e is one lt strategy gt directory for each installed strategy Each lt strategy gt directory also holds the corresponding template methods if installed Strategy files are copied to the local strategy directory according to the systems installed on the local station UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA E 3 E File organisation E 4 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Troubleshooting F Troubleshooting F 1 Start up problems F 1 1 Warning signal from computer If the computer beeps at start up after the O S 2 desktop Is displayed this may indicate that you have a local system started In STARTUP CM D that cannot be initiated Either edit STARTUP CM D see Section 10 7 and reboot the computer to eliminate the system start up or define the system in Administration System in the M ain menu see Section 12 1 F 1 2 UNICORN OS fails to start Start up parameters are incorrectly set or UNICORN OS files are damaged or missing Check the O S 2 CON FIG SYS file in the root directory of the boot drive The following statements are required LIBPATH drive UNICORN DLL SET DPRADR 10 SET DPRVEC 5 DEVICE drive UNICORN DLL DPRCTL SYS where drive isthe letter of the drive where UNICORN OSIs installed Check UNICORN OS program settings Click on the icon for UNICORN OS with the right hand mouse button on a two button mouse the mi
125. e local network In general each user should have a separate home directory Important In a network installation always create home directories on a network drive which is accessible from all computers If you create a home directory on the C local drive it will only be accessible from the computer on which it was created Enter the user s password in the Password field The minimum number of characters in the password is defined when UNICORN OS is installed see Section 11 3 The program may also be UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 12 11 7 2 Administration installed without password protection The case of letters in passwords is not significant Warning Leaving the password as default can constitute a serious security risk All user passwords are visible in the User setup dialogue box For security reasons make sure that access to this function Is restricted 4 Select a home directory from the pull down list in the Home directory field Y ou may choose any directory from this list even if you created a home directory in the New user dialogue box 5 Inthe Access panel select other directories to which the user will have access from the Directories list Selecting a directory here will give the user access to all files and subdirectories therein Directories that are not selected in this list will not be visible in the methods or results panel of the M ain menu
126. e one of two policies concerning access to the FAILED directory eeGrant access to the FAILED directory to all users so that each user can retrieve his or her own result files This places the responsibility for retrieving result files and deleting old files from the FAILED directory on the individual users N ote that with this policy any user 9 2 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Security features 9 will be able to examine copy and move the other users results in the FAILED directory This policy has the advantage that the FAILED directory can conveniently be used to temporarily store methods and results from runs performed from the local station when the network is not running eeGrant access to the FAILED directory only to one or a few users who share the responsibility for retrieving results files and deleting old files from the FAILED directory This policy should be used if the installation requires restricted access to users result files N ote that the user s with access to the FAILED directory should also have access to other users home directories to be able to copy or move result files to suitable destinations In granting access to the FAILED directory it is sufficient to grant access to C UNICORN OS LOCAL FIL FAILED since this Is the path to the FAILED directory on each local station 9 4 Security recommendations for control stations Synthesis systems may be contro
127. e possible run data items can be defined for convenient switching between different presentations To define a run data layout 1 2 3 4 Select View Run data contents from the System control menu Click on New layout in the dialogue box Enter a name for the layout and click OK Select the run data items to be included in the layout and click OK To edit a layout definition select the named layout in the View run data dialogue box and change the selected run data items in the list UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 6 5 6 Performing a run To delete a layout definition select the named layout in the View run data dialogue box and click on Delete layout Y ou will be asked to confirm the delete operation Y ou can choose run data items to display without using named layouts simply be selecting or deselecting items in the list in the View run data dialogue box N ote however that this will change the definition of the currently selected layout 6 2 3 Curves Displays monitor signal values graphically Click on the button on the left of the panel or choose View Curve contents to open a list of available curves The curves in this list are those for which STORE is set to ON in the system settings see Section 13 4 together with any reference curves defined in the method Curve contents Press Ath isl Figure 6 6 The Curve contents dialogue box In the curve conten
128. eate the method Click on the Save As button in the Sequence page if the sequence should be saved for future use Enter the method variables Read the method notes Save the method Run the method es oe ee UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 4 1 4 Creating methods from method templates 4 1 Creating a new method D New method To create a new method click on the New Method toolbar button or choose the File New Method menu command in the M ethod editor or choose File New Method or New Method in the M ain menu These alternatives are equivalent W hen you choose the command from the M ain menu the M ethod editor is opened automatically TiTa TETEL Figure 4 1 The New method dialogue box l Choose the system for which the method is intended The instructions available for a given system are determined by the system strategy A method developed for one system may not be valid on another Select Any as the technique A list of ready to run method templates is displayed for the selected technique Available templates are determined by the system strategy Select one of these templates to create customized methods either by adjusting variable values see Chapter 4 or changing method instructions For your first run you are recommended to select the method template fixrec Click on a template to display information about the particular template in the Method notes field
129. eave a clear note on the computer that it should not be turned off 9 4 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Network setup 7 0 10 Network setup UNICORN OS version 1 10 can be installed in a N ovell network environment allowing synthesis systems to be controlled from any PC in the network where UNICORN OS is installed This chapter describes how to set up the network environment before installing UNICORN OS You can skip this chapter if you are running UNICORN OSon a stand alone PC not connected to a network Note Thesameversion of UNICORN OS must be installed on all computers in the network M ixing different versions will not work 10 1 Summary of network setup procedures Procedures for setting up a networked UNICORN OS environment are summarized in this section and described in more detail in the following sections 1 Set up a shared hard disk on the server M ake sure that this disk can be accessed with the same drive letter from the PCs where UNICORN OS will be installed 2 Setup the PC whereUNICORN OS isto be installed as a named pipe server Section 10 6 Define a suitable pipe server name for the PC 3 Reboot the PC log in to the network and map up to the server disc and directory where UNICORN OS network components are to be installed 4 Install UNICORN OS for network environment with the installation program for UNICORN OS Section 11 4 5 Set up automatic log in
130. echanging selected instructions in the method templates e g changing the method base eeadding blocks and instructions e g WATCH instructions eechanging method instructions to adapt to non standard system configurations eecreating new methods for applications which are not covered by the templates supplied Advanced editing facilities can be used at three different levels with the level chosen being dependent on the type and extent of method modifications to be made T hese are eemodifications at the sequence editor level i e creating custom methods by assigning a specific base in the sequence editor to a freely chosen method block see Section 5 1 eeadding new blocks to an existing method and or modifying instructions in existing blocks see Section 5 2 eewriting anew method from scratch i e selection of No Template in the New Method dialogue box see Section 5 9 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 5 1 5 Creating and editing methods 5 1 The sequence editor The sequence editor in the Sequence page of Run setup is a user interface which allows methods to be easily created The bases in a sequence are each cross referenced to a specific block in the method template with each block representing a series of instructions to be performed Thus in entering a sequence into the sequence editor and clicking on the Create button the specific blocks that are cross referenced t
131. ectory since the installation program for UNICORN OS is going to create a directory structure under it with the root name UNICORN OS see Appendix F M ap this drive for all UNICORN OS users to a common drive letter e g Q to match the manual otherwise any unused letter is fine All UNICORN OS users must have the same drive mapping letter Ideally this mapping should be done to the users log in script 10 6 Named pipes N amed pipes are a feature of N ovell N etware for OS 2 for peer to peer process communication which permit transmission of real time data over the network UNICORN OS stations are set up as named pipe servers so that results are displayed in real time even on remote stations The pipe server name identifies the station in UNICORN OS system definition see Section 12 1 N amed pipes are established with the program N ovell N etware Workstation for O S 2 Installation utility version 2 1 A brief summary of the procedure involved in setting up a named pipe server ona computer where N ovell N etware is already installed is given below Pay particular attention to step 8 Refer to the program documentation from N ovell for more details This procedure must be followed on each local and remote UNICORN OS station in the network 1 Doubleclick on the Novell icon on the O S 2 desktop and then on the Install icon in the Novell window 2 Choose the menu command Installation Requester on workstation 3 Click on OK in the Set ta
132. ed format can be applied to any new report by selecting it from the list of Report Formats Note A report format saved with the Current chromatogram does not necessarily print the chromatogram as it appears on the screen The layout curve axis etc that will be printed is the one which is found under Define If you want to print the chromatogram as it appears on the screen click on the Current layout button and then on Print Some global report formats are provided with the installation Do not delete these formats since you will then be unable to run the corresponding procedures If you want to print a number of results with the same report format create a procedure to print one result and then perform a batch run for the required results see Section 8 3 6 7 6 3 Printer set up To change your choice of destination printer for Evaluation printouts select File Printer setup and select the desired printer Within the Printer setup dialogue box you can change the job properties by clicking on the appropriate command button A new box will open which allows you to choose from the standard printer options for example paper size portrait or landscape printing form feed control Print set ups are saved on clicking OK N ote that printing from other parts within UNICORN OS is performed via the default O S 2 printer 7 6 4 Printing active chromatograms To print out the active chromatogram select File Print If the Documentation is
133. editor These may be modified to suit specific evaluation needs and be saved for later use 1 Click on Procedures Edit Open and select the desired evaluation procedure from the list The Procedure editor dialogue box will open and display the procedure steps 2 To view a description of the components and parameters of a specific instruction in the procedure select this from the listed procedure There are two main fields of interest in the Procedure Editor box namely the Instructions field and the Parameters field Proce aa Linde REJECT FEMA OFF OF OFF OFF N EID MEGATAE Peaks UF ET OOL HEIGHT 1 0 Pear JHITEGRATE 47 j Iraina hear AC FFT CHET SMO TH oF SMOOTH id henge 3 SMOOTH MECH Figure 8 18 Editing procedures in the Procedure editor dialogue box Several radio buttons e g Curve op erations Integr ation File op erations Export Chrom atogram op erations and Other are located in the Instructions field which can be individually selected to list a set of related instructions An instruction selected in a procedure is automatically selected in the Instructions field A definition of a selected instruction is shown in blue text at the bottom left hand corner of the dialogue box A list of procedure instructions with descriptions and parameters can be found in Appendix C 3 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 8 17 8 Evaluating results 1 Instructions
134. efined by the hysteresis setting above and below the warning limit This prevents repeated warnings from noisy or oscillating signals close to the warning boundary Hysteresis is only relevant for warnings since an alarm puts the system in PAUSE at the first alarm Flow rate warning If the software has calculated a flow rate that is more than the maximum capacity for a pump a warning is displayed and the maximum flow rate will be used This could mean a longer contact time than is set in the variables this is mostly relevant for larger columns UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 13 3 1 3 System settings 13 2 Specials Figure 13 3 Special settings in System control 13 3 Monitors M onitor settings determine how baseline and peaks in the monitor signal are handled when WATCH instructions are used see Section 5 8 7 as well as a number of other settings for certain monitors such as whether the local keypad on the monitor can be used Figure 13 4 Monitor settings in System control 13 4 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA System settings 1 3 13 4 Curves Curve settings determine which monitor signals will be stored as curves in the result file Check that Store is set to ON for all signals that are to be monitored Warning If a curve is set to Store OFF data from the monitor concerned cannot be displayed in the curv
135. egration C 4 3 File O perations C 4 4 Export C 4 5 Chromatogram functions C 4 6 O ther N I NMONWONPE eS 8 Pro gt rPr gt gt rP i I I I I I I I I I I I m moO WO WOWdDWOWOOOWYNO UWE I m m me HDUWWNCONODUOAWWNEE www ANNDANDAAONOON Co 5 oouv vovwv o Vov v v v v wD ANDA AND NDO Pepe UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Contents D Feedback tuning D 1 Flow rate tuning D 2 Gradient tuning E File organisation E 1 Stand alone installations E 2 Network installations E 2 1 Local and remote computers E 2 2 Network server F Troubleshooting F 1 Start up problems F 1 1 Warning signal from computer F 1 2 UNICORN OS fails to start F 1 3 O S 2 cannot create swap file F 2 Login problems F 2 1 Unable to loginto UNICORN OS F 2 2 Error message Strategy file error F 3 UNICORN OS access problems F 3 1 Unable to access certain UNICORN OS functions F 3 2 Connections are not available F 4 M ethod and run problems F 4 1 Cannot switch from control to view mode F 4 2 Monitor signals do not appear in the System control Curves panel F 4 3 Error message Couldn t create result file F 5 Evaluation problems F 5 1 Incorrect date and time F 5 2 Evaluation procedure aborts F 6 Other problems UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA D 1 D 2 D 3 m i 1 mim m m 1 1 i h e e a on Tl a Aan Aan aT Aaa
136. em settings in System control The illustration shows the Alarms group of settings ee o change settings select a setting in the text panel and make the required changes in the parameters in the instruction box The text panel will be updated when you select another setting ee o restore a setting to its default setting as defined in the system strategy select the setting in the text panel and click on Default UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 13 1 1 3 System settings When all required changes have been made click on OK Choosing Cancel will cancel all changes made since the dialogue box was last Opened Important Changes made to settings do not take effect until you click on OK to close the dialogue box Settings are divided into groups as listed in the instructions panel at the bottom of the dialogue box Click on the appropriate group button or move to the first line in the respective group in the text window Y ou can also scroll between the groups in the text window 13 1 Alarms Alarms define upper and lower alarm and warning limits for process monitor signals eelf the signal exceeds the ALARM limits a buzzer sounds and an alarm message is displayed and the process is paused i e method execution is suspended and all pumps are stopped The situation must be acknowledged and corrected before the process can be restarted eelf the signal exceeds the WARNING limits a
137. es window during a process run and will not be recorded in any way Wrfa t i D Damira 010 Paak Cis Fraai pixe QH Tie bekase ainaka 1I 4 iier Taraji i Cure Figure 13 5 System settings in System control UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 13 5 7 3 System settings 13 6 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Introductory material Methods and Runs Evaluation System management Appendices This page is deliberately left blank Technical specifications A A Technical specifications A 1 System requirements A 1 1 Hardware requirements Computer type IBM compatible PC Compaq with at least one free full length expansion slot CPU Pentium 75 M Hz or higher RAM minimum 24 Mb Hard discrecomm 16Gb Floppy disc drive 3 5 1 44Mb Printer or plotter As supported by OS 2 Warp operating system M ouse M icrosoft mouse or compatible M onitor Colour SVGA Compag Q vision The M CU for communication between UNICORN OS and the synthesis system s is supplied as part of the system s and is not included with UNICORN OS TheM CU is available for E ISA and M CA bus computers A 1 2 Software requirements IBM Operating System O S 2 Warp version 3 0 or later A 1 3 Network requirements The following are minimum network requirements for running UNICORN OSin a network installation
138. essarily pass through them The baseline will thus be a smoothly curved function passing close to or through the points To reduce the effect of noise on the peak integration the created baseline is adjusted by forcing it equal to the source curve In every position where the difference between the baseline and the source curve is small enough If the Accept negative peaks option is off the baseline will be forced down to the level of the source curve whenever the created baseline goes above the source curve C 2 4 Estimating the baseline parameters from the source curve The baseline parameters can sometimes be difficult to set Rough estimates can be found by analyzing the source curve M easuring the Shortest baseline segment using curve co ordinates If you are uncertain of the length of the Shortest baseline segment you can try to measure it directly on your chromatogram Locate the shortest segment of the curve that you consider as a part of the baseline and measure the length of the segment using the XY icon on the chromatogram Insert this value as the Shortest baseline segment value M easuring the noise level using curve co ordinates As for measuring the Shortest baseline segment curve co ordinates can be used in exactly the same manner to measure noise levels on the source curve First use the Zoom function to select a part of the curve representative of the baseline noise M easure the Y axis co ordinates using the appropriatel
139. ether several windows can be open at the same time The recommended setting with Autominimize selected reduces the active window to the corresponding icon if another module is opened reducing potential confusion on UNICORN OS desktop UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA ra Maximize ISI Restore F1 Help Help UNICORN OS concepts 2 ee rag the window border to change the size of the window eoU se the scroll bars to scroll the window contents Scroll bars are only shown when the window contents do not fit in the current window size eeEach window has a title bar which identifies the program or folder in OS 2 or module in UNICORN OS associated with the window In UNICORN OS the title bar for the System control window can be set to different colours according to the identity of the oligonucleotide synthesis system connected to the window see Section 14 1 eeAt the right hand end of the title bar are buttons for quickly adjusting the size of the window Clicking on the Maximize button enlarges the window to fill the desktop At the same time the button changes to the Restore button which restores the window to its previous size If the window is not maximized you can drag the window borders to adjust the size of the window N ote Depending on how UNICORN OS has been installed there may also be a minimize button to reduce the window to an icon eeAt the left hand end
140. ethods uses T ext instructions This involves modifying the instructions within the blocks of an existing method and or adding new blocks to a method Note Users with the appropriate access authorization have global access to methods created by all users In such circumstances an already saved method can be used as the basis for generating a new method with a different sequence This is particularly useful if for example a method was previously saved with specially modified blocks This section introduces you to the T ext Instruction editor and the following sections present the components of a method and how to edit and create blocks SI The Text instruction editor is used for entering and editing method instructions Click on the Text instructions icon on the toolbar or Text instructions choose View Text Instructions to display the Text instruction editor 5 6 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Windows Text instructions Run setup View windows Creating and editing methods 5 Matha edlir Teei FES Heap hade f m TR ie r rela on Digit went ic Liis Dd Dait poak Dot paak 1900 0E Bd Cs iim oe i hase saji Fil aa wiih De JT support 5 Sar Padese Pred CONTINUE when red Soin Pau 1 all ermal STAAT pararet r Cul Nomeal paspa phin ow ahlini Cal l a Flipa Corbrae J Adsense Eve_rvectty on TI HN Evoluote ul af Wich Hold Cher Loup
141. etup procedures 10 2 Introduction 10 3 N etwork organization Poe Q0 co co te eg ee E Gee NMONINNONORFPRP RP RP RP RRP ke FPP re BNNIF OW OO CO WY UWI UW Be erP own us Q2 90 a L WON aut gt T k m m m nd NN UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 10 4 N etwork requirements 10 5 Shared network resources 10 6 N amed pipes 10 7 Startup files 10 8 System and user definitions 11 Installation 11 1 Installation summary 11 2 System requirements 11 2 1 H ardware requirements 11 2 2 Software requirements 11 3 H ardware installation 11 4 Software installation 11 4 1 Installing UNICORN OS for the first time Contents 10 4 10 5 10 5 10 7 10 8 11 1 11 1 11 1 11 1 11 2 11 2 11 3 11 3 11 4 2 Installing selected software components after the initial installation11 9 11 4 3 Installing software upgrades 12 Administration 12 1 System definitions 12 1 1 Defining and editing systems 12 1 2 Deleting system definitions 12 1 3 Changing strategies 12 2 Access levels 12 2 1 Defining access levels 12 2 2 Access level examples 12 3 User administration 12 3 1 Defining new users 12 3 2 Changing user passwords 12 3 3 Viewing and changing user definitions 12 3 4 Deleting users 12 3 5 Defining new home directories 12 3 6 Deleting home directories 12 4 Audit trails 12 4 1 Examining audit trails 12 4 2 Renewing audit trail files 12 4 3 Backing up audit trail file
142. ewee START piirati al ETTE FA art z F PU mmp gE io Figure 5 15 Displaying and hiding block instructions Instructions are indented for each call level see illustration above General oligosynthesis instructions are listed in Appendix B 5 4 2 Adding instructions To add a new instruction 1 Inthetext instruction window display the instructions in the block where you want to add a new instruction see above 2 Select an instruction in the block where you want to add the new instruction If there are several instructions at the same breakpoint where you want to add the new instruction select the instruction immediately before the position you want to add UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 5 17 5 Creating and editing methods New instruction at breakpoint 0 TT will be added here yng Furge_cappingAB Base Volume Flow_Reag 10 00 ml min solvent Cap_aA vaste solvent Cap_B vaste Solvent ACM 3 1 vaste Flow_Reag 0 00 ml min End block Figure 5 16 Instructions added at the same breakpoint as existing instructions are inserted after the highlight N ote M ake sure that the current instruction in the text window isin the block not the call to the block 3 In the Instruction dialogue box a Set the desired breakpoint in the Breakpoint panel b Choose the instruction group and instruction in the Instructions panel Instructions are divided into
143. f the pages in Run setup are only relevant for UNICORN version 2 10 used for chromatographic separations These pages are not included in the descriptions 5 6 1 Variables The Variables page lists all variables used in the method with their default values organized in order of the method blocks Y ou can change the default values to create a variant of the method see Chapter 4 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 5 25 5 Creating and editing methods gt SSeS eee SSeS SSS Figure 5 26 The Variables page in Run setup To change the default values simply enter new values in the appropriate fields Use the scroll bar to display additional variables if the variables occupy more than one screen Click in the scroll bar to move one screen at a time or on the arrows to move one variable at a time Figure 5 27 Scroll bar functions The changed values will be displayed for the corresponding instructions in the T ext instruction window Remember to save the method with the changed variables Note The Variables box must be checked in the start protocol if you want to be able to change variable values at the start of a method see Section 5 6 8 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Creating and editing methods 5 5 6 2 Questions Questions provides a means for entering structured run specific information at the start of a run M ethod
144. file The copy must have a different name from the original Y ou cannot copy or move files between the M ethods and Results panels of the M ain menu Explicit authorization is required to copy or move files see Chapter 9 3 8 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Login and file handling 3 3 2 6 Deleting files To delete a file or directory 1 Select the item s to be deleted in the M ethods or Results panel of the M ain menu To select multiple files hold down the lt Ctrl gt key while you click on the file names or icons 2 Click with the right mouse button on any file icon and choose Delete from the pull down file menu or choose Delete from the File menu in the M ain menu bar 3 Confirm the deletion in the dialogue box Notes Home directories cannot be deleted by this method see Chapter 12 Explicit authorization is required to delete files see Chapter 12 A file that has been deleted cannot be recovered except by restoring a back up copy or using a third party utility for recovering lost files 3 2 7 Backup security To protect Important data against accidental deletion or loss in the event of hard disk failure backup copies should be taken at regular intervals TO make back up copies you can either copy files to diskette using the File Copy command in UNICORN OS choose the a folder as the target diskette for the copy operation or use O S 2 backup functions from t
145. fined in the method Note Ifa column diameter has not been defined in the method linear flow will not be able to be used 5 8 7 Conditional instructions Conditional WATCH instructions allow the progress of a run to be determined by the events during the run eg wash steps to be continued until a specific conductivity has been reached The system strategy includes a WATCH instruction for each monitor defined in the system A watch is active from the point at which it is issued until either UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 5 43 5 Creating and editing methods eethe watch condition is met eea New watch is set for the same monitor eea WATCH OFF instruction is issued for the monitor Watch conditions The conditions for which a watch can be set areas follows for most monitors Greater_than The signal exceeds a specified value Less than The signal falls below a specified value Slope greater than The rate of change of the signal exceeds a specified value expressed in monitor units minute The slope is calculated from the average signal over a number of data points between 1 and 10 set when the system is configured Slope less than The rate of change of the signal falls below a specified value expressed in monitor units minute The slope is calculated from the average signal over a number of data points between 1 and 10 set when the system is configured Less than_or valley The
146. followed and the rest ignored If it is important that instructions at the same breakpoint are not executed simultaneously e g when HOLD or PAUSE is used place the instructions at breakpoints separated by 0 1 base units see Section 5 8 2 for further details 5 4 3 Deleting instructions To remove an instruction 1 Select the instruction in the Text instruction window 2 Press Delete in the instruction dialogue box An instruction which has been deleted can only be recovered by re inserting the instruction Note You cannot delete the BASE instruction at the beginning of a block If you delete the END_ BLOCK instruction the block will end at the last instruction in the block 5 4 4 Changing instructions There are three possibilities for changing an instruction eechange the breakpoint eechange parameters including variables see Section 5 5 eeselect another instruction To change an instruction 1 Select the instruction in the Text instruction window The instruction with its current parameters will appear in the Instruction box 2 Inthelnstruction box make the required changes to the breakpoint or parameters or choose a new Instruction UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 5 19 5 Creating and editing methods 3 Press Change or Replace These buttons are equivalent unless changes are made to the breakpoint or the length of a gradient Changing breakpoints Change
147. for a connection you may be running a scouting method or a sequence T hese functions require a control mode connection in order to start subsequent cycles correctly F 4 2 Monitor signals do not appear in the System control Curves panel For monitor signals to be displayed in System control they must be set to STORE ON in system settings Signals for which STORE ON Is set can be chosen from the View Curve contents dialogue box for display in the curves panel F 4 3 Error message Couldn t create result file If you receive an error message Couldn t create result file Destination path could not be found at the end of a method the local computer was unable to access the directory specified in the result file path This may arise if the specified directory is on the network server and network communication has been lost see Section 9 3 1 The result file is saved in the FAILED directory on the local station UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Troubleshooting F 5 Evaluation problems F 5 1 Incorrect date and time The date and time recorded in the result file are taken from the PC system clock setting If these are not correct check the system clock setting F 5 2 Evaluation procedure aborts Instructions in an evaluation procedure address curves by identification number irrespective of curve names M ake sure that the curves processed when the procedure is executed are co
148. g curve Resulting curve The y axis of the Resulting curve will be a normalized First Order or Second scale without unit Order DIV Divides two curves to gain a third curve which is the First Curve source quotient of the two curves The two curve sources Second curve source must have the same y axis unit or one of them must Resulting curve have AU scale and the other Activity scale and not be fraction or injection curves The y axis of the Resulting curve will be a normalized scale without unit HISTOGRAM Creates a histogram from any non fraction curve First curve source Curve source 1 and a fraction curve Curve source Second curve source 2 frac and stores the result in the Resulting curve If Resulting curve Curve source 2 is not a fraction curve a run time error will occur The y axis of the Resulting curve will be the same as that of the first curve source C 10 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA INTEGRATE POOL_ FRACTIONS RET MUL RET SHIFT SMOOTH AR SMOOTH_MA SMOOTH MEDIAN UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Evaluation functions and instructions C Performs a mathematical integration of the _ frac curve in the Curve source and stores the result in Result curve This instruction is not the same as Peak integrate which performs a real peak integration Pools fractions from the curve in Curve source and stores the resu
149. g on the New Method toolbar button or choose the File New Method menu command in the M ethod editor or by choosing File New Method or New Method in the M ain menu These alternatives are equivalent When you choose the command from the M ain menu the M ethod editor is Opened automatically The New Method dialogue box will be displayed 2 Choose the system for which the method is intended e g OligoPilot or OligoProcess From the Technique pull down list select OligoPilot Il or OligoProcess N ext select one of the available method templates in the Template pull down list Finally choose Any in the Column pull down list 3 Click on OK once you have made your selections The method template will now be opened as an untitled method 4 Click on the Run setup button or select View Run setup Select the Sequence page 5 Click on the Cross references button to display the Cross reference list dialogue box UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 5 3 5 Creating and editing methods Figure 5 2 Cross reference list dialogue box Select the Coupling list radio button in the List field In the Block field you will see the all the bases and the block name that each is cross referenced to in the method template For example A Add_DNA_A To change the assignment of a base to a different block in the method template first select the base in the Block field N ext click on the Block pull
150. gnment W Chromatogram Name C Right Alignment W Curve Name Top Alignment Figure 7 9 Curve Name dialogue box 7 8 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Presenting results 2 Choose the components of the legend name to be displayed either Result name Chromatogram name and or Curve name 3 Choose the display alignment of the names in the active chromatogram window 4 Click on OK to return to the Chromatogram Layout box It is usually sufficient to display only the curve name if a single chromatogram Is being evaluated H owever confusion may arise when more than one chromatogram is shown so more complete names may be necessary C hanging the curve or chromatogram name Sometimes it may be desired to change the name of a chromatogram or curve To do this leave the Chromatogram Layout box and then 1 Select Edit Rename 2 Select the appropriate option to be renamed Chromatogram or Curve 3 From the panel that appears select the appropriate object for renaming and type in the new name The new name will replace the old one rather than creating a new curve or chromatogram Note Theoriginal raw data curves cannot be renamed 7 2 6 Changing the colour and style of curves By default the curves positions within a chromatogram are each represented by a default colour and line style Curves imported into the chromatogram or newly created curves are thus designated
151. grate menu evaluation 8 5 edit global evaluation procedures access 12 7 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Index edit peak table adjusting peak end and start 8 9 deleting a peak 8 8 identification tags 8 10 Integrate menu evaluation 8 3 8 7 Joining a peak 8 9 splitting a peak 8 8 editing evaluation procedures 5 33 method instructions 4 12 method notes 5 31 questions 5 30 run data layout 6 5 system definitions 12 2 systems 12 2 user profiles 12 13 variables 4 6 editing instructions overview 4 13 End button system control 6 9 END BLOCK instruction 5 12 evaluation access 12 5 evaluation overview 2 11 evaluation procedure report 8 20 evaluation procedures 8 15 batch runs 8 19 deleting 5 33 editing 5 33 editing procedures 8 17 executing automatically after a run 5 32 importing 5 33 in start protocol 5 37 menu procedures 8 21 page in run setup 5 31 Procedure Editor dialogue box 8 17 recording a procedure 8 15 renaming 5 33 reports in methods 5 31 running procedures 8 19 saving procedures 8 16 executing automatic evaluation procedures 5 32 manual instructions 6 11 exporting from evaluation 8 22 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA vii Index F FAILED directory 9 2 features of UNICORN OS 1 1 feedback tuning D 1 file display 2 6 file filter 3 5 file management 3 3 file names method
152. h gives in most cases a very accurate measurement This is the most common alternative and it is recommended that you read the information contained within Appendix C 2 which describes the principles of baseline creation eeA blank run with the same chromatographic conditions as the sample can be used as the baseline for peak integration e In addition to blank runs it is possible to select any curve present In the current chromatogram e g an edited baseline see Section 8 1 4 as baseline ee sing a Zero baseline i e no baseline subtraction at all eelf a baseline for the selected source curve already has been calculated using Integrate Calculate baseline it can be reused by selecting the Correlated baseline option This is the default alternative whenever possible UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 8 1 8 Evaluating results 8 1 2 Performing a basic integration To perform a basic integration 1 Select Integrate Peak integrate T he Integrate box will open wi Jc SE 1 Pre ed IF aai 41 Frani CH 14 SHI HA Cerna ree Ce EF ZE Couch jen T e Figure 8 1 Integrate dialogue box 2 Choose the source curve to be analyzed usually the Cond1 curve and a peak table destination in the peak table list Any one chromatogram can contain up to eight peak tables designated A H 3 Makethe appropriate Baseline selection from the above mentioned possibilities Th
153. he O S 2 Workplace Shell A tape streamer is however recommended for efficient backup handling from OS 2 If you are working in a network you can copy files to the network server make sure that the network administrator runs server back ups regularly Backup copies should be stored in a safe place physically separated from the PC The responsibility for making backup copies rests entirely with the user Amersham Biosciences cannot undertake to replace method programs lost as a result of computer failure or other incident UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 3 9 3 Login and file handling 3 3 Logging out 3 4 Quitting UNI 3 10 UN To log out of UNICORN OS press function key F12 or click on the Logout button at the bottom of UNICORN OS desktop Processes that are running when you log out will continue to run and may be left locked with a locking password or unlocked see Section 6 5 for more details Any modules that were active at the time of log out e g unsaved methods or evaluation sessions will be reopened when the same user logs in again UNICORN OS will still be open after a user has logged out and another user may log in We recommend that you always log out when you leave the computer to prevent other users from accidentally changing or deleting your files or disturbing your runs CORN OS To quit UNICORN OS and close the program choose File Quit program Or Quit Quit progr
154. he list 5 32 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Creating and editing methods 5 Importing procedures To import an evaluation procedure 1 Select the EvalProc page and click on Import 2 Choose a procedure from the Select list You can also choose to import a procedure from another method Select a method in the Methods panel to show the procedures stored in the method If you have chosen a method click on Eval Procedures to return to the complete list 3 If desired change the procedure name in the Import as field 4 Click on Import The dialogue box remains open until you click on Exit so that you can continue to add procedures from the same or different method files D eleting procedures To remove one or more procedures from the method 1 Select the EvalProc page and click on Delete 2 Select one or more procedures from the list and click on OK Note Procedures that you delete from the method are removed from the method file when you save the method Renaming procedures To rename a procedure in a method 1 Select the EvalProc page and click on Rename 2 Select a procedure from the list and change the name in the Rename item to field 3 Click on Rename The dialogue box remains open until you click on Exit so that you can rename more than one procedure without closing the dialogue box Editing procedures To edit a procedure in a method 1 Select a procedu
155. her file decompression utility UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 11 3 7 7 Installation 11 4 Before installing UNICORN OS use the O S 2 DISKCOPY command to make working copies of the distribution diskettes and store the Originals in a safe place See your O S 2 documentation for further details of how to use DISK COPY Amersham Biosciences does not undertake to replace lost original diskettes without charge Follow the procedure below to install UNICORN OS For a network installation follow this procedure on each computer in the network Y ou can quit the installation at any point by pressing function key F3 or clicking on the Exit button If you do this however the installation will be incomplete and the software cannot be used 1 For network installations log in to the network and check that you are mapped up to the server disk and directory where UNICORN OS network components are to be installed To view and change your mapping click on the Novell icon run Netware tools and select Tools Disk drives 2 Insert diskette no 1 labelled Installation in diskette station A 3 Open an OS 2 command prompt window on the OS 2 desktop Command prompts can be found by default in a Command prompt folder in the OS 2 System folder you may also have set up command prompt icons elsewhere on the O S 2 desktop 4 Enter thecommand a install 5 Select the items to install then click on OK
156. highlighted in the text window 5 3 2 Calling blocks Blocks are activated in a method with the CALL instruction A call to a block passes process control to the instructions in the block when the block either finishes or executes an END_ BLOCK instruction control returns to the point from which the call was made per Figure 5 9 Illustration of the flow of process control through method blocks UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 5 9 5 Creating and editing methods Calls may be of two types eeU nconditional calls are made with the CALL instruction Unconditional calls can be made in Normal or Restore mode depending on how the system is to behave on return from the block N ormal mode retains any settings made in the block when the block is completed so that the method continues using the settings which apply at the end of the block Use of the Restore mode is not recommended eeC onditional calls are made with a WATCH instruction which sets a watch on a particular monitor signal and transfers control to a specified block if the signal meets a given condition As long as the condition is not met the block is not activated There are different WATCH instructions for each process monitor signal and each WATCH instruction can use various conditions to respond to absolute signal values or to rate of signal change N ote that the breakpoint when the WATCH instruction is issued determines whe
157. hoose Administration Audit trail from the M ain menu Enter the required interval in the Renew every field and click on Set The new setting will take effect from the time the change is made e g if the setting is changed to 7 days at 10 a m on a M onday the file will be renewed at 10 a m every M onday Y ou can also start a new audit trail file at any time by clicking on Now in the Renew log file panel This will not affect the automatic setting e g if the audit trail is set to renew at 10 a m every M onday and you click on Now on a Friday a new file will be started immediately and another new file will be started on the following M onday morning UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 12 17 7 2 Administration 12 18 12 4 3 Backing up audit trail files Click Backup in the audit trail window to make copies of audit trail files on diskette Choose whether to copy or move the files to diskette and click on Backup The Move alternative is recommended to save disk space Backup operations are recorded in the audit trail Note The Backup command simply copies the audit trail file to diskette It does not use the O S 2 backup command 12 4 4 System run hours In the system audit trail click on Run hours to display the accumulated run time for the system i e the time the system has been in manual or run mode To reset the run hours to Zero click on Reset in the dialogue box The reset event is
158. iFCY_ detrit wash Flow ACH 0 00 rlsrmin 10 00 Message Integrate OFF Nascreen 10 00 WasteQut Waste ACh 10 00 End_block Figure 5 22 Default values for variables appear in parentheses in Text instructions eewhen the instruction is shown in the Instructions dialogue box the Var button beside the parameter field is labelled with upper case letters VAR 5 22 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Creating and editing methods 5 Figure 5 23 Parameters with variable definition are identified by upper case letters on the VAR button In this example the breakpoint is defined as a variable and the valve position is fixed All variables are also listed on the Variables page of the Run setup see Section 5 6 1 grouped according to the block in which they appear 5 5 2 Defining variables To define a new variable i e convert an existing fixed value to a variable 1 IntheT ext instruction window select the instruction where you want to define the variable The parameters for the instruction are shown in the instruction dialogue box 2 Locate the breakpoint or the required parameter in the instruction box Click on the Var button 3 Enter a name for the variable in the dialogue box and click on OK Varable name detinition Variable name Figure 5 24 The Variable name definition dialogue box Variable names may be up to 32 characters long and may contain letters
159. ialogue box and perform the next evaluation step s Stop recording by reopening the iconised dialogue box and clicking on either the Stop command button or Control Record off The new steps are added to the previous procedure Note Ifa linein the list is highlighted the new instruction will be 8 inserted after this line M ake any changes required in the evaluation procedure see Editing evaluation procedures below Select File Save or File Save as from the menu bar in the dialogue box Give the procedure a name The evaluation procedure is saved within UNICORN OS and is specific to your user name If you mark the Save as global procedure the procedure will be available for all users Such procedures are marked with Global before the name Even if the results of an evaluation session are not saved the created evaluation procedure s are saved Choose File Exit from the menu bar in the dialogue box R epeat this process from step 2 onwards if you want to create other new procedures Note You can also create new procedures by selecting either 8 16 File New Procedure Or Procedures Edit New In either case the Procedure Editor dialogue box will open and you should than press the Record button UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Evaluating results 8 8 3 2 Editing an existing procedure Evaluation operations are represented by instructions see Appendix C 4 in the procedure
160. iameter in mm Diameter used in Lflow functions 10 100 mm Sets the column volume for calculation of CT flows and volume of Capping and Beaucage Volume 1 200 ml B 9 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA B Strategy for Oligo Synthesis CV_Cap Column volume of capping 1 2 0 1 10 CV CapA and 12 CapB CV_Thiolat Sets the column volume for Volume thiolation 0 1 10 CV DelayVol The dead volume from port1 to the Volume column inlet 0 1 10 ml Concentration of amidite Eq 0 1 10 Eq OX Equivalents of oxidation dependent Eq on the scale 1 10 Int_Values Sets the level where an integration StartLevel starts and ends 2000 uS cm StopLevel 2000 uS cm A scaling factor to calibrate the flow Gain of the P50 pump 0 75 1 25 Enables disables the alarms from ACN Detrit and the pumps Reagent each Enabled Disable d Loading mol g weight g Weight 0 1 1000 g Loading 10 250 umol g CT_Thio Sets the contact time for Beaucage 0 1 10 min CT_Oxidate Sets the contact time for Oxidate 0 1 10 min CT_Capping Sets the contact time for Capping 0 1 10 min B 3 3 Monitors Settings Cond1Keys Keyboard Enabled Displayed Peak_Det Sets the alarm and warning MinPeak Peak ACN limits for the pressure on the Det 0 20 bar and ACN pumps An alarm will D_ Baseline set the system in Pause A 0 20 bar warning will issue a warning message with the system in Run B 10 UNICORN f
161. ic instructions are listed in Appendix B 8 If necessary create the necessary cross references in the Sequence page of Run setup as described in Section 5 1 9 Savethe method as written or as a method template as described in Section 5 7 10 Run the method as described in Chapter 6 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 5 49 5 Creating and editing methods 5 50 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Performing a run 6 6 Performing a run This chapter describes how to perform and monitor a run from the System control window It is recommended that for your first run you use the 20 base pair sequence already supplied with the fixree method template and which you should have saved as a method called test20 See Section 4 2 This and other methods that you have created should be present in the Methods box of the M ain M enu window 6 1 Starting a method Y ou can only start a method if the system is connected and no method is currently running You must have Run method authorization on the appropriate system to start a method Before starting a method make sure that ee he correct system is connected in control mode see Section 6 5 The name of the connected system is shown in the title bar of the System control window and the connection status is shown on the toolbar If the correct system is not connected open a disconnected System control wind
162. ication tags that can be set in the Integrate Edit Peak table function The choice of label type is made via the Tag command button in the Chromatogram Layout box N ote that the labels may be displayed vertically for each peak by deselecting the Horizontal text option Filtering peaks from view It is possible to temporarily remove peaks from view in a peak table based upon the criteria you determine In the Chromatogram Layout box click on the Filter peaks command button in the Peak tables field A box will appear which is similar to the Reject peaks function in the Peak Integrate function dialogue box Select check the criteria and parameters by which peaks will be excluded from your peak table view Y ou are able to define the minimum height width and area the maximum width as well as a specified number of the largest size peaks Click on OK If you later want to Include the peaks again you have to deselect the criteria in the Filter peaks dialogue T he difference between Filter peaks UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 8 3 8 Evaluating results and Reject peaks is that the latter function permanently excludes peaks from the integration and affects the calculation of total peak area etc 8 1 3 Optimizing peak integration If the results from the peak integration are unsatisfactory there are several possibilities to improve the results 1 Changing the parameters used in the Calculate baseli
163. ich is recorded in the log book Mode Screen and may be displayed on the Noscreen screen New _ Opens a new chromatogram icon in Chromatogram chromatogram the result file All data collected name after the instruction will be stored under the new icon until another New_chromatogram instruction is issued Places the system in the Pause Time state for the specified length of 1 infinite time 9999 9 in minutes Indicates that the next step ina process sequence may start UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA B B Strategy for Oligo Synthesis End_ Block Terminates a block and returns control to the point from which the block was called Inserts a comment in the method Comment text below the marked instruction B 2 Manual control instructions B 2 1 Pump This group contains the same instructions as the Pump group in method instructions Section B 1 1 B 2 2 Flowpath This group contains the same Instructions as the Flowpath group in method instructions Section B 1 2 with the addition of the following instruction B 2 3 Alarms amp Monitors This group contains the same Instructions as the Alarms amp M on group in method instructions Section B 1 3 in addition to the following Instruction Description Parameters Efficiency_ Can be used to set the watch on Threshold efficiency from off to on Pause at_ This sets the system to Pause at the Cycle_End
164. ick on the connection mode button to establish a control mode connection you must supply the locking password or your login password if you have Unlock locked systems authorization View mode System is A user has left the system in an available unlocked state Click on the connection mode button to establish a control mode connection In all modes you can choose System Disconnect to disconnect the system from the control window UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 6 15 6 Performing a run Control mode 6 16 6 5 3 Leaving and locking a system A running or idle system with a control mode connection can be left and locked by clicking on the control mode button or selecting System Leave system When the system is left the connection becomes a view mode connection After leaving and locking scouting or sequence runs it is not possible to establish a control mode connection from another computer Leave control of SYSTEM Leave system C Unlocked Locked Username default Cancel Help Figure 6 15 The dialogue box for leaving a system Y ou may leave the system unlocked or locked eeUnlocked leaves the system unlocked Any other user may establish a control mode connection to the system Use this option if you do not intend to use the system in the near future eeLocked locks the system with the password specified in the dialogue box A control mode c
165. ick on Operations Multiply and select the curve to be multiplied Select check the appropriate axis for multiplication either Multiply retention and or Multiply amplitude Insert the appropriate multiplication factor and click on OK 7 26 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Presenting results Tage doman parm 1 Di Sebi M41 _Cordi IHHT Preset 360414 Pressel 03 3604 IDE Frari 039 SSD Pt Frera 04 9604 Foe Gon HSM Cos igs T Candia RAUL Figure 7 28 Multiply dialogue box 7 4 4 Mirror images of curves A very useful way of comparing the features of two curves is to produce a mirror image of one curve To achieve this 1 Select Operations Multiply 2 Select the desired curve to be mirrored and select Multiply amplitude in the Multiply type field 3 Typeina multiplication integer of 1 and click on OK 4 Shift the mirror image curve downwards for better presentation See Alternative B above N ow the mirror image of the original curve will be displayed in the active window Select deselect for the other curves wanted in the active chromatogram window in the Chromatogram Layout box UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 7 27 Presenting results TEE TT oT a i f i l Aiii Te a oh 1036 a rid Figure 7 29 Two curves displayed in mirror image 7 5 Saving results Any changes to chromatog
166. ilea parga ahn ree Baak Khasi Pungo_sehesvis mii Figure 5 5 Cross reference list of the purge solvent blocks available in a method 7 Click on Exit to implement the new cross references 8 Enter the appropriate sequence in the sequence editor field of the Sequence page Create a method as described in Section 4 2 9 Savethe method see Section 4 6 Users with the appropriate access authorization have global access to methods created by all users In such circumstances an already UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 5 5 5 Creating and editing methods saved method can be used as the basis for generating a new method with a different sequence This is particularly useful if for example a method was previously saved with a specially modified cross reference list In such circumstances open the appropriate method containing modified cross reference list and access the Sequence page in Run setup Delete the existing sequence in the sequence editor and enter the new sequence Click on the Create button Note Do not open a saved sequence from your personal sequence list in the Sequence page otherwise the cross reference list corresponding to the saved sequence will replace the modified cross reference list present in the current method Always manually enter your sequence over the original method sequence 5 2 Text instructions editor The next level of advanced editing to create new m
167. in startup cmd Section 10 7 6 Reboot the PC and log in to the network 7 Start UNICORN OS asauser with administration authorization and set up system definitions and user profiles see Chapter 12 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 10 1 7 0 Network setup 10 2 Introduction Establishing and maintaining a networked UNICORN OS installation requires some understanding of the working of N ovell networks and the use of network administration functions W e recommend that the network is maintained by a competent network administrator who will also be involved in the installation of UNICORN OS software The network administrator is not necessarily the same person as the system administrator for UNICORN OS Once the network is set up network functions are entirely transparent to UNICORN OS users and the network administrator does not need to understand the use of UNICORN OS for controlling synthesis systems Security is very important in a networked UNICORN OS installation Three aspects of security can be distinguished and the responsibility for maintaining security is shared by the network administrator and the system administrator for UNICORN OS N etwork administrator UNICORN OS administrator Data storage Back up routines for Controlled user access to security back server and local disks home and shared directories up routines e g placing all home directories on a shared disk t
168. ing to one of eename alphabetical order eetype directories and files eesize Smallest files first eecreation date oldest files first eelast saved date least recently modified files first To change the sorting order choose Sort from the pull down panel menu or from the File menu and choose the appropriate sorting order from the sub menu Changing the sorting order affects only the currently active panel Filter To restrict the files displayed according to file name or the system with which they are associated choose Filter from the pull down panel menu or from the File menu M ark the system s for which you want to display files and enter a file name specification if required Click on OK to activate the filter The filter affects the display in both panels UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 3 5 3 Login and file handling 3 6 Filter Systems Oligo Pilot EJ EJ Mame View all Figure 3 5 The Filter dialogue box Y ou can use standard O S 2 wildcard characters in the file name specification stands for any number of characters for any single character For example test will display only files named test test will display all files with names beginning with test test will display all files with names ending with test tet will display only 5 character names ending with test If a filter is active this is indicated in the title bar of the panel e g
169. into the method Similarly for the following base C this is cross referenced to the block named Add_DNA_C which is also copied into the method Note that each block contains a self contained set of instructions for the complete coupling cycle of the specific base 5 2 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA il New method Run setup Cross references Creating and editing methods 5 5 1 1 Modifying cross references in the sequence editor Cross referencing a base to a method template block lends much flexibility to modifying the method creation process UNICORN OS allows the user to change the block that is cross referenced to any specific base type For example you may decide that the DNA sequence 5 ACTGGT 3 should have a column wash step after the addition of each base in the sequence By cross referencing a base not used in the current sequence e g A to the column wash block in the method template this can be directly incorporated into the sequence to signal a column wash T hus mean that a thiolated DNA A base will be added to the oligonucleotide but rather a column wash procedure is performed after the addition of the preceding base Alternatively you may modify the instructions in an existing block or create a new block It is thus a straightforward task to assign the new block to the base in question To change the assignment do the following 1 Create anew method by clickin
170. ion 1 10 User M anual 18 1118 52 Edition AA Performing a run 6 For methods which are used frequently it may be convenient to define the methods as commands In the File menu To do this choose File Menu and select the required method The method name will appear as a command in the File menu and choosing the command will start the method 6 1 3 Start protocol M ethods should be defined with a start protocol which will be displayed before the method actually starts Work through the start protocol answering questions as required As each screen is completed click on Next to move to the next screen or Prev to return to the previous screen The last screen has a Start button to start the method At any stage click on Cancel to abort the method start Y ou are recommended to include the following start protocol items in the display see Section 5 6 8 for more details Method Variables Prerun Questions Notes Method Information Oligo Synthesis sequence Result Name Changeable All the variables defined in the method instructions organized by block Values for variables can be entered or changed here for the current run Questions are data entry fields which are filled in by the operator when the run is started Some questions may be mandatory and some may require authorized confirmation M ethod notes are displayed and start notes can be entered Information about the method being run The sequence of oligon
171. ions 5 37 notes 5 37 page in run setup 5 36 questions 5 37 result name 5 38 system settings 5 37 variables 5 36 starting UNICORN OS 3 1 starting a run from system control 6 2 from the main menu 6 1 overview 4 11 startup notes 5 37 problems F 1 start up files 10 7 status indicator in system control 6 9 store setting for curves 13 5 strategy 2 1 12 3 changing in system definition 12 4 strategy for Oligo Synthesis B 1 style colour Chromatogram Layout box 7 9 swap file F 2 system in administration 12 2 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA XVII Index system administrator 12 7 responsibilities 12 1 system audit trails 12 16 system available 6 10 system connections see connections see connections system control 2 10 6 1 alarms and warnings 6 12 auto scaling 6 7 control bar 6 9 curves panel 6 7 flow scheme 6 8 logbook 6 8 panel techniques 6 4 run data panel 6 5 status Indicator 6 9 system settings 13 1 toolbar buttons 6 9 system definition 12 2 autosave 12 4 changing strategy 12 4 deleting 12 4 pipe server name 12 4 strategy 12 3 system name 12 3 title bar colour 12 3 system fonts 11 3 system name 12 3 system run hours 12 18 system settings 13 1 access 12 6 alarms 13 2 curves 13 5 monitors 13 4 warnings 13 2 system setup access 12 5 system strategy 2 1 1 tag Chromatogram Layout box 8 3 templa
172. is organized into 13 chapters and 6 appendices Introductory material 1 Introduction 2 UNICORN OS concepts Login and file handling Methods and runs Creating methods from method templates Creating and editing methods 3 4 5 6 Performing a run 7 8 9 Evaluation Presenting results Evaluating results System management Security features 10 N etwork setup 11 Installation 12 Administration 13 System settings Appendices A Technical specifications B Strategy for Oligo Synthesis C Evaluation functions and instructions D Feedback tuning E File Organization F Troubleshooting UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Preface Assumptions Two broad assumptions are made in this manual 1 You should be familiar with the oligonucleotide synthesis systems in your installation Refer to the appropriate system user manuals for details 2 You should be familiar with the general principles of using IBM s O S 2 operating system on your PC Although UNICORN OSisa self contained program package and does not require any direct interaction by the user with O S 2 the user interface principles follow the conventions set by the OS 2 operating environment Typographical conventions M enu commands and dialogue box prompts are Identified in the text by a Helvetica typeface A colon separates menu levels thus File Open refers to the Open command in the File menu A typewr
173. ite coupling etc Blocks are called either from M AIN the top level of the method or from other blocks using CALL instructions unconditional or WATCH instructions conditional see Section 5 8 7 Calling a block executes the instructions in the block A single method can contain up to 250 blocks Use text instruction mode to view and edit blocks 5 3 1 Viewing blocks In the block window The organization of blocks in the method is shown graphically in the block window in Text instruction mode Figure 5 8 The Method editor block window 5 8 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Creating and editing methods 5 Each block is represented by a grey line with the block name and the length of the block The line is shifted down to indicate calls to other blocks In the example above the blocks are called in sequence from the M ain block at breakpoint 0 Blocks to which there is no valid call are not shown in this window Conditional WATCH instructions are indicated by a green line showing the start and duration of the watch In the T ext instruction window In the text instruction window each block is represented by a CALL or WATCH instruction in blue text Double click on the blue instruction to show the instructions in the called block see Section 5 4 1 If you click on the line representing a block in the block window the first instruction in the block will be
174. iter like typeface is used for instructions as they appear in the text editor for methods and evaluation procedures T hese are normally entered automatically by UNICORN OS II UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Contents Contents Introductory material 1 Introduction 2 UNICORN OS concepts 2 1 UNICORN OS user interface 2 1 1 UNICORN OS control software 2 1 2 Stand alone and network installations 2 1 3 Software modules 2 1 4 O S 2 terminology 2 1 5 On line help 2 2 Files and directories 2 2 1 Method files 2 2 2 Result files 2 3 M ethods 2 3 1 M ethod structure 2 4 System control 2 4 1 Control facilities 2 4 2 System connections 2 5 Evaluation 2 6 N etwork considerations 2 7 Security 3 Login and file handling 3 1 Logging in 3 2 UNICORN OS desktop 3 2 1 Icons and windows 3 2 2 UNICORN OS Main menu panels 3 2 3 Presenting files 3 2 4 Finding files 3 2 5 Copying moving and renaming files 3 2 6 Deleting files 3 2 7 Backup security 3 9 3 3 Logging out 3 10 3 4 Quitting UNICORN OS 3 10 N ond I I nk h NNNNNNNNNNWN I I I a I PNF EF OONDADADOUABRNNE oy a p h ww ww wy COM BWN KN ws O Methods and Runs 4 Creating methods from method templates 4 4 1 Creating a new method 4 4 2 Saving and running a test sequence method 4 4 3 Creating a sequence and method 4 4 4 Editing method variables 4 UNICORN for Oligo Synthesis version 1 10 User
175. ities for designing and editing your own customized methods Y ou will also use the instruction editor for refining and modifying methods based on the standard templates e g eeC hanging the method base column volume volume or time eeC hanging method specifications if the templates do not suit your system configuration eeA dding or removing variables eeA dding conditional events such as starting peak integration when a certain peak level is detected eeA dding or removing instructions to change the method functionality eeA dding or removing blocks to change the method structure To gain an understanding of how method templates are built up and can be modified work through Chapter 5 which gives a full description of method editing facilities UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 4 13 4 Creating methods from method templates 4 14 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Creating and editing methods 5 5 Creating and editing methods This chapter describes the complete facilities for creating and editing methods in UNICORN OS Refer to Section 2 3 for an overview of method concepts For many applications suitable methods can be created by changing the default variable values in one of the templates supplied with UNICORN OS see Section 4 4 N ew methods can also be created using the more advanced editing facilities These can be used for e
176. ition AA 6 7 6 Performing a run 6 2 4 Flow scheme The flow scheme is a graphical representation of the oligonucleotide synthesis system During a run the flow scheme displays open flow path s in colour and monitor signals with numerical displays The flow scheme thus shows the current status of the run at a glance T E E TEE Oat J chanot pE bezierhe Bachiri AE sirus i ta eh dal tata hth TYF Tere hee ia f T ak oji Ba a Tia E a ae mre HH 1 2 be riri A A m Feii G Pe E a i a ee HF JERAT H Figure 6 9 The flow scheme for a run 6 2 5 Logbook All actions including method start and end method instructions and manual interventions such as Pause or H old and unexpected conditions such as warnings and alarms are logged for every run with date time and current username where appropriate the date and time are taken from the system clock in the PC The logbook thus provides complete history of any given run including trityl data which is a measure of the coupling efficiencies The log can contain up to 12000 lines and is saved in the result file Figure 6 10 The logbook panel for a running method 6 8 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Performing a run 6 6 3 Manual control 6 3 1 The control bar The control bar at the top of the System control window contains a set of buttons for starting and stopping the run accessing run notes and locking
177. ive chromatogram window in the available workspace area click on the Maximize button in the top right corner of the window aim re EHTO 01 Alternatively select Windows Tile This option is particularly useful when you want to simultaneously view all of the opened chromatograms in the maximum available space Selection of Windows Cascade will stack all of the active windows like a deck of cards 7 6 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Presenting results 7 2 2 The Chromatogram Layout dialogue box M ost of the changes that you are likely to make regarding chromatogram presentation are made in the Chromatogram Layout dialogue box This is opened in one of two ways either click on the command button containing a black cross in the top left corner of the active chromatogram or select Edit Chromatogram Layout Chraratogram Laut gt z ae HT g a QO BAH Heri oo eee iHa Figure 7 7 Chromatogram Layout dialogue box The basic operation of the Chromatogram layout box is described in the following sections 7 2 3 Selecting a layout If there is a pre defined layout that you want to apply to the chromatogram select it from the Apply layout pull down list If the same layout is to be applied to all chromatograms on the Evaluation workspace select check the Apply to all option Click on OK to apply the layout To create pre defined layouts see Section 7 2 9 7 2 4 Cho
178. k installation an oligonucleotide synthesis system can be controlled from any computer in the network provided that the user has sufficient access rights in UNICORN OS UNICORN OS software has to be installed but not necessarily running on the computer to which the system is physically connected The computer has to be logged in to the network eeA system which is controlled by another user through a control mode connection can be viewed through a view mode connection by any user with sufficient access on any number of computers in the network This allows runs to be monitored from multiple display stations although only the active connection can control the system eeA system which Is locked by a user can be unlocked on any computer in the network by any user with sufficient access rights see above eeA UNICORN OS user may login to UNICORN OS on any number of computers in the network Each successive multiple instance of the user automatically establishes the same System control connections as the first instance when the login is performed M ultiple instances are however treated by UNICORN OS as separate users although they are not distinguished in the System control window display and only one of the instances may maintain a control mode connection to a system M ultiple instances may also disconnect and connect their System control windows independently of each other once the login is performed UNICORN for Oligo Synthesis version 1 1
179. ks in the method templates unless otherwise stated in the specific block is column volume To use absolute volume or time as the method base change the Base instruction in the Main block using the text instruction editor See Chapter 5 for a description of how to do this 4 8 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Text instructions Windows Run setup Creating methods from method templates 4 Y ou can get a graphical view of the length of the blocks in a method as follows 1 Click on the Text instructions button on the toolbar 2 Click on the Windows button on the toolbar and select only Graph The method is now displayed graphically 3 Click on the Scale button in the graphical display to select a base for the graphical display Changing the display base will not affect the base in the method 4 To return to the Variables page in Run setup click on the Run setup button on the toolbar 4 5 Method notes Print ol Maximize notes Click on the Notes thumb tab in the run set up to show the Notes page and read through the method notes Y ou can maximize each section in the notes page to fit more of the text on one screen Click on the printer icon or choose File Print to print the method notes The method notes provided with each template describe the important information about the template and if relevant how the system should be connected for the method to work correc
180. l 18 1118 52 Edition AA Contents 6 Performing a run 6 1 6 1 Starting a method 6 1 6 1 1 Starting from the M ain menu 6 1 6 1 2 Starting from System control 6 2 6 1 3 Start protocol 6 3 6 2 M onitoring a run 6 4 6 2 1 General panel techniques 6 4 6 2 2 Run data 6 5 6 2 3 Curves 6 6 6 2 4 Flow scheme 6 8 6 2 5 Logbook 6 8 6 3 M anual control 6 9 6 3 1 The control bar 6 9 6 3 2 M anual instructions 6 11 6 3 3 Alarms and warnings 6 12 6 4 If communication fails 6 13 6 4 1 Network failure 6 13 6 4 2 Local station failure 6 13 6 5 M anaging system connections 6 13 6 5 1 Establishing a connection 6 13 6 5 2 Connection modes 6 15 6 5 3 Leaving and locking a system 6 16 6 5 4 Disconnecting a system 6 16 6 5 5 Network considerations 6 17 6 6 Calibrating monitors 6 18 Evaluation 7 Presenting results 7 1 7 1 O pening a result file 7 1 7 1 1 Chromatogram 7 2 7 1 2 Documentation icon 7 2 7 1 3 Temporary icon 7 5 7 2 Basic presentation of chromatograms 7 5 7 2 1 Viewing chromatograms 7 5 7 2 2 The Chromatogram Layout dialogue box 7 7 7 2 3 Selecting a layout 7 7 7 2 4 Choosing the curve s you want to see 7 7 7 2 5 Curve and chromatogram names 7 8 7 2 6 Changing the colour and style of curves 7 9 7 2 7 Changing and fixing the axes 7 10 7 2 8 Viewing information about the run 7 11 7 2 9 Saving a layout 7 12 7 3 Additional presentation possibilities 7 12 7 3 1 Showing part of a curve 7 13 7 3 2 Entering text in the chromatogram 7 16 7 4 Comp
181. ld 4 Click on OK The templates for each system are common for all users Be restrictive in saving methods as templates We recommend that only methods that are useful for all users are saved as templates 5 7 3 Deleting a template Y ou can delete templates if you have Save templates and global evaluation procedures authorization 1 Choose File Delete template UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 5 39 5 Creating and editing methods 2 Select the system and the template to delete and click on OK Note Thetemplates for each system are common for all users Be restrictive in deleting templates 5 8 How to use selected method instructions This section provides recommendations for how to use some common programming features in UNICORN OS methods 5 8 1 BASE and METHODBASE instructions Every method block must start with a BASE instruction defining the base for calculating breakpoints Note Do not confuse the concept of a method base instruction with the bases in a sequence The method base may be volume ml or depending on the scale defined in the system strategy time min column volume CV defined as a numerical value or taken from the column definition For all blocks other than the main block the base may also be defined as SameA sM ain which means that the block will inherit the base defined in the main block Different blocks may use different bases Use the method b
182. le To open a result file double click on a result file icon in the M ain menu Alternatively click on the Evaluation icon on the M ain menu workspace and then select File Open in the Evaluation module Select the desired result file from the panel that appears All contents of a result file are transferred to the Evaluation workspace By default the first chromatogram in a run is shown as an opened active window Any other chromatograms are represented by icons on the workspace O ther icons are username Temporary and Documentation Eva tion Shi m4 Fie Fail imegae Operaiang Precedurc Windows F lapale F S ae i I Sond Coa Tod Pree leet 44144 Figure 7 1 Evaluation module view UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 7 1 Presenting results Icons can be neatly arranged on the workspace by selecting Windows Arrange icons Icons representing the various components of a result file are as follows 96041704 Chromatogram Documentation Temporary default Temporary Documentation 7 1 1 Chromatogram Curves that have been created during arun such as the conductivity and pressure of the Detrit and ACN pumps are stored together in a chromatogram The original raw data curves cannot be deleted or modified although they can be used as the basis for evaluation procedures and subsequent creation of new curves A chromatogram also contains the curves
183. le Click on the Fixed option 3 Enter the desired minimum and maximum values for the y axis If you click on All with this unit other curves that have the same y axis units as the current curve will be similarly scaled Click on OK Note All with this unit will only be applied to existing curves It will not be applied to new curves created after this function was last used N ew curves are automatically scaled x axis 1 Select the appropriate method base from the Base field in the Ee Chromatogram Layout box Time of retention Volume or Column Q Time volume may be selected T he default setting is Volume After i 7 certain operations e g integration the base cannot be changed 2 Click on the Axis command button For the x axis click on the Fixed option Type in the desired minimum and maximum values for the x axis Click on OK 7 2 8 Viewing information about the run you may wish to display header information at the top of a chromatogram detailing the Variables Questions and or N otes H eader information cannot be displayed for imported chromatograms 1 Inthe Chromatogram Layout box click on Header 2 A box is displayed that allows you to select any of the four header items Variables Scouting variables Questions and or Notes Click on OK Note Scouting variables are not relevant for oligonucleotide synthesis methods UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 7 11 Presen
184. ll be the same and will occur at the same retention point The curve to be normalized is automatically moved along the x axis and stretched along the y axis 4 When all operations have been performed click on OK to save the new normalized curve O pen the Chromatogram Layout box to select the curves you want to see UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 7 25 Presenting results 5 Repeat the procedure for other curves you want to stack or stretch Alternative B M oving a curve using the Shift function If more precise positioning of curves is required then Operations Shift should be used Use of this function is logged in the evaluation log 1 Select Operations Shift and then the curve to be shifted in the displayed dialogue box 2 Select the axis along which the shift will be made i e along the x axis Shift retention or y axis Shift amplitude Enter the shift value Click on OK 1 M 5041741 _ Conall UA GE Ti Preece Cet Sa _ ee Dri 07 9004 17040 Presao ct mAH ee 4 IAHI T Dard Da a Eeri Cunag nee Condi ai SHFT Figure 7 27 Shift dialogue box Alternative C Stretching and shrinking a curve using Multiply Curves can be stretched or shrunk in the x or y plane using the Multiply function This function is similar to Normalize Size except that each curve is repositioned with precise numbers instead of by eye and the is instruction logged in the evaluation log Cl
185. lled without running the user interface modules the UNICORN OS application program on the OS 2 desktop In a network installation this situation can arise if a system is controlled from a remote station without starting UNICORN OSon the local station In a stand alone installation the situation can arise if a user quits UNICORN OS after starting a run In both cases it is not apparent from the O S 2 desktop that UNICORN OS control software is actually running and there is therefore a risk that O S 2 may be shut down and the computer turned off in the belief that it is not in use To prevent this ee Do not quit UNICORN OS if you are controlling a system ee Do not turn off local station computers in a network installation ee f possible start UNICORN OS application program on all local stations In a network installation and establish a view mode connection as an indication that a connected system might be running A similar risk arises if UNICORN OS desktop Is minimized in installations where this is permitted since it is not immediately apparent on the OS 2 desktop that the icon Is hatched indicating that the application may be running For this reason UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 9 3 9 Security features ee Do not permit UNICORN OS application to be minimized on local computers in a network installation ee f you minimize the application in a stand alone installation l
186. lows you to specify which chromatograms and or chromatogram components that are to be included in the report 1 Click on the radio button for Chromatogram 2 Makea selection from the item list Current chromatogram The chromatogram that is currently active on the screen All chromatograms All chromatograms on the evaluation desktop including those that are shown as icons 1 2 etc An individually named chromatogram Report contents Select tems 4 Header Q gt Curent chromatogram All chromatograms 4 Method Q gt 2 _j Documentation C gt _ Evaluation log Q gt 4 Chromatogram gt Synthesis data Q m Chromatogram layout Figure 7 31 Selection of chromatogram content in the Generate Report dialogue box 3 Ifyou want the chromatogram s to be printed out as they currently appear on the screen click on the Current button The layout will be the present layout for each chromatogram on the evaluation desktop 7 30 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Presenting results If you want the chromatogram s to be printed out with a different layout as they currently appear on the screen click on the Define button This opens the Chromatogram Layout box and enables changes to be made to the layout N ote that the changes made here within the Chromatogram Layout box are only with respect to the report format and not the layout of the chromatograms displayed
187. lt in the Resulting curve The fractions are pooled from the first selected fraction to the last selected fraction If Curve source is not a fraction curve or First or Last is not an existing identification a run time error will occur Multiplies the retention of the curve in Curve source by the Multiplication factor and stores the result in the Resulting curve Shifts the retention of the curve in Curve source by the Shift factor and stores the result in the Resulting curve Smooths Curve source with an autoregressive filter and stores the result in Resulting curve The Filter parameter decides the strength of the filter Smooths the curve in Curve source with a moving average filter and stores the result in Resulting Curve The Filter width parameter decides how many samples wide the filter is Smooths the curve in Curve source with a median filter and stores the result in Resulting curve The Filter width parameter decides how many samples wide the filter is Subtracts two curves to gain a third curve which is the difference of the two curves The two curve sources must have the same y axis unit and not be fraction or injection curves Divides two curves to gain a third curve which is the quotient of the two curves The two curve sources must have the same y axis unit or one of them must have the AU scale and the other the Activity scale and not be a fraction or injection curves The threshold values are used to avoid division of
188. m or in the peak table 2 Click on Join left or Join right if you want the peak to be joined with the peak to its left or right respectively T he original intervening drop line will now have been taken away and the peaks all renumbered Adjusting peak start and end points The beginning of each peak is marked with a drop line above the curve and the end of each peak is marked with a drop line below the curve a Hiz McA i Hirem i inil Figure 8 9 A drop line at the start and end of a peak W here there are two peaks beside one another the end of the first peak will be at the same point as the beginning of the next peak Thus there will be a drop line below and above the line respectively at the same point Poa ond ee ar Figure 8 10 A drop line between two peaks It is possible to move the drop lines for a selected peak and thus affect the area beneath the peak UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 8 9 8 Evaluating results Click on the peak of interest on the curve or in the peak table and two vertical cursor bars will become superimposed on the left and right drop lines that delimit the selected peak Figure 8 11 Active bars delimiting left and right drop line for a peak Drag the left and right drop line bars to define the new left and right limits respectively for the selected peak The drop lines can never be moved beyond any other drop line The new left and right
189. m the company included in the conditions of delivery is valid only if the product has been installed and used according to the instructions supplied by Amersham Biosciences AB Preface Preface About this manual This manual provides reference to UNICORN for Oligo Synthesis version 1 10 referred to as UNICORN OS from Amersham Biosciences AB UNICORN OS is a complete package for control and supervision of oligonucleotide synthesis systems suitable for use with Amersham Biosciences O ligoPilot II and OligoProcess All functionality associated with UNICORN OS consists of software which runs on an IBM compatible PC under the O S 2 operating environment and hardware for interfacing the controlling PC to the synthesis module The control platform and functions within UNICORN OS are essentially the same as for UNICORN version 2 10 the latter developed by Amersham Biosciences for chromatographic separations UNICORN OS is supplied with system strategies and templates for oligonucleotide synthesis although it is possible to perform chromatographic separations associated with UNICORN by obtaining the appropriate strategies and templates from Amersham Biosciences In the writing of this manual those functions that are directly relevant for UNICORN OS are described Functions that are more relevant to chromatographic separations such as Scouting BufferPrep and some evaluation functions are not discussed in this manual This manual
190. mber for the synthesis system on the local computer see Chapter 11 If you only have one system physically connected to the local computer this number should be l 6 Enter a value in the Autosave interval field if you want UNICORN OS to save a copy of the result file at preset intervals during a run This minimizes loss of data in the event of a computer failure The recommended interval for most systems is 5 minutes A shorter interval may slow down the user interface response The control functionsin UNICORN OS performance will however not be impaired Note Normally you should define systems before defining users If you add system definitions after you have defined users remember to grant access to the new systems to the appropriate users see Section 12 3 12 1 2 Deleting system definitions To delete a system definition select the definition in the System s list and click on Delete A system definition can only be deleted if the system is idle and no user has a control mode connection to the system 12 1 3 Changing strategies Y ou can change the strategy assigned to a system provided that the system is idle and no user has a control mode connection to the system Choose Administration System check the system in the System dialogue box and click on Edit Choose the required strategy and click on OK Remember that the arrangement of tubing pumps columns etc may need to be changed on the synthesis system if you change the s
191. me dialogue box to repeat this step for as many other curves you want from the same or different chromatograms When you have copied all desired curves click on Exit 3 Open the target chromatogram and its Chromatogram Layout box Select the curves that you want to view Curves can be scaled individually or all with the same scale using the All with this unit function see Section 7 2 7 Figure 7 22 Edit Copy curve dialogue box Alternative B Importing curves using O pen related Result files contained in the same directory can be automatically searched to locate all curves of a specified type for example all Cond1 curves UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 7 21 Presenting results 1 Select File Open related Curves Select the search criteria for the directory result chromatogram and curve name using the respective Browse command buttons Alternatively click on the respective All button to include all result files chromatograms and or curves Wildcard characters and can also be used to specify the search parameters see Section 7 4 1 under Alternative A TEDA IFE i Coad MAL OS i Coed 7 TH IFE JE TEITE Figure 7 23 File Open related curves dialogue box 2 Click on Search and a list of found curves will be displayed based on the designated search criteria A new search can be performed with new search criteria without erasing the chromatogram
192. measured at 10 of the peak height HETP an HETP L N N 5 54 Vp wp 17 puss 4 3 where habi F SN N no of theoretical plates L bed height in cm Vp peak elution volume or time Wh peak width at half height expressed in the same units as Vp UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA C 9 C Evaluation functions and instructions C 4 Evaluation procedure instructions C 4 1 Curve operations Instruction Description Parameters Adds two curves to gain a third curve which is the sum First curve source of the two curves The two curve sources must have Second curve source the same y axis unit and not be fraction or injection Resulting curve curves or else a run time error will occur AMP_MUL Multiplies the amplitude of the curve in Curve source Curve source by the multiplication factor and stores the result in the Resulting curve Resulting curve Multiplication factor AMP_SHIFT Shifts the amplitude of the curve in Curve source by Curve source the shift factor and stores the result in the Resulting Resulting curve curve Shift factor the computer Resulting curve CUT Cuts out the part of the curve in Curve source Curve source between Left and Right Limits and stores the result in Resulting curve the Resulting curve Left limit Right limit DERIVATE Differentiates the curve in Curve source first or Curve source second order and stores the result in Resultin
193. member that the synthesis always proceeds in the reverse direction i e 3 5 direction By default a DNA sequence consisting of oxidated bases taken from the standard reagent positions is created This is evident by the default selected radio buttons ONA 9 5 Modified DJRNA 0 Standard Figure 4 3 Radio buttons in the Sequence page used for choosing the base type to be included in the sequence By changing the radio button combination you can freely change the component bases contained within the sequence Y ou are able to select DNA or RNA whether the base is oxidated or thiolated and whether the base is taken from the standard or modified reagent position There are two extra physical reagent positions in OligoPilot II labelled X and Y The extra characters Z and Q are also provided The available combinations are as follows 4 4 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Creating methods from method templates 4 Radio button combination Bases as represented on the screen DNA O xidated Standard A A Shi seca ICG eo G T X Y Z Q DNA S thiolated Standard DNA S thiolated Modified A C G T RNA O xidated Standard a C 9 U X y Z q RNA O xidated Modified a c g u RNA S thiolated Standard a C O U X Y Z q RNA S thiolated Modified a c g u Group 5 Click on the Group button if you want the sequence to be displa
194. method to align curves with respect to the x axis or the y axis is to use Operations Normalize To select the curve to be moved within a chromatogram 1 Select Operations Normalize A panel will appear 2 Select the curve to be normalized and a reference curve to be normalized against For example if you want to stack curves select the curve at the bottom of the stack to be normalized against and the curve to be moved as normalized Click on OK 7 24 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Presenting results Figure 7 26 Normalize chromatogram window 3 The Normalize window is displayed A box surrounds the curve selected to be normalized Y ou can now use the following functions Move Allows the selected curve to be moved to any position on the chromatogram Axes are automatically re scaled to accommodate the new position This function is useful for stacking curves Click on Move and then move the curve with the mouse pointer Click on the mouse button when the curve is in the correct position Size Allows the selected curve to be stretched along its y axis or x axis Click on Size and then drag the box along either one of its axes This is useful for example to stretch a curve along Its y axis for comparison of different gradient lengths Normalize The curve to be normalized will be adjusted to the reference curve Thus the height of the highest peak on both curves wi
195. ministration User setup in the M ain menu F 3 2 Connections are not available Check the connection between the PC and the synthesis system Check that the power to the synthesis system is turned on If the connection appears to be correct and the power is turned on switch off the synthesis system and quit UNICORN OS Shut down and switch off the computer then restart the entire system If a system is not available when you attempt to establish a connection check that you have access rights to the system Access rights are not automatically assigned for a newly defined system If you receive the error message Cannot connect to system in a network installation check eethat the local computer to which the system is connected is turned on and logged in to the network eethat the computer from which you are trying to establish a connection is logged in to the network eethat the limit of 8 connections to the system has not been exceeded If you can establish a connection but cannot control the system the manual menu commands In System control are grey check that no other user has a control mode connection and that you have sufficient access rights to control the system manually UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA F 3 Troubleshooting F 4 Method and run problems F 4 F 4 1 Cannot switch from control to view mode If you are unable to switch from control mode to view mode
196. mode connections to one system on different computers This allows a running process to be monitored from several locations at the same time The actual control of running processes is handled by the local computer A user may disconnect a control mode connection without affecting the running process The Evaluation module provides extensive facilities for presentation and evaluation of synthesis results Essential features of evaluation include ee rityl data This is stored in the result file and can be printed in a report as a table eeC urve manipulation A wide range of operations can be performed on curves such as addition and subtraction of two curves differentiation normalization and scaling The original raw data curves are always kept unmodified in the result file eeC urve comparisons Curves from different result files can easily be compared in the Evaluation module eeE valuation procedures O perations performed in the Evaluation module can be recorded as an evaluation procedure and repeated for other result files with a single menu command Evaluation procedures may be executed either automatically on completion of a method run or interactively from within the Evaluation module UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 2 11 2 UNICORN OS concepts eeR eports Comprehensive reports of the evaluation results can be generated for hard copy documentation of the synthesis process Genera
197. mpatible with those processed when it was recorded An evaluation procedure aborts if you try to store resulting curves at the position of an original raw data curve F 6 Other problems If system errors arise during a run these are displayed in a dialogue box with a description of the error If you do not understand the error description or are unable to correct the error situation contact technical service Note Some information warning and error displays arise from OS 2 not from UNICORN OS If you have installed a different language version of OS 2 these displays will bein the O S 2 language UNICORN OS displays will however still be in English UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA F 5 Troubleshooting F 6 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Index Index gt access levels 12 5 security 2 14 9 1 to FAILED directory 9 3 12 12 to systems 12 12 access level administration 12 1 Access levels Administration menu main menu 12 5 ActionIfPC Fail 6 13 actions for watch instructions 5 47 adding alternative answers for multiple choice questions 5 29 evaluation procedures 5 33 method instructions 5 17 methods to the system control file menu 6 3 procedures to evaluation file menu 8 21 questions 5 29 administration 12 1 User setup 12 10 advanced method editing 5 1 air sensor conditions 5 45 alarms 6 12 alarms 13 2 arr
198. n the System control window during arun Accumulated Block Instruction time vol time vol Main 0 00 Call Normal Purge_G 0 00 Base Volume 1 00 End_block 0 00 Call Normal Purge_C 0 00 Base Volume 1 00 End_block 0 00 Call Normal Purge_A 0 00 Base Volume WNNNNFFREF OOO FPOoOoOocoOoOrRFrOOOrF OO O 1 00 End_block Table illustrating the relationship between accumulated and block time volume for a simplified method fragment Depending on how conditional calls are used see Section 5 8 8 the overall method time or volume may vary according to watch events during the run 5 8 4 Messages Use messages to inform the operator of the progress of the run It isa good idea to issue messages at critical points in the method The example block below instructs the operator to fill the column with DNA T support Main 0 00 Base CV 6 30 Column_volume ml Any 0 00 Message Fill your column with DNA T support Screen 0 00 Message Press CONTINUE when ready Screen 0 00 M essages which are set to Screen will be displayed on the screen during a run and will remain there until acknowledged by the operator M essages can also be set to N oscreen these will be recorded in the run log but not displayed on the screen UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Creating and editing methods 5 5 8 5 Pausing a method A method can be programmed to pause at critical points There are
199. n the watch begins not when the block is activated The block will in fact never be activated if the watch condition is not met during the run Once set a watch remains active until the condition is met or a new WATCH instruction is issued for the same monitor The watch is cancelled automatically when the condition is met A watch can also be turned off with the WATCH_OFF instruction See Section 5 8 7 for more details of WATCH instructions 5 3 3 Adding blocks To add a new block to a method make sure the editor is in Text instruction mode then click on the New block toolbar button or choose New block File New Block from the menu 5 10 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Creating and editing methods 5 New Block Volume Column volume x Cancel Hep Figure 5 10 The New block dialogue box Block name Enter a name for the block in the Name field Block names may be up to 30 characters long and may contain letters A Z digits 0 9 and underscore characters Use underscore characters instead of spaces If you want to separate words in a block name e g Add DNA W This field may not be left blank Block names must be unique within the method T he case of letters is retained but is not significant the names Add DNA _W and add _dna_w are treated as identical Base Choose a base for the block If you choose SameAsMain the new block will inherit the base from the M
200. n will be listed in the Select curves field 4 Select the desired curve and click on the Select command button The selected curve will now be displayed in the Selected curves field 5 If you want to choose curves from the same or other chromatograms repeat steps 3 4 for each curve When all the desired curves have been selected click on OK 6 Doubleclick on the icon for the new chromatogram and access the Chromatogram Layout box Select the curves that you want to view Curves can be scaled individually or all with the same scale using the All with this unit function see Section 7 2 7 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 7 23 Presenting results 7 4 3 Stacking and stretching curves W hen two or more curves from different runs are displayed on the same chromatogram it is often difficult to visualize the differences between them due to the high degree of overlapping This makes it difficult to align the curves and to make comparisons between individual features Several tools are available to overcome such problems namely normalizing curves shifting curves and stretching curves The operations presented below require the curves to be present in one chromatogram see Section 7 4 2 Figure 7 25 Stacking curves Alternative A Stacking and stretching curves using the normalize function The simplest
201. ne instruction can help to optimize the integration result The baseline parameters can be altered in two possible locations i in the dialogue box which appears after selection of Integrate Calculate baseline or ii by selection of the Baseline settings command button in the resulting dialogue box from Integrate Peak integrate T hese are the same function except that in the latter case the baseline is immediately used in an peak integration 2 Itis possible to exclude peaks from integration based upon criteria you determine Click on the Reject peaks command button in the Integrate Peak integrate dialogue In the box that appears select check the criteria and parameters by which peaks will be excluded from the integration Y ou are able to define the minimum height width and area the maximum width as well as a specified number of the largest size peaks The default criteria is to include only the 20 largest peaks Aeject Peaks _ Height less than 0 000000 _ Width less than 0 000000 _ Area less than 0 000000 _ Width more than 0 000000 Peak must be one of 20 largest Cancel Help Figure 8 3 Reject Peaks dialogue box 3 To select only a part of a curve for integration click on the Peak window command button in the Integrate Peak integrate dialogue box A chromatogram will open containing the curve and two vertical cursor lines These lines can be dragged to define a region between them that will be anal
202. nection to the system where the method is to be run See Section 6 5 for details Y ou cannot start a method without a control mode connection to the appropriate system 2 Choose File Run from the System control window for the required connection and select the method to run Alternatively select the method in the methods panel of the M ain menu Do not double click on the method icon in the M ain menu this will open the M ethod editor 3 Click on Run This will display the first page of the start protocol UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 4 11 4 Creating methods from method templates 4 6 Method Variables Prerun Questions Notes Method Information Oligo synthesis sequence Result name changeable 4 8 Editing text instructions e Text instructions 4 12 Change the method variable values if required such as loading weight CV column volume etc The suggested values are those saved in the method Any changes you make will apply only for the current run and will be recorded in the run documentation Go through the rest of the start protocol entering information where appropriate Use the Next and Previous buttons to move through the start protocol If you click on Cancel on any page in the start protocol the method will remain loaded in the System control module but will not start Start protocol pages for most method templates are AIl the variables
203. next cycle start Active only one time B 2 4 Other Next_ Jump to the next breakpoint in the breakpoint current method only relevant when a Record_on Begins recording a run that has been started manually A result file will be generated method or block is running a B 8 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Strategy for Oligo Synthesis B B 3 System settings instructions B 3 1 Alarms Settings Cond1Error Enables disables all alarms Enabled Disabled Sets the alarm and warning limits for the signal from the respective monitor Cond1Alarm Efficiency __ Threshold pAlarm_Det pAlarm_ACN Sets the threshold for an acceptable coupling efficiency An efficiency below this threshold will pause the synthesis Sets the alarm and warning limits for the pressure on the Det and ACN pumps An alarm will set the system in Pause A warning will issue a warning message with the system in Run B 3 2 Specials Settings Instruction Description Parameters Tetrazole Percent tetrazole of the column volume High alarm 0 99900 uS cm Low alarm 0 99900 uS cm High warn 0 99900 uS cm Low warn 0 99900 uS cm Hysteresis 0 99900 uS cm Enabled Disabled Threshold 0 100 Enabled Disabled High alarm 0 20 bar Low alarm 0 20 bar High warn 0 20 bar Low warn 0 20 bar Hysteresis 0 5 bar Enabled Disabled Conc 1 100 CV AmiditeConc Conc 0 01 0 5M ColDiam Sets the column d
204. nfirm Unlock authorization 5 27 connect system 6 15 connecting a system 6 14 connection interface module 10 4 connection security 2 14 9 1 connections 2 11 6 13 disconnecting 6 15 6 16 establishing 6 14 from a remote station 6 14 modes 6 10 6 15 multiple 6 14 status 6 10 Cont button system control 6 9 control network installations A 2 stand alone installations A 2 control bar 6 9 control mode connections 2 11 control unit number 12 4 controlled system 6 10 copy to clipboard 8 24 copy chromatogram 7 21 copy curve 7 21 copying blocks within a method 5 15 copying files 3 8 access 12 6 to diskette 3 8 create button functions 4 6 create curve 8 13 creating blocks in a method 5 10 curves in evaluation 8 13 method instructions 5 17 methods 4 2 5 3 variables 5 23 creating a method 4 4 creating a new method from scratch 5 47 modifying text instructions 5 6 no template 5 48 sequence editor 5 2 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Index creating a sequence 4 4 cross reference list modifying 5 3 current method instruction 5 17 curve evaluation copying 7 21 curve window layout in system control 6 6 curves changing the axes 7 10 Chromatogram Layout box 7 7 creating a curve 8 13 cutting 7 14 deleting 7 28 differentiation 8 12 mirror images 7 27 normalizing 7 24 presentation 7 5 resizing with multiply 7 26 shifting 7 26
205. ng algorithms C 1 1 Moving Average For each data point in the source curve the processed target curve Is calculated as the average of the data points within a window centred on the source data point The width of the window is determined by the parameter value expressed as number of data points W hen the source point is less than half the window size from the beginning or the end of the curve the average Is calculated symmetrically round the source point over as many data points as possible Increasing the window width increases the smoothing effect The filter algorithm only accepts odd integer parameter values between l and 51 If an even number has been given it is incremented by one C 1 2 Autoregressive The first data point in the source curve is copied to the processed target curve For each subsequent data point the previous processed target point is multiplied with the parameter value and added to the current source data point The result is then divided by the parameter value plus 1 according to the following formulae UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA C 1 C Evaluation functions and instructions h S t p t _ s p 1 where t current processed target point t previous processed target point S current source point p smoothing parameter value Increasing the parameter value increases the smoothing effect T he filter algorithm accepts integer parameter
206. nistration To define edit or delete users choose Administration User setup from User setup the M ain menu To use this menu command you must have User Access levels setup Levels authorization see above All user administration is Audit trail Performed from this dialogue box System Hew Password arnald Home directory Access Directores EJ System s E teval OE Figure 12 7 The User set up dialogue box Access level 12 10 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Administration 7 2 12 3 1 Defining new users l To define a new user click on New and enter the username in the dialogue box The username can be up to 15 characters long and can contain any combination of letters digits or other printable characters Hew User W Create home directory Home directory name malone Drive Cancel Figure 12 8 Dialogue box for defining a new user To create a new home directory for this user check the Create home directory box and enter the directory name and drive in the appropriate fields If you choose to create a home directory ona network drive make sure that the drive is always accessible Click on OK to create the user and return to the User setup dialogue box Note A homedirectory can always be created on a network drive even if UNICORN OS is not installed for network control The computer only need to be connected and logged in to th
207. nistration concerns authorization of access to the system and should at least in larger installations be the responsibility of one person or a small group These activities are described in the present chapter In a network installation maintenance of the network functions will normally be carried out by the computer staff responsible for the company s network Aspects of network administration relevant to UNICORN OS are considered in Chapter 10 After installation the following operations should be performed in UNICORN OS by the administrator before other users can use the program 1 Set up system definitions for the synthesis systems Section 12 1 2 Define access levels for the installation Section 12 2 3 Define new users with home directories and access profiles Section 12 3 Note These operations can be performed on any station in a network installation It is however important that the administrator is logged in to the network on the station being used so that the changes will apply globally throughout the network UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 12 1 7 2 Administration 12 1 System definitions Administration User setup Access levels Audit trail system 12 2 System definitions set up the synthesis systems which are connected directly to the local computer in UNICORN OS installation This must be done for each new system installed In a net
208. ntScreen gt key on the keyboard 7 4 2 Overlaying curves Curves from different runs can be overlaid in one chromatogram Curves can be copied into one chromatogram either from other chromatograms that are present on the Evaluation workspace or be imported from other result files using File Open related or File Open Figure 7 21 Comparison of different curves 7 20 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Presenting results Alternative A C opying curves into one chromatogram For effective comparison of curves it is suitable to transfer all relevant curves to a single chromatogram This can be achieved by creating a new chromatogram using File New Chromatogram and copying curves into it from other chromatograms present on the Evaluation workspace Alternatively you can copy an existing chromatogram using Edit Copy Chromatogram and import more curves into it or else copy curves into the Temporary chromatogram see Section 7 1 3 You can create the desired curves perform evaluations in the Temporary chromatograms and transfer the final curves to other destination chromatograms The unwanted contents remaining in the Temporary chromatogram can then be removed using Edit Clear temporary chromatogram 1 Select Edit Copy Curve 2 Select the source chromatogram and the curve of interest Select the target chromatogram Click on the Copy command button to effect the copy Stay within the sa
209. ntegration 1 Select Integrate Edit baseline 2 Choose the desired baseline from the panel that appears and click on OK A window will appear displaying the baseline and the UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 8 5 8 Evaluating results curve from which it was calculated Additionally blue crosses are displayed the baseline points and their co ordinates in the Point list SHUG La 13 Cer aa rd 73 faii y nd A BAE a ag 0 000 fo J TETE nw AMT Pn L_ amp oYr ULE LEJ LL 78 L17 0 000 Ba 6e7 0 000 i f L L75 UDG E E iT 646 0 000 0 16 14 155 Ci Cad Ca Ci iLD 66E 0 wim f IL sd Lis Di Lee lz BE Ci Cal Ca oe mn jan 795 Col Cai ie 136 Hg D Cay Ca Ce 142 214 D Go 14 724 D Ce Tre j iG cod D Caj fed H iii 162 742 E mmg lit 52 Ci wan irt THE D wiw a jm ari hin eee a Dewa Cine E ea ee Dure cgi reipi E o me Ijzer ma Ok Caan Hisi Figure 8 5 Edit Baseline chromatogram window Insertion of baseline data points Select the Point mode radio button Click on the left mouse button to place the point on the chromatogram Each new point is represented by a cross and its co ordinates are automatically entered into the Point list This is useful when for example you want the baseline to go up to a high valley between two peaks Use the right mouse button if you want to use the zoom function to magnify a region of the chromat
210. number of home directories in the system In network installations home directories may be created on the server disk as well as on local disks method H olds method files sequence directories and user created method directories result H olds result files scouting result directories and user created result directories failed H olds result files in the event of network communication failure strategy H olds subdirectories for each system physically connected to the local computer system0 System information for systems physically system1 connected to the local computer H olds a copy of system2 the strategy flow scheme and templates and files system3 for the system settings and system audit trail Directory systemo corresponds to installed System 1 Directories for systems that are not installed are empty UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA File organisation E server H olds the original file for system definitions system tab This file is copied to the local directory fil H olds the original files for user definitions users20 mpm and global procedures globproc gpl global report formats globproc grf The user definition file is copied to the local fil directory strategy H olds the directory structure for installed strategies lt strategy gt H olds the original files for installed strategies Ther
211. nvalid instructions are marked in red in text instruction mode in the M ethod editor see Section 5 4 1 and must be deleted or replaced before the method can berun T The method remains open in the M ethod editor when it has been saved so that you can continue editing if you wish If you edit any of the Save method instructions or change the method variables you can save these changes by selecting File Save or clicking on the Save button in the toolbar If you want to save a copy of the method under a new name choose File Save As and enter the details as described above If you close an open method or the M ethod editor module and there are unsaved changes you will be prompted to save these changes 4 7 Starting a run This section briefly summarizes how to start a run with a method The method must be named and saved before it can be started See Chapter 6 for more details of how to run a method Note Ifyou are editing the method in the M ethod editor and have made changes that you have not yet saved these changes will not apply during the run Similarly if you edit the method while it is running the run will not be affected It is the version of the method which is saved on disk at the time when the method is started that controls the run Y ou are able change the method variables in the Start Protocol at the beginning of a run but these will only be valid for the current run and will not be saved 1 Establish a control mode con
212. o prevent data from being scattered throughout the network N etwork M aintenance of user access security passwords and access rights to shared resources UNICORN M aintenance of user profiles OS access see Chapter 12 security 10 3 Network organization Figure 10 1 illustrates how a networked UNICORN OS installation can be organized There are two kinds of PC where UNICORN OS software is installed eeA local station isa PC to which synthesis systems are physically connected UNICORN OS software must be installed on all local stations in the network 10 2 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Network setup 1 0 eeA remote station is a PC to which no systems are physically connected but which can control systems over a network link UNICORN OS software is installed with the Remote Only option on remote stations see Section 11 4 Local station a E lamigi Hermae Grr be atatia stabon amp io i Network sener Renee Local ation ig station 1 seriami a Figure 10 1 A network installation with 3 synthesis systems and 5 stations 3 remote and 2 local The systems can be controlled from any computer except the network server The network server requires N ovell N etware version 4 02 or higher The server in a Novell network is a dedicated server and cannot be set up as either a local or a remote UNICORN OS station Synthesis systems in the network
213. o the bases are copied from the method template into the method The method must be saved before it can be then used to run an instrument For example in a sequence containing the bases 5 CA 3 theDNA base A is assigned to a specific block in the method template called Add_DNA_A Consequently by clicking on the Create button in the Sequence page the A base in the sequence causes the block Add_DNA _A to be copied into the method The next base in the sequence DNA base C is assigned to the block name Add_DNA _C Thus the block cross referenced to base C will next be added to the method see Figure 5 1 In the standard method templates each block that is cross referenced to a base is a Self contained set of instructions to perform a complete coupling cycle of one base i e detritylation detrit wash coupling oxidation or thiolation and capping see Figure 5 1 Ca gore eos o p Gal Homaat OHA A Se SS OELE aa i es 2 WNI Emsa lime mat Call Hor a Caio Hit Cal Hormel Dart wash OIL Deal Homia Or mic ace Dibi i oli Cal ford Deion na OT si Homa o aspnr UEU bal Loms r Pls l e wdd DEA al eri s DIL Dal Hour a Diii ii Call horna Cari aai OT Lal Moma Coda ende _ OA L Cal Homa L i 2 TEFA I Lal Mnre a re Figure 5 1 The relationship between the bases entered into the sequence editor and the blocks copied into the method In the example the sequence base A is cross referenced to the block name Add_DNA_A which is then copied
214. ode From preview mode press Edit to return to question editing mode Inserting a question To insert a new question after an existing question 1 Click on the existing question in the questions list 2 Enter the text status type and answer for the new question as required The Answer panel depends on the type of question eelnput field questions Enter a default answer if required eeM ultiple choice questions Click on the input field under Alternatives enter the answer and click on Add Delete R epeat this procedure to add other alternatives N ew alternatives are always added at the end of the list To remove an alternative mark the alternative in the scroll list and click on Add Delete ee V alue questions Enter maximum and minimum limits Check the Integer box to if the question is to accept only whole numbers as answers 3 Click on Insert to add the new question to the list If the list is empty the Insert operation creates the first question in the list UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 5 29 5 Creating and editing methods Editing an existing question To change the definition of an existing question select the question to be changed Change the text status type and answer as required and click on Replace D eleting a question To remove a question select the question and click on Delete To remove all questions click on Delete All 5 6 3 Method notes N otes
215. of 10 calls allowed in a method There is no limit for calls outside a loop Press OK to add the new block Strategy for creating blocks For blocks which are to be called unconditionally it is usually easiest to create the CALL instruction at the same time as you create the block by selecting another block name in the From field in the New block dialogue box UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Block Hew Import block as Rename block Delete block Creating and editing methods 5 For blocks which are to be called conditionally with a WATCH instruction do not create a CALL instruction at the same time as you create the block Leave the From field empty in the New block dialogue box When you later insert the WATCH instruction in the method the new block name will appear in the list of possible actions for the watch See Section 5 4 2 for details of inserting instructions 5 3 4 Deleting blocks To delete blocks from a method choose File Delete Blocks Delete blocks Select the blocks to delete Figure 5 11 The Delete blocks dialogue box Select one or more blocks to delete and press OK then confirm or abandon the deletion Double clicking on a block name is equivalent to selecting the block and pressing OK If you delete a block by mistake you can restore the block by importing from the saved version of the method see Section 5 3 6 provided that you have no
216. of the title bar on OS 2 windows is a button for window control functions This button is not used in UNICORN OS windows ee he Launchpad is located on the OS 2 workspace and allows applications to be launched with a single click of the mouse button It is possible to link the UNICORN OS icon to the Launchpad eeA pplication program windows including UNICORN OS havea menu bar with menu commands see Figure 2 1 Click on a menu item or press lt Alt gt followed by the underlined letter in the item to open the menu Click on a command in the menu or press the underlined letter in the command to activate the command 2 1 5 On line help A comprehensive on line help utility is included in UNICORN OS software Choosing Help from the menu or pressing function key F1 provides an entry to the help texts for the currently active module Contents Search and Index functions help the user to find information for all modules regardless of which module is currently active M ost dialogue boxes have a Help button for help on how to use the currently open dialogue box UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 2 5 2 UNICORN OS concepts In the instructions boxes for method Instructions procedure instructions and system settings pressing F1 when an instruction is highlighted will display an information box with short help on the function and usage of the selected instruction 2 2 Files and directories 2 0 Metho
217. ogram Selecting Zoom mode switches the mouse buttons for the two functions i e the left mouse button controls the zoom function and the right mouse button is used to enter points Before Figure 8 6 Baseline before and after editing UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Evaluating results 8 D eleting baseline data points If you want to delete a data point from the Point List select the appropriate point in the Point list and press the Delete button Alternatively double click on the unwanted data point entry in the list to delete it Delete all removes all baseline data points D rawing the new baseline When you are satisfied with your baseline point selection click on the Draw Baseline command button The new baseline curve will be drawn as a spline function based on the previous and the new points The spline function is guided by the points but does not necessarily pass through them Y ou may also force a straight baseline between two points by selecting the first of the two points in the point list and then clicking on the Draw straight to next point command button Click on OK and the new baseline will be saved with the default name Edited Baseline This may now be used as the baseline in a new peak integration 8 1 5 Adjusting the peak boundaries Once a peak table has been generated using the appropriate baseline it is now possible to split or join peaks and to manually adjus
218. on Pra cena W Weti annia Harwrerce Ceran jaa Bey a mmm Capes j Y Frun haa of Pama roms ct eapi Cokes BErdawa Bong iiad proiscal aiai mo ei Lari Halip Figure 5 37 The StartProt page in Run setup O ptions that are most relevant for UNICORN OS areas follows M ethod information If this box is checked the method information including creator target system and date and time of creation and latest change will be displayed at the start of the run Y ou cannot edit the method information M ethod variables If this box is checked values for method variables will be displayed and can be changed at the start of the run T hese values will override the default values for the particular run and will be recorded in the result 5 36 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Creating and editing methods 5 file The defaults stored in the method are however not affected Variables defined for scouting cannot be changed in the Variables page If the Variables box is not checked the run will be executed with default values for all variables as defined in the method M ethod Displays the method instructions Double click on a blue Call instruction to display or hide the instructions in the called block M ethod instructions cannot be changed from this display Prerun Q uestions If this box is checked questions defined in the method will be displayed at the start of the run
219. onnection can only be established by providing the correct password N ote that this password is independent of the user s login password A locked system can also be unlocked with the log in password for a user with Unlock locked systems authorization This authorization should be restricted to a small number of users to prevent indiscriminate unlocking of locked systems 6 5 4 Disconnecting a system To disconnect a oligonucleotide synthesis system from a System control window choose System Disconnect If you are disconnecting a control mode connection you will be asked to leave the system first Logging out or quitting UNICORN OS automatically disconnects all connected systems displaying the Leave control dialogue box for each UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Performing a run 6 system Systems which are disconnected in this way will be reconnected automatically when you login to UNICORN OS again Note You may however have disconnected from a control mode connection but establish a view mode connection on re connect if another user has taken control of the system in the meantime Note You can disconnect a system during a run and the run will continue It is not recommended to do this without locking the system since this can leave a run on the system with no responsible user You cannot however disconnect from scouting or sequence runs 6 5 5 Network considerations In a networ
220. or Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Strategy for Oligo Synthesis B PeakCond1 Sets the minimum peak MinPeak MinPeak 0 99900 uS cm Not to be regarded as a D_ Baseline disturbance when using the 0 99900 uS cm instruction Watch and the limits D_ Baseline used by the instruction Watch Stable_baseline B 3 4 Curve Configuration PressDet tor storing inthe Resultfile The the frequency with which curve data samples Tisrecorded 05 0008 B 3 5 Method variables in alphabetical order Column diameter for linear flow rates Column_Volume Column volume for text method Conc_of DNA _ Sets the DNA amidite concentration amidite Conc_of RNA _ Sets the RNA amidite concentration amidite CT Oxidation DNA Sets the oxidation contact time CT Oxidation RNA Sets the oxidation contact time V CT_Thio Sets the Beaucage reagent thiolation contact time CT_Capping V C Sets the capping reagent contact time Column volume for calculation of functions in the strategy CV_CT Capping C Sets the number of column volumes that the CT_Capping will last UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA B 11 B Strategy for Oligo Synthesis CV_CT Ox DNA CV Sets the number of column volumes that the oxidation will last CV_ CT Ox RNA CV Sets the number of column volumes that the oxidation will last CV_Capping CV Sets the number of column volumes of Capping reagent CV
221. orded sequence of interactive operations in the Evaluation module which can be executed for automated data evaluation and report generation It can be used for single chromatograms and for a number of chromatograms in different result files The concept is analogous to the macro facility provided in many word processing and other programs Evaluation procedures can also be called from methods making run execution evaluation and documentation fully automatic Automation is achieved using the Procedures menu 8 3 1 Recording a procedure F 2 3 O pen the appropriate results file in the Evaluation module Record a procedure by clicking on Procedures Record on Perform the evaluation steps that the procedure is to contain These steps are recorded as you perform them W hen you have finished the appropriate procedures click on Procedures Record off T he Procedure editor dialogue box will open and lists the steps in the procedures you have just created UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 8 15 8 Evaluating results Py ucehoes mi paimi Fie Dorini uar El ABJECT PEs OFF OFF OF DFF S ERY gi VS dF j HEGATES FE ACS DFF SET DILUA HE HT FEAR _WWTEGASTE 47 Figure 8 17 Procedure editor dialogue box It is possible to add subsequent steps to the listed procedure by clicking on the Rec command button or else select Control Record in the dialogue box menu Iconise the d
222. osing the curve s you want to see In the Curves field in the Chromatogram Layout box is a list of all curves contained within the chromatogram numbered from 01 onwards By default a chromatogram is displayed with all curves presented and hence all of the curves in the list are highlighted Select only those curves you want to see in the chromatogram UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 1 1 Presenting results J2 Se Freia Ja S014 Freesat J WHI Conde as 16 ifs 16 19 Ii ka Figure 7 8 Curves field in the Chromatogram Layout dialogue box Click on OK to return to the active chromatogram window 7 2 5 Curve and chromatogram names By default each curve legend consists of three components namely the result name chromatogram name and curve name see Figure 7 6 For example a curve with the name 96041704 1_Cond1 is derived from the result named 96041704 The chromatogram name is a number automatically given during the run e g 1 The curve name corresponds to the curve type e g Cond1 is the conductivity If two or more curves of the same type were created within a result file they are numbered sequentially e g Cond1 Cond2 etc C hanging the legend name view If you do not want to display the full legend name in the dialogue boxes and chromatogram windows 1 Click on the Curve Names command button in the Chromatogram Layout box Curve Name J Left Ali
223. other result files into an already opened result file namely File Open related or File Open The former option is most useful in searching for many chromatograms In a specific directory based on defined selection criteria The latter option is best used to import any individual chromatograms from result files in different directories The imported chromatograms will be sequentially numbered for identification purposes Up to 10 chromatograms can be made available at the same time on the evaluation workspace UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 7 17 Presenting results She tA agi l ahidi 13 0 3h Lid 14 Peed HOLT Oa L Cori 1 TU lS Od 14 _ Ferenn lirt w04 7045 l Fret JALE L1 Eoad l MeL LL LI PEGG LT JEL LID 13 Coad L JEH LT O44 13_Premelet ay HU nie im fie m E b Liia bi B 4 Figure 7 18 Windows Tile function to display many chromatograms To print a picture like this click on the Evaluation title bar at the top of the window and then press the lt PrintScreen gt key on the keyboard Alternative A Import chromatograms using O pen Related 1 Click on File Open related and select Chromatograms Figure 7 19 File Open related chromatograms dialogue box 2 Select the directory and or result file to be searched using the Browse options A single asterisk indicates that All has been selected N ote that you cannot sear
224. ow or disconnect the system from an occupied window and choose System Connect If you cannot establish a control mode connection because the system is controlled or locked by another user you cannot start a method on the system ee he system monitors are correctly calibrated see Section 6 6 Calibrating system monitors requires Calibrate tune authorization in the user profile see Section 12 2 6 1 1 Starting from the Main menu Y ou start a method from the M ain menu by selecting the method in the M ethods panel and choosing Run from the pull down file menu or clicking on the Run button UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 6 1 6 Performing a run testi oligapilot Figure 6 1 Starting a method from the Main menu Do not double click on the method as this will open the M ethod editor with the method loaded 6 1 2 Starting from System control Y ou can also start a method from the System control window Double click on the System control icon at the bottom of the screen If you have more than one system installed make sure you open the System control window for the correct system From the System control window choose File Run Double click on the method icon in the resulting dialogue box or select the icon and click on Run C D esti ETHE wa oe en cower nee Figure 6 2 Starting a method from the System control menu 6 2 UNICORN for Oligo Synthesis vers
225. ow rate is determined by CV and CV_Thio Starts the flow with acetonitrile from Pump P6000 to the column or waste Starts the flow with detrit solution from Pump P6000 to the column or waste Starts the flow with reagent connected to valve x x to the column or waste Starts a linear flow of acetonitrile to the column or waste and is dependent on column diameter Starts a linear flow of detrit solution to the column or waste and is dependent on column diameter Starts a linear flow of reagent to the column or waste and is dependent on column diameter Pressure controlled flow of acetonitrile from Pump P6000 to the column or waste UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Strategy for Oligo Synthesis B PFlow_Det Pressure controlled flow of detrit solution from Pump P6000 to the column or waste Vol_Amid Set amidite and tetrazole with respect to the Scale loading weight Tetrazole AmiditeConc Eq AM Vol_Cap Set capping with respect to CV and CV_Cap Vol_Oxid Set oxidation with respect to Scale loading weight Eq Ox using the contact time flow rate Vol_Thio Set Thiolation with respect to CV and CV_Thio using the contact time flow rate B 1 2 Flowpath Means to column 1 1 5 2 6 valve pos to column 1 2 p1 0 p2 1 5 2 6 port1pos to column 1 4 p1 0 p2 1 5 2 6 port2pos1 to column 1 3 p1 0 p2 1 5 2 6 valve5pos2 to column 1 4 p1 0 p2 1 5 2
226. ox On the detrit pump press and hold the SET button and manually set the pressure to the desired level While holding the SET button on the pump you can observe in the UNICORN OS Calibration dialogue that an internal Measured 6 18 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Performing a run 6 value is assigned Click on the Read value 2 button and then release the SET button on the pump Click on Save to save the calibration Refer to the P 6000 Pump Instruction M anual for more detailed instructions Note A value for the actual calibrated pump pressure can be obtained by multiplying the values obtained in the Measured value and Measured 1 fields ACN solvent pump LiF Ce pihs Fee aL a e ee pa Gees i Abeerce vel 1 MINEN ENT 2 1 Relinde vakat 2 ow emm Hissi TEEF Figure 6 17 Dialogue box for pressure calibration of the ACN pump Select PressACN in the Monitor pull down list to calibrate the pressure reading of the ACN P 6000 solvent pump The calibration is based on the maximum set pressure of the pump 1 Thevaluein the Reference value 1 box should be zero 0 0000 bar If not enter 0 0000 Click on the Read value 1 button 2 Enter the desired maximum pressure value lt 20 0 bar in the Reference value 2 box On the ACN pump press and hold the SET button and manually set the pressure to the desired level W hile holding the SET button on the pump you can observe in th
227. printer currently set up on the local station not on the remote station If however you execute the procedure interactively from the UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 5 31 5 Creating and editing methods Evaluation module on the remote station the results will be printed on the printer set up on the remote station where you are working Method editor tesi28 Vin Fi Help ics I 3 j i Gaika Ewen Procedures wall ran Sd a red oo Oe rated rerio rr I i 3 I i d Firen CHi Flaite at Gide a ee EF L i E H Figure 5 34 The EvalProc page in Run setup Defining and viewing procedures Evaluation procedures are normally defined in the Evaluation module Procedures imported to a method can also be viewed and edited in the M ethod editor select the required procedure in the list and click on Edit Note Evaluation procedures which process chromatogram data rely on consistent identification of curves in the result file for correct operation If you include evaluation procedures with a method make sure that references to curves in the procedure will be valid when the procedure is executed at the end of the run Selecting procedures to run The EvalProc page lists all evaluation procedures associated with the method Click on the procedure s which are to be executed at the end of the run The procedures will be executed in the order they appear in t
228. r UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 2 7 2 UNICORN OS concepts Different blo cks in the same method may be written with different method bases for example a column wash block might be written in terms of column volumes while a purge block might be best expressed in absolute volume The sequ N ote Instructions The method term method base should not be confused with bases in a ence or an oligonucleotide is a call to blocks with each block containing a series of instructions or sub routines see Figure 2 3 Each instruction is a request for specific operations in the system A block may also contain other blocks which in turn contain their own series of instructions Double click on a block to expand collapse the view of the instructions kiiri a y E A ml pii ARI paren eee 000 Message Fil pour colina with OFL4 T suppa a COC H sa Tirra 2 ii ail 000 Stale 2 Healt of supper fy 000 Mesage Pues CONTINLIE when mani Losing si support umimi 000 Fais 10 F O00 Dalay 2 00 gml 000 Call Normal START parameters at 100 Clim Coe alum n_ Cem fen 0 00 Calihomai Puge Tw OO Bed bok 000 Call Mona Purge C _ a 000 Calima Purge A Purge 0 00 Call Normal Purge_G DOD Base volumne 000 Calomnia Purge Tatars a 0 00 Fire Frag 500 imimn 000 Call Normal Purge igberi os 000 Ari dic ri Wizi UO Gal Homa Column wash 1100 End bck 000 Call Horna Add
229. r disk if the computer itself is connected to a network see Section 11 4 1 2 1 3 Software modules UNICORN OS control software consists of four integrated modules ee he Main menu with functions for file handling and administrative routines such as definition of available oligonucleotide synthesis systems and maintenance of user profiles ee he Method editor where methods for pre programmed control of oligonucleotide synthesis systems are created and edited ee he System control module which permits manual or method based control of oligonucleotide synthesis systems and on line monitoring of synthesis processes There may be up to four independent System control modules on one computer for controlling up to four separate systems ee he Evaluation module for viewing and presenting stored results from synthesis processes 2 2 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA UNICORN OS concepts 2 UHC RH bial mermi File Mew Administration User Multidethodiun Quit Adviser Fl Help x id thoes 2 o m lt a caa ___ A Hia Edi cdas H a a A Co egc ket i Erill pede acdace Eth Shs Tisd E lt e Figure 2 1 UNICORN OS desktop with the Main menu shown as a window and the other modules as icons These modules are represented by either windows or icons on UNICORN OS desktop see Figure 2 1 UNICORN OS desktop itself occupies the entire screen According to how the software i
230. r Int_Start_Level to begin integration of the trityl peak CT_Thio Sets the contact time of Beaucage CT Oxidate Sets the contact time of oxidation CU CT_Capping Sets the contact time of capping O B 1 4 Watch Watch_PressDet Watch on Pump P6000 for detritylation 0 20 bar Watch_PressACN Watch on Pump P6000 for detritylation 0 20 bar Watch_Cond1 Watch on conductivity monitor Watch_IntStatus Watch on integration ON OFF Watch_ Off Cancels a watch on a specified monitor UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Strategy for Oligo Synthesis B B 1 5 Other Base Defines the base for a block for Volume calculating breakpoints Each block Time must have a defined base Column volume Call Calls a block unconditionally Continue Resumes execution of a paused or held method This instruction has the same effect as clicking on the Cont button in System control End Method Terminates method execution equivalent to clicking on the End button in System control Evaluate Calls an evaluation procedure The Procedure name procedure must be stored together with the current method Places the system in Hold state This instruction has the same effect as clicking on the Hold button in System control Loop Runs the instructions between a No of loops start and a number of loops 1 9999 Loop_end Marks the end of a loop e Message Generates a user defined message Message wh
231. r with the SET_ DESTINATION CHROM instruction Saves the specified chromatogram in the specified Name file If is used for Name then the current default To file chromatogram is saved If is used for the ToFile parameter the default file is used SET _ Opens the named chromatogram as destination for Chromatogram name DESTINATION the subsequent curve and peak operations Used in _CHROM pair with the RESTORE_DESTINATION_CHROM instruction UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA C 15 C Evaluation functions and instructions C 4 6 Other Sets the x axis base in which the following X axis base calculations will be done in If the value of x axis base is DEFAULT then the default base is used usually the base the method was run in This instruction should be the first in the evaluation procedure otherwise it will have no effect at all COMMENT Inserts a comment below the marked instruction Comment text ENDLOOP Marks the end of a LOOP statement Pe ne LOOP The instructions between this statement and the n Number of loops ENDLOOP statement are repeated n times It is possible to have loops within loops as long as the number of LOOP statements matches the number of ENDLOOP statements REPORT Prints a report with the specified named report layout Report layout and title If Title is then the title in the report layout Report title is used If ReportLayout is then a default layout is
232. rams including all new created curves and all imported or created chromatograms can be saved in two ways either using File Save which saves all changes in the original result file or using File Save as which allows you to create a copy of the result file under a new name Note All curves created during the manipulations will also be saved This may not always be desirable Before saving remove unwanted curves from a chromatogram using Edit Delete Curve T he original curves can never be deleted 7 6 Printing chromatogram and documentation reports A printed record of all documentation concerning a run including the method and Run Log can be obtained as follows 1 Select File Report and a dialogue box will appear If you have predefined report formats you can choose one and then press Print 2 Select the main items to be included in your report by placing a check mark in the box beside each of the relevant options under Report contents 7 28 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Presenting results a had Paces EEn LAGG Cydonia hi Figure 7 30 Synthesis data options in the Generate Report box 3 Associated with each report content item is a radio button Selection of a radio button lists a subset of options linked to the specific report contents option Select the options which are to included in the report Note Itis possible to press a radio button and select items even
233. re on the EvalProc page and click on Edit 2 Edit the procedure UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 5 33 5 Creating and editing methods 3 Choose File Exit from the procedure editor menu File Save is not available in the procedure editor window when you edit procedures in a method Changes are saved automatically when you close the procedure editor Note Report formats in procedures cannot be edited or viewed 5 6 5 Info The Info page displays information about the method including method name username date for creation target system and most recent modification The Info page is for information only and cannot be edited Figure 5 35 The Info page in Run setup 5 6 6 Sequence T he Sequence page contains the user interface between the desired sequence to be synthesized and the method created to run the synthesis procedure The properties of this interface are described in Chapter 4 and Section 5 1 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Creating and editing methods 5 5 6 7 Result Use the Result page to specify how the result files will be named for the results of arun and where the result file will be saved Figure 5 36 The Result page in Run setup The result file name is constructed by adding a 2 digit serial number to one of the base options listed below T he serial number is incremented automati
234. recorded in the audit trail for the respective system Run hours 9 0 hours Figure 12 13 Run hours in the system audit trail UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA System settings 1 3 13 System settings The System Settings command in the system control menu allows you to define global system settings for alarms and warnings and to choose which data will be stored in result files Configuration access is required to change settings parameters see Section 10 2 To access system settings double click on the System control desk top icon for the appropriate system and choose Settings from the System menu Details of the settings for each system are defined in the system strategy This section gives an overview of the principles of system settings The system settings should be established when the system is installed Certain settings may need to be adjusted if system components are changed e g alarm and warning limits or for specific run purposes e g monitor and curve settings Some settings may also be set with an instruction in a method overriding the global setting for the duration of the method See Appendix B for settings defined in general synthesis strategies re zi u Hagh akra ALO an pr lems a l N High ama 10 EE ki Los seam D AN Ei Hahna l TE bear Unde Friston pede he brdih nTa ip r else SEET i b raki Lpa Mees P Dih Figure 13 1 Syst
235. rget directory box 4 Choose Only edit config sys 5 Choose the appropriate network driver name e g 3C5x9 SY S for Etherlink III 6 Choose PX support on or off as appropriate for your network UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 10 5 7 0 Network setup 7 Choose Cancel in the Suggested default settings for Autoexec bat box if you do not want to change your AUTOEXEC BAT file 8 In the Choose optional protocol box select SPX support for OS 2 sessions Choose Remote named pipes support and Client and server support Specify a name for the pipe server in the Machine name field this is the name that will identify the station in UNICORN OS system definition see Section 12 1 M ake a note of the pipe server name for each station The name is required during installation of UNICORN OS Section 11 4 and system definition Section 12 1 Click on Save Note Two computers in a network must not be given the same pipe server name Pay special attention to this if you change the computer to your system 9 Click on OK to save changes to CON FIG SYS 10 Choose the menu command Configuration This workstation Edit NET CFG to include the following lines PROTOCOL STACK SPX ABORT TIMEOUT 60000 LISTEN TIMEOUT 12000 VERIFY TIMEOUT 6000 SESS LONG 353 NAMED PIPES CLIENT SESSLONS 32 SERVER SESSIONS 300 11 Save NET CFG and reboot the computer 12 Login to the network and map up to the
236. riables supported by the standard strategy for OligoPilot II M ost of the instructions apply to other synthesis systems for example O ligoProcess The user is referred to the appropriate synthesis system manual for specific details B 1 Method instructions B 1 1 Pump CT_Coupl Start flow with the reagent pump to create the contact time with the oxidation reagents The flow rate is determined by scale Eq_Amidite Tet CV Delay volume CapCT5 Flow Start flow with the reagent pump to create the contact time with the oxidation reagents The flow rate is determined by CV and CV_Cap OXCT5 Flow Start flow with the reagent pump to create the contact time with the oxidation reagents The flow rate is determined by the Scale and Eq Ox ThioCT5 Flow Start flow with the reagent pump to create the contact time with the oxidation reagents The flow rate is determined by CV and CV_Thio UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA B 1 B Strategy for Oligo Synthesis B 2 CapCT6 Flow OXCT6_Flow ThioCT6_Flow Flow_ACN Flow_Reag Start flow with the Pump P6000 to create the contact time with the oxidation reagents The flow rate is determined by CV and CV_Cap Start flow with the Pump P6000 to create the contact time with the oxidation reagents The flow rate is determined by the scale and Eq Ox Start flow with the Pump P6000 to create the contact time with the oxidation reagents The fl
237. root directory on the drive shown in the UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 8 23 8 Evaluating results Directory field It is possible to change the destination by pressing the Set command button 8 4 2 Copying results to the clipboard A related activity is Edit Copy to Clipboard where the contents of the active window are copied to the OS 2 clipboard and can be later easily pasted into other programs eg M icrosoft Word Curves and Documentation are copied as bitmaps while Peak tables are copied as text In the latter case only the columns selected in the Chromatogram layout box will be copied 8 24 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Introductory material Methods and Runs Evaluation System management Appendices This page is deliberately left blank Security features 9 9 Security features This chapter considers security features in UNICORN OS under the topics eeaccess security eesystem connection security eediata security eesecurity recommendations for control stations 9 1 Access security Operation of UNICORN OS is restricted to authorized users defined in the system by the system administrator see Section 12 3 User access may be protected by a password The minimum password length for users is defined when UNICORN OS is installed see Section 11 4 In installations where access security is important pas
238. s To minimize a window to an icon click on the Minimize button at the right hand end of the window title bar Depending on how UNICORN OS is installed see Section 11 4 you may also be able to minimize UNICORN OS window to an icon on the OS 2 desktop Note Minimizing a module window to an icon does not close the module Once opened UNICORN OS modules remain open until you quit the program A minimized System control icon may thus be actively in control of a running process 3 2 2 UNICORN OS Main menu panels The two M ain menu panels display method and result files respectively Use the panels as follows eeC lick anywhere in the panel outside an icon or file name with the right mouse button to pull down a menu for setting the panel presentation eeC lick on an icon or file name with the right mouse button to pull down amenu for opening copying moving renaming or deleting the file M ethod files can also be opened for either System control Run or editing Edit from this menu Figure 3 3 The M ethods panel showing pull down menus for panel presentation left and file management right UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 3 3 3 Login and file handling 3 4 eeA Iternatively double click on an icon to open the file For method files this opens the file in the M ethod editor For result files this opens the file in the Evaluation module eeSelect a method file and click on
239. s Alternative B Importing using O pen 1 Click on File Open and then select Chromatogram 2 Select the desired result file by double clicking on it and all of the chromatograms contained within will be displayed ent bret ce z qihb hape THP Ce ety E Ho p b l Dran Jimi kiah tw Cire Has Figure 7 20 File Open chromatogram dialogue box 3 Select in turn the chromatogram s of interest and press the Select command button for each chromatogram Selected chromatograms will be added to the Selected Chromatograms field Chromatograms can be deselected by using the Remove command button UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 7 19 Presenting results 4 Repeat steps 2 3 for chromatograms in other result files 5 Click on OK Viewing all chromatograms 1 Doubleclick on the imported chromatogram icons to open them 2 Simultaneously display the chromatograms by selecting Windows Tile or stack them by selecting Windows Cascade 3 a Chromatogram windows can be individually sized and the presentation of the curves changed b If you want to have the same scale on all of the chromatograms open the Chromatogram Layout box for any chromatogram make the changes and select check the Apply to all chromatograms option 4 Toprint all chromatograms on one page as they appear on the screen click on the Evaluation title bar at the top of the window and then press the lt Pri
240. s 12 4 4 System run hours 13 System settings 13 1 Alarms 13 2 Specials 13 3 M onitors 13 4 Curves UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 11 10 12 1 12 2 12 2 12 4 12 4 12 5 12 5 12 7 12 10 12 11 12 12 12 13 12 13 12 13 12 14 12 15 12 15 12 17 12 18 12 18 13 1 13 2 13 4 13 4 13 5 Contents Appendices vi A Technical specifications A 1 System requirements A 1 1 Hardware requirements A 1 2 Software requirements A 1 3 Network requirements A 2 Control capacity A 2 1 Stand alone installations A 2 2 Network installations A 3 Data sampling B General strategy for Oligo Synthesis B 1 M ethod instructions B 1 1 Pump B 1 2 Flowpath B 1 3 Alarms amp M onitors B 1 4 Watch B 1 5 O ther B 2 M anual control instructions B 2 1 Pump B 2 2 Flowpath B 2 3 Alarms amp M onitors B 2 4 O ther B 3 System settings instructions B 3 1 Alarms Settings B 3 2 Specials Settings B 3 3 Monitors Settings B 3 4 Curve Configuration B 3 5 Method variables C Evaluation functions and instructions C 1 Smoothing algorithms C 1 1 Moving Average C 1 2 Autoregressive C 1 3 M edian C 2 Baseline calculation theory C 2 1 Finding baseline segments C 2 2 Selecting baseline points C 2 3 Drawing the baseline C 2 4 Estimating the baseline parameters from the source curve C 3 Peak table column components C 4 Evaluation procedure instructions C 4 1 Curve operations C 4 2 Int
241. s 4 10 file structure E 1 files backup protection 3 9 copying 3 8 copying to diskette 3 8 deleting 3 9 filter 3 5 finding 3 7 moving 3 8 renaming 3 8 sorting 3 5 filter peaks Chromatogram Layout box 8 3 finding files 3 7 flow rate for volume base 5 40 flow scheme in system control 6 8 window 5 21 e global audit trails 12 15 graphical display of methods 4 9 Greater_than condition 5 44 grouped sequence 4 5 H hardware requirements 11 1 header Chromatogram Layout box 7 11 help 2 5 help on method instructions 5 18 H ETP 8 10 HETP Chromatogram Layout box 8 11 hiding block instructions 5 17 hold 5 43 Hold button system control 6 9 viii UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Index holding a method 5 43 6 9 home directory 12 11 hysteresis 13 3 I icon view 2 6 3 4 icons 2 4 3 2 temporary 7 5 identifying variables 5 22 importing blocks 5 15 importing chromatograms individual 7 19 importing curves evaluation individual 7 23 several 7 21 info page in run setup 5 34 input field questions 5 28 inserting questions 5 29 insertion point for new method instructions 5 18 installing default user 11 9 installing MCU 11 3 installing UNICORN 11 1 in network environments 11 2 network installation 11 5 selecting items to install 11 4 selective installation 11 9 stand alone installation 11 5 strategies 11 8 systems 11
242. s located in the previous search 3 Deselect the curves you do not want to be imported 4 If wanted select check the Store in new chromatogram option Enter a name for the new chromatogram This option is recommended since all searched curves can be kept separate from the original chromatogram 5 When you have made your selection click on OK If you selected for a new destination chromatogram to be created its icon will now appear in the workspace Otherwise the chosen curve s will instead have been imported into the active chromatogram 6 Doubleclick on the icon for the target chromatogram and open the Chromatogram Layout box Select the curves that you want to view Curves can be scaled individually or all with the same scale using the All with this unit function see Section 7 2 7 7 22 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Presenting results Alternative C Importing curves using O pen Using the File Open Curves function individual curves may be imported into the active chromatogram 1 Ensure that the destination chromatogram for the imported curve s is active on the screen Figure 7 24 Open curves dialogue box 2 Select File Open Curves A dialogue box will appear containing all results files 3 Click on the result file of choice and where appropriate the specific chromatogram containing the desired curve The chosen chromatogram and curves contained therei
243. s determined when the software is installed see Chapter 11 each of which may be connected to one oligonucleotide synthesis system at a time Connections are managed using the Connect and Disconnect commands in the System menu A network installation may have more than four systems in total but each computer in the network can establish a maximum of four connections Connection management is the same for stand alone and network installations 6 5 1 Establishing a connection To connect a System control window to an oligonucleotide synthesis system open a disconnected window identified by the text Disconnected below the window icon on the desktop and choose System Connect The dialogue box lists the systems to which you have access Select the system to which you want to connect and click on OK To connect to a system from a remote station in a network installation the local station i e the computer physically connected to the oligonucleotide synthesis system must be logged in to the network UNICORN OS does not however need to be running on the local station UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 6 13 6 Performing a run 6 14 System connecton Available systems OligaPilot Il OligaPilot Il 2 Figure 6 14 The dialogue box for connecting to a system A local station can be used to control the oligonucleotide synthesis systems directly connected to the PC without logging in to the net
244. s installed you may be able to minimize UNICORN OS to allow access to other programs on the OS 2 desktop UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 2 3 2 UNICORN OS concepts 2 1 4 OS 2 terminology This section explains some of the basic terms relating to the O S 2 user interface Refer to your O S 2 documentation for a complete description Onr m _ 2 a Figure 2 2 Main components of the OS 2 user interface ee he desktop is the area of the screen in which windows can be opened UNICORN OS has its own desktop which may occupy all or part of the screen eeAn icon is a symbol on the desktop or in a window representing an object in the interface usually a file or program In general double click on an icon with the left mouse button to open theicon to a window Double clicking on an icon on the OS 2 desktop generally starts the program associated with the icon Icons on UNICORN OS desktop represent modules in UNICORN OS software and double clicking on a module icon opens the window for the module eeA window is an active area of the desktop used by a program or an open folder displaying its contents There may be several windows open at the same time on the O S 2 desktop but only one is active at any onetime The active window Is generally identified by the colour of the title bar and border In UNICORN OS the Autominimize setting under Preferences in the M ain menu determines wh
245. s list to return to the M ain menu with the selected file highlighted in the appropriate panel If you click on Exit with or without selecting a file you will return to the M ain menu with the panel display unchanged 3 2 5 Copying moving and renaming files Y ou can copy move or rename files and directories using commands from the pull down file menu or from the M ain menu bar If you copy or move directories all files within the directories will also be copied or moved 1 Select one or more files in the M ethods or Results panel of the M ain menu To select multiple files hold down the lt Ctrl gt key while you click on the file names or icons Rename is not available if you select multiple files 2 Click with the right mouse button on any file icon and choose the appropriate command from the pull down file menu or choose the command from the File menu in the M ain menu bar 3 For Copy and Move choose the directory to which you want to copy the file and click on OK For Rename enter a new name for the file or directory Notesi To copy or move a method or result file to a diskette choose the a folder as the target directory A file that has been stored on a diskette cannot be opened directly from the diskette To use a file from a diskette first copy the file back to an appropriate folder on the hard disk To copy a file within the same directory use the File Save as command in the appropriate module after opening the
246. s version 1 10 User M anual 18 1118 52 Edition AA Creating and editing methods 5 2 By default the method will be saved in your home directory To change the directory double click on the appropriate directory icon in the Methods panel 3 Ifyou have more than one system available choose the system for which the method is intended The method can only be run on the system for which it is saved Remember that different systems may have different configurations and control capabilities 4 Click on OK The method remains open in the M ethod editor when it has been saved i so that you can continue editing if you wish If you edit any of the Save method instructions or change the method variables you can save these changes by selecting File Save or clicking on the Save button in the toolbar If you want to save a copy of the method under a new name choose File Save As and enter the details as described above If you close an open method or the M ethod editor module and there are unsaved changes you will be prompted to save these changes 5 7 2 Saving as a template Y ou can save the method as a template if you have Edit global lists authorization 1 Choose File Save as template 2 Enter a name for the template in the Name field or choose an existing template name from the list If you choose an existing name your will overwrite the existing template 3 Choose the system for which the template is intended in the For system fie
247. scillation is satisfactory note the P value Po and the oscillation period in seconds T 0 4 Calculate suggested PID values for the required regulator type from the table below Regulator type P D P 0 5 Po PI 0 45 Po 0 83 To PID 0 6 Po 0 5 To 0 125 To 5 Adjust the PID parameters from these suggested starting values until the feedback behaviour is satisfactory 6 When satisfied press Save to save the PID parameter settings During tuning adjustments UNICORN OS displays the effects of the current parameters graphically The Output signal is the signal to the pump The Input signal is the actual flow rate D 2 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Feedback tuning D Hiem corral Meihets View Mya Conligesten Eni Fi Help Few Habi Pause Comi Pad C Heia i E oa ip Cui Satpont cues Tuning of er dbeir Feedback conmkul Paremneleia fg EE q tonne yyy l LUGI USERRA E Oe PUL Figure D 2 PID tuning in UNICORN OS D 2 Gradient tuning Tune gradient settings in the same way as flow rate settings with the exception that the Setpoint parameter is B The Input signal is the actual eluent concentration B as determined by the conductivity UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA D 3 D Feedback tuning D 4 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1
248. signal falls below a specified value or a valley is detected A valley is defined by a local minimum followed by an increase to 102 of the local minimum value plus the M in_ peak value See below Peak_max The signal falls to a specified fraction of the most recent peak maximum minus the M in_peak value see below Factor 1 detects peak maximum Stable baseline The signal is stable within the limits of the D_ Baseline value see below for the period specified by the minutes parameter Int_ Status Equal to 0 or 1 to Indicate if peak integration is switched off or on respectively To determine suitable values for watch conditions it is often most convenient to examine data from a test run For slope values use the Differentiate function in the Evaluation module to measure the slope of the test chromatogram 5 44 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Creating and editing methods 5 Note The slope criteria operate on the arithmetic value of the slope so that a value of 3 is less than a value of 2 The end of a peak is thus detected by Slope greater_than with a negative value the slope is negative but increasing Two conditions apply for air sensors not available for OligoPilot II Equal0 Air isnot detected by the sensor Equall Airis detected by the sensor Note Tousethe WATCH AS instruction for air sensors the ASALARM setting in Alarms amp Mon must be disabled use the M ethod edi
249. start of the new interval Watch actions The following actions can be taken when a watch condition is met Block name Call the named block The call is always made in N ormal mode see Section 5 3 2 Pause H old Pause or hold the method Continue Continue the method if paused or held End_block End the current block and return to the point from which the block was called End_method End the method 5 9 Creating methods from scratch This is the most advanced level for creating methods allowing you to create methods from the very beginning in an empty M ain block without the benefit of an established method template nor the sequence editor interface in the Sequence page of Run setup A method is built up entirely using the Instruction Box and Text instructions in the M ethod editor All blocks for the entire method must be created and breakpoints instructions and variables inserted into the blocks UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 5 47 5 Creating and editing methods 5 48 Relevant information must then be inserted into the appropriate pages of Run setup such as Variables Questions N otes EvalProc Results and StartProt The method can then be saved and run from System control as described in Chapter 6 There is no real need to use the Sequence page in Run setup since in creating a method from scratch you have bypassed the need for a sequence editor interface
250. structions which apply to a different system strategy these can arise if a method is written for one system and saved for another All red instructions must be deleted or changed before a method can be run see Sections 5 4 3 and 5 4 4 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Creating and editing methods 5 Grey Instructions which will not be executed because they are either after the end of a block or method or constitute a block to which there is no call The current instruction is highlighted with a grey bar and is also displayed in the instruction dialogue box where it can be edited see Section 5 4 4 In working with instructions you should display both the instruction dialogue box and the Text instruction window Use the View Windows command or toolbar button see Section 5 2 to select windows to display Alternatively if the instruction dialogue box is not displayed double click on a black instruction in the T ext Instruction window to display the box Double click on a CALL or WATCH instruction to display the instructions in the called block If the instructions are already displayed double click on the call to hide the instructions in the called block Double clicking on the M AIN keyword at the beginning of the method will show or hide instructions in all blocks in the method T ea coders sah OT ecco Goes i Peseage Wace Bi ete l F I a B ae Cal head TAAT pay Ss Daji Pe
251. swords should be enforced and should be changed regularly see Section 12 3 2 Each user is assigned an access level and a directory access profile defining the operations that the user can perform and the directories the user is allowed to access See Section 12 3 For system security administrative routines such as user and system definition should be permitted only to the system administrator Installation of UNICORN OS establishes a default user with full access rights in the system It is important for system security that this user is deleted when the site specific users have been created 9 2 Connection security To prevent conflicts in System control requests each system in an installation may have only one control mode connection at any one time A running system may be left in a view mode connection and locked with a password independent of the user s login password to prevent other users from establishing a control mode connection and interfering with controlling the synthesis process If the system is left unlocked in a view mode connection any user may establish a control mode connection to the system UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 9 1 9 Security features W e recommend that systems are always locked when a user leaves the system For controlled and locked systems the responsible user is identified in the System control window for view mode connections A system which is
252. systems that are installed in the network The username and password for logging in to the network are entirely independent of the those for logging in to UNICORN OS 3 2 UNICORN OS desktop 3 2 1 Icons and windows Each of the four modules in UNICORN OS is represented by an icon Or an open window on UNICORN OS desktop see Figure 3 2 On start up the M ain menu is presented as a window and the other modules as icons There may be several icons for System control according to how the program was installed There is however only one icon for the M ain menu M ethod editor and Evaluation module SSS L e UIT CE C Bio few Aciministration User MultiMethedhun Quit Adviser FisHap BE hitaat Ax T j i TE Loo Leap kp Figure 3 2 UNICORN OS desktop with the Main menu active 3 2 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Login and file handling 3 To expand a module to a window double click on the corresponding icon If User Autominimize is checked in the M ain menu only one window can be expanded at once and expanding oneicon to a window automatically minimizes the previously active window If User Autominimize is not checked any number of windows can be expanded at the same time Y ou can drag window borders to adjust the size of module windows and of panels and windows within the module window
253. t block as vavirmints Dinietdemon pe i s penk end Li k k Dei pan siai lia beet assii el wore i j OHS minm Gedy drpaisi abgepalri eee OF f DRLE szaraka import black er Swit nh bee Frim EFTE Figure 5 13 The Import block dialogue box Choose the method from which you wish to import and mark the block to import The name of the selected block is suggested in the Import block as field Click on Import to import the block The dialogue box remains open until you click on Exit so that you can import more than one block without closing the dialogue box Note Ifyou usethe import function to copy blocks within a method the blocks are copied from the saved version of the method on disk Any changes you have made in the method but not yet saved will not be copied The imported block may not have the same name as an existing block in the method If the default name is not allowed for this reason the Import button will be grey and locked Change the name of the imported block so that the Import button becomes available The block is imported exactly as it appears in the source method and is placed after the last instruction in the text window unless there is a call to the imported block already in the method If any CALL or WATCH instructions in the imported block refer to non existent blocks the instructions will appear in red in the text window If the base of the imported block is defined as SameA sM
254. t saved the method with the block deleted Note Any CALL or WATCH instructions which call deleted blocks remain in the method but are shown in red in the Text instruction window to indicate that they are no longer valid A method cannot be run if it contains invalid instructions UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 5 13 5 Creating and editing methods 5 3 5 Renaming blocks Block To rename blocks choose File Rename block Hew Import block as franc ince Rename blocks Delete block Template block Select the block to rename Rename black to Add DNA_G_ special Figure 5 12 The Rename blocks dialogue box Select the block to rename enter the new name in the Rename Block field and press Rename T he dialogue box remains open until you press Exit so that you can rename more than one block without closing the dialogue box If the block you renamed is used ina CALL or WATCH instruction the block name in these instructions will be changed automatically 5 14 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Block Hew Import block as Rename block Delete block Template block Creating and editing methods 5 5 3 6 Importing and copying blocks Y ou can import blocks from other methods to the method you are developing Y ou can also use the import function to copy blocks within the method Choose File Impor
255. t the peak start and end points The peaks will then be renumbered and the peak areas will be recalculated It is Suggested that you first open the Chromatogram Layout box press the Peak Label command button and choose either Number or Retention for labelling the peaks The former option will sequentially number each of the peaks in the chromatogram which is opened during the edit mode of a peak table The latter option will display the retention value for each peak Peak label Retention CI Number i Horizontal text Peak name Cancel Help Figure 8 7 Peak Label dialogue box Select Integrate Edit peak table Choose the desired peak table from the dialogue box that appears N ote that name of the baseline on which the selected peak table was based is displayed at the bottom of the dialogue box Double click on the desired peak table in the list or click on OK A chromatogram will appear containing the selected peak table UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 8 7 8 Evaluating results 8 8 with corresponding curve and baseline The various editing features are described below As an aid it is possible to use the zoom function on the chromatogram in the Edit Peak Table window Once you have completed your changes click on OK and verify the destination of the new edited peak table Tage A e 6041708 18d ee GEM Oa 24 Cond lao Ba TT mA TI is ail Aii
256. te button deletes selected instruction from the current list Only one instruction can be deleted at a time If you close the box by clicking on the Exit button without choosing Execute Commands in the list will not be executed and will be deleted from the command list M anual instructions can also be issued while a method is running A manual setting applies until the next method instruction of the same type is executed e g a manual Flow instruction will set the flow rate until the next Flow instruction in the method Is executed M anual instructions that you issue during a method are recorded in the logbook for the method run UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 6 11 6 Performing a run 6 3 3 Alarms and warnings The system settings see Section 13 1 determine the acceptable limits of monitor signals during a run The limits can also be set for the current run using an instruction in the method Limits set with a method instruction override the limits set in system settings If these limits are exceeded in arun a warning W or alarm A box with a message is displayed on the screen ee he run continues if a warning is issued eeAn alarm pauses the system Warnings and alarms are displayed regardless of the activity currently in progress in UNICORN OS you will be notified of an exceeded limit in a running system even if you are developing a method evaluating data or monitoring a run on a
257. ted to Sr 95 06 23 09 14 59 Connection change default at LOCAL disconnected fror 35 06123 09 15 24 Connection change default at LOCAL connected to 5 r g5 06 23 09 23 27 Connection change default at LOCAL disconnected fror Renew log file of Sequence 95 06124 10 10 17 Login default 95 06124 10 10 44 Connection change default at LOCAL connected to Sr Renew every day Set Now T e ml Figure 12 10 Global audit trails User Displays all user creation deletion and redefinition operations Backup Displays backup operations for global audit trail files Log in out Displays all login and logout attempts with the name of the user logging in or out including failed login attempts UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 12 15 7 2 Administration Define system Displays all system definition deletion and redefinition events Sequence Displays sequence start operations System audit trails Choose the system for which the audit trail is to be displayed and check the items you want to display in the System panel All items are recorded in the audit trail the check boxes in the System panel only control which items are displayed System audit trail files are saved on the local station to which the system is physically connected and may be examined from the local station without logging in to the network System audit trail files can be viewed from any computer in a network installation
258. tegies Accept the suggested name or enter a new name for the strategy This will not affect any existing method or result files in the system Strategies are installed on the network server in a network installation M ethod templates Check the Method templates button in the installation program to re install method templates or to install method templates for a new strategy Normally templates are installed together with strategies Y ou will be asked to enter the strategy name to which the method templates correspond if you are not installing a strategy at the same time Templates are installed on the network server in a network installation 11 4 3 Installing software upgrades Instructions for upgrading UNICORN OS installations to new versions will be provided with the upgrade software 11 10 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Administration 12 12 Administration There are three main aspects of administration of aUNICORN OS installation eeSystem administration eeA ccess level and user administration eeN etwork administration where appropriate System administration concerns maintenance of software aspects of the installation including definition of connected systems and monitoring of system usage audit trails These activities are described in the present chapter System administration duties may also include routine monitor calibration Section 6 6 Access level and user admi
259. tes 2 7 4 1 4 2 deleting 5 39 method notes 5 31 typical variables 4 7 using with custom strategies 4 11 temporary 7 5 7 21 temporary icons 7 5 test sequence 4 3 text 7 16 XVIII UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Index text instruction editor 4 13 5 6 flow scheme window 5 21 text instruction window 5 16 blocks 5 9 text instructions creating anew method 5 6 tile 7 6 7 18 7 20 time base 2 7 time volume base in system control display 6 7 title bar colour 12 3 toolbar system control 6 9 tuning D 1 access 12 6 U unconditional calls 5 10 Undo 7 12 unlock access 12 6 unlocked systems 6 16 user interface module 10 4 User setup Administration menu main menu 12 10 user setup levels access 12 5 username 12 11 users defining 12 11 deleting 12 13 VIW value questions 5 29 valve positions in method pause 5 43 variable list 4 7 variables 2 9 5 22 affecting block length 5 24 changing default values 5 24 default value 5 22 defining 5 23 editing 4 6 identifying 5 22 in instruction box 5 22 in instructions and run setup 2 9 in start protocol 5 36 in text instructions 5 22 naming 5 22 5 23 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA X X Index XX page in run setup 5 25 removing 5 24 renaming 5 25 typical variables in templates 4 7 warnings 6 12 13 2 watch actions 5 47
260. the Peak Table Columns list of the Chromatogram Layout box Every peak will havea HETP value A narrow peak gives a low value corresponding to a well packed column A broad peak gives a high value indicating a column that is not optimally packed H ETP is calculated as follows HETP L N N 5 54 Vp wp where N no of theoretical plates L bed height in cm Vp peak elution volume or time Wh peak width at half height expressed in the same units as Vp 8 1 7 Measuring peak asymmetry This function can be used in combination with H ETP to help assess column performance A perfect peak will have no asymmetry and after peak integration give a value of 1 0 Any value less than 1 0 means that there is a left skew i e the asymmetry falls on the leading side left of the peak The reverse is true for values greater than 1 0 where the asymmetry comes on the tailing side right of the peak To view the asymmetry data open the Chromatogram Layout box and select asymmetry from the list in the Peak Table Columns list Click on OK and return to the chromatogram Asymmetry width B width A where A and B are measured at 10 of the peak height 8 2 Other evaluations 8 2 1 Finding the slope values for Watch instructions It is possible to set up conditional W atch instructions which allow the progress of a run to be determined by the events during the run e g start integration when the first peak emerges W atch instructions m
261. ther define the synthesis scale i e Weight of the support Loading of the support and Column diameter CV column volume is also defined and is used for the calculation of special instructions such as Vol_Cap Vol_amid and CT5 Cap eeCV_ column_wash The number of column volumes CV to wash the column is set here If zero is entered no wash will take place eeDetrit_peak_start The flow rate of the detritylation solution is set here eeDetrit_wash The pressure of the detritylation wash and the number of column volumes of detritylation solution to be used are set here eeDNA_ Parameters T hese variables together define the coupling of a base to the oligonucleotide sequence i e how many equivalents of amidite should be added to the column with respect to the scale the percentage volume of tetrazole to be used with respect to column volumes and the concentration of the amidite eeDNA_ Recycle The amidite recycling flow rate and time used are set here eeOxidation DNA Oxidation stabilizes the phosphite group of the coupled amidite The variables determine how many equivalents of iodine are used in the oxidation solution and the contact time between the oxidation solution and the support eeCapping The unreacted 5 hydroxyl groups on the oligonucleotide are capped to prevent further participation in the synthesis reaction The column volumes of capping solution and the contact time are set here Note The base for the all the bloc
262. ting results ie She fod 1 Colunn_ o cy Meiaght_of_support 1 30fqg Loading_of_support 14 104 umo dm t Col ism if OG mm delayv_yvolume L 24ml Datrit_ flow 200 em h Le_Wasah_ Fressure ae BIDAT Oetrit waen Lo of 470d 1 Cond f a Lo Le a La Us 3 HUH BBE mi Figure 7 12 Chromatogram with header information displayed 7 2 9 Saving a layout All changes that you make in the Chromatogram Layout box can be saved as a layout It is possible to apply saved layouts to other chromatograms All saved layouts are user specific Note A layout saved with the name DEFAULT in upper case letters will be used as the default layout for all chromatograms when they are opened for the first time Once a layout has been selected in the Chromatogram Layout dialogue box save it as follows Click on the Save as command button Type in the name of your layout and click on OK Several different layouts can be saved in this manner Layouts are saved for use with other result files You can now apply your layout s as described in Section 7 2 3 7 3 Additional presentation possibilities The Evaluation module allows you to perform operations on the curves to optimize the presentation Note Thelatest evaluation operation that is performed can be undone using Edit Undo or reapplied using Edit Redo 7 12 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Presenting results 7 3
263. tion and printing of reports may be included as an operation in an evaluation procedure to automate process evaluation and documentation The Evaluation module is described in detail in Chapters 7 and 8 2 6 Network considerations Figure 2 6 illustrates how a networked UNICORN OS installation can be organized There are two kinds of PC whereUNICORN OS software is installed eeA local station isa PC to which oligonucleotide synthesis systems are physically connected eeA remote station is a PC to which no systems are physically connected but which can control systems over a network link Eb 25S Ge CE jl Local station 2 systems ae i l E Nebwork Server a E Renate Local station station 1 swotem ia Figure 2 6 A network installation with 3 oligonucleotide synthesis systems and 5 stations 3 remote and 2 local The systems can be controlled from any computer except the network server Ramote Remote Baten station Oligonucleotide synthesis systems in the network can be controlled from either remote or local stations A local station in a network can also be used as a remote station to control other systems 2 12 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA UNICORN OS concepts 2 Note Ifa run is being controlled from a remote station and a network communication error occurs the run will continue under the control of the local station Results will be saved in the FAILED
264. tion gives directions on how to access result files and optimize the presentation of a chromatogram and its curves via the Chromatogram Layout dialogue box 7 2 1 Viewing chromatograms Chromatograms can be selected and made active in two possible ways either click on the relevant icon on the Evaluation workspace or select the desired chromatogram in the Windows menu To minimize the active chromatogram to an icon click on the Minimize button in the top right corner of the window Minimise 96041704 Di What you get to start with The first time a chromatogram is opened and viewed a default layout will be applied where all of the original curves are displayed The default layout can be changed by the user see Section 7 2 9 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 7 5 Presenting results Result file Chromatogram Curve name name name Figure 7 6 Displayed chromatogram in a newly opened result file Each curve has its own colour as denoted by the legends in the chromatogram header Each curve legend consists of the result file name chromatogram name and curve name Each coloured curve has a correspondingly coloured y axis To choose the relevant y axis scale click repeatedly on the y axis until the desired scale is displayed By default the x axis is shown in volume ml To change the x axis units see Section 7 2 7 O ptimizing the workspace To maximize the size of an act
265. tly Adise FI Halp File Bok Wire O oe 5 j GE i ii j Skikes Hela L oo Fae Tha a a apla ba Fo chara Cooder ilo The recht ee Sarmrecycing d capia Heyes H rim A E upis E ge Tee pet hs ere ol T EnF J p cer babes bee eed on eae eth Fi beak Ceci hee Neti gii ka borane cubed pEr BL Phe biir na keel pequence eh of ead DHA tje head ine Pea a pabi oi at baari H bal iagh padac amd n i barre than S aira AFi rete rod af Hull D Hoed weal amadis ponhon red intact Sua es call H E LU E E r E a Figure 4 7 The Notes page in run set up with the method notes maximized UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 4 9 4 Creating methods from method templates If your system does not correspond to the description either rearrange the valves and tubing connections in accordance with the method notes description or edit the method instructions see Chapter 5 in accordance with your system set up 4 6 Saving the method When you click on the Create button in the Sequence editor page UNICORN OS checks the sequence for invalid combinations generates the method updates the method variables and automatically opens the Save As dialogue box The method must be saved before it can be run _ e os me E whe Sion ohagopal cl i Ieee 1 Figure 4 8 Dialogue box for saving a method The Methods panel will be shown in icon or detail view according to the
266. tor to disable the alarm locally in a method or the system settings to disable the alarm for all methods see Section 13 1 The ASALARM setting overrides any WATCH _AS instruction and if the alarm is enabled the method will pause when air is detected System settings for watch conditions For continuous monitoring the following parameters in the Peak Monitor instruction e g PeakCond1 in system settings affect the interpretation of certain watch conditions MinPeak T he MinPeak setting affects only the Less than_or_valley and Peak _max conditions This setting a sets the threshold for signal increase after a local minimum which will be interpreted as a valley for the Less than_or_valley condition A valley and a new peak are detected when the signal increases to 102 of the local minimum plus the M inPeak value b sets the threshold for signal decrease after a local maximum which will activate the Peak_max condition Peak_max is detected when the signal falls to the specified fraction of the most recent peak maximum minus the M in_Peak value UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 5 45 5 Creating and editing methods Figure 5 40 Illustration of the MinPeak setting Peak max is detected when the signal falls by MinPeak from a local maximum if the Peak_max factor is set to 1 Valleys are detected when the signal rises by 2 of a local minimum value plus MinPeak value The M inPeak
267. tory Practice GLP In addition UNICORN OS offers a comprehensive security system e Password control for all users with access authorization for other users method and result files e Customized definition of access control levels e Audit trail for system operation Note UNICORN OS must be correctly installed for stand alone or network operation before the software can be used N etwork considerations software installation and administration of system and user definitions are described in Chapters 10 11 and 12 of this User M anual UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 1 1 7 Introduction 1 2 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA UNICORN OS concepts 2 2 UNICORN OS concepts This chapter introduces the basic concepts that are needed to use UNICORN OS For a description of general concepts of how to work with the O S 2 operating system see your O S 2 system documentation M aterial in this chapter is divided into 7 sections dealing with ee UNICORN OS user interface eeFiles and directories eeM ethods and method structure eeSystem control eeE valuation eeN etwork considerations eeSecurity and administration 2 1 UNICORN OS user interface 2 1 1 UNICORN OS control software UNICORN OS runs under the O S 2 operating system and provides facilities for method controlled operation of oligonucleotide synthesis systems as well as real
268. trategy An attempt to control a system using the wrong strategy may cause malfunction and damage to the synthesis system 12 4 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Administration 7 2 12 2 Access levels Administration User setup Access levels Audit trail system Access to UNICORN OS software is controlled by username and password authorization see Sections 12 3 Within the program each authorized user is assigned an access level within the system which determines which functions the user can perform 12 2 1 Defining access levels Up to 10 different access levels can be defined Initially all levels are the same with access to all functions To edit an access level choose Administration Access levels from the M ain menu and choose the level you wish to edit To use this menu command you must have User setup Levels authorization see below The levels are named Level 1 Level 10 by default to change the name of a level simply type a new name in the Level name field Check the items to which users at this level are to have access and click on OK If you change the definition of a level to which users are already assigned the changes will apply to all users at this level At least one access level must have User setup Levels authorization UNICORN OSwill not allow you to remove this authorization from all levels The authorization items are eeMethod Editor
269. ts box panel click on Layout to adjust the scales for curve presentation 6 6 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Performing a run 6 Tenn jer Figure 6 7 Curve Window layout dialogue box The y axis can be set to either auto mode or to a fixed scale with user defined maximum and minimum values In auto mode the scale is adjusted continually according to the highest and lowest values of the curve in the current run The y axis scaling can be set independently for each curve The x axis can be set to show the total run from time 0 or to show a fixed time or volume window The window stretches back in time from the present moment The curves panel displays graphs for the selected curves in different colours with any reference curves included with the method as dashed lines Values on the y axis apply to the curve with the same colour as the axis markings Click on the y axis to change the axis markings between the displayed curves Single click on the x axis to switch the display between time and volume units The run is controlled according to the time volume base defined in the current block regardless of the base in the curves display mt L ler i l i 4 p l nh a ns 4 oc wu io I A a1 a Les Figure 6 8 The curves panel UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Ed
270. two instructions for this purpose Hold Suspends execution of the method but continues to pump eluent at the current flow rate and concentration settings Pause Suspends execution of the method and stops the pumps so that the system comes to a standstill In OligoPilot Il valves remain in the position they were in before the pause The pause may be defined as indefinite or for a given number of minutes This instruction is most useful for stopping the system in the event of an unexpected condition In both cases the method may be resumed by pressing the Cont button in the System control toolbar see Section 6 2 Note Never select Pause or Continue during a system Hold Vol_amid Vol_Ox Vol_Thio Vol_Cap as this will interrupt the adding of reagent 5 8 6 Linear flow rates Linear flow rates cm h for ACN detrit solution and reagent can be specified by the instructions LFlow ACN LFlow_Det and LFlow_Reag respectively To use these instructions it is necessary for a column diameter to be defined in the Variables page of Run setup The volume flow rate is calculated from the specified linear flow rate and the column diameter The calculated volumetric flow rate is shown during runs piperi hargi eir a ai t ri roi Harm an Few Eaj i AF impak i La ee Ct je Te A mh ale var A gers r4 TEE Figure 5 39 Setting linear flow rate in a method block The linear flow rate option is only available if the column diameter is de
271. ual instructions as well as start methods and sequences Supervisors are also allowed to calibrate monitors configure system parameters unlock running processes evaluate run data and quit UNICORN OS 12 8 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Administration 7 2 Detine access levels Supervisor PI LILI LI LI A L Figure 12 5 Suggested authorization profile for process supervisors Process operators Process operators are allowed to run and pause methods and sequences but may not perform any other operations Define access levels E E m E x m x E x E E E m Figure 12 6 Suggested authorization profile for process operators UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 12 9 7 2 Administration 12 3 User administration All UNICORN OS users are identified by a username and password A new installation is provided with a default user username default password default This user provides unrestricted access to all UNICORN OS functions Important As part of the installation procedure new users should be created with passwords and restricted access rights as required T he default user should be deleted or redefined to prevent unauthorized access to the system M aintenance of user authorization information is the responsibility of the system administrator In a newly installed system log in as user default Admi
272. ucleotide to be synthesized This cannot be altered The name of the result file is specified here This page is always displayed and the name may be changed if this is permitted in the start protocol If any questions in the start protocol require authorized confirmation you will be asked for a username and password when you attempt to leave the screen containing the questions Only users with Confirm Unlock authorization may authorize answers to such questions One authorization is valid for all questions in a single screen UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 6 3 6 Performing a run 6 2 Monitoring a run Windows El Hide panel RME Maximize Restore Contents 6 4 The System control window displays the status of the current system There may be up to four System control windows available on UNICORN OS desktop which may be connected to different systems Icons are identified either as Disconnected or by the name of the connected system Separate systems may be controlled and displayed independently of each other Each System control window displays up to four panels for monitoring different aspects of the run Click on the Windows toolbar button or choose View Windows from the menu to select which panels to display Windows aeeeescescossasscansas hy Run data W Curve data _ Flow scheme _ Logbook ox Cancel Help Figure 6 3 Dialogue box for
273. used RUN _ Starts a program as a separate process The Program Program name PROGRAM name string contains the program name and parameters to start it with C 16 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Feedback tuning D D Feedback tuning This appendix describes the principles of PID feedback tuning of pump flow rate UNICORN OS process control software provides a graphical interface for feedback tuning Feedback control is aimed at eliminating discrepancies between the actual value and the requested value in this case flow rate OligoPilot II and OligoProcess use the measured flow rate to control overall pump speed and the measured conductivity to control the relative speeds of pumps A and B in gradient formation PID tuning uses three parameters to determine feedback control ee he P parameter reduces the effect of an error but does not completely eliminate it A simple P regulator results in a stable discrepancy between actual and requested flow rate a stationary error ee he parameter eliminates the stationary error but results in a slight instability leading to oscillations in the actual flow can have values between 0 and where smaller values have greater effect and a value of has no effect The value is set as 9999 in UNICORN OS e n certain cases the D parameter can reduce the oscillations introduced by a Pl regulator D can have values between 0 and where l
274. ustrating peak parameters See the parameter list below for explanations Retention time or volume base Retention at the peak maximum C in Figure C 4 Width time or volume base Difference in retention between the peak end and peak start G A in Figure C 4 Area time or volume base Calculated as the area between the curve and baseline between the peak start and peak end shaded in Figure C 4 Height se amplitude above the baseline C F in Figure 4 Peak endpoint time or volume base Retention value at peak start and retention peak end A G in Figure C 4 Width at half height time or volume base Calculated by taking the maximum height of the peak above the baseline then determining the peak width at half this value above baseline D B in Figure C 4 where BD bisects CF Percent of total area time or volume base Peak area as a percent of the total area under the curve above the baseline Note that this value may differ in time and volume base if the flow rate is not constant throughout the method Percent of total peak time or volume base Peak area as a percent of the area sum of all integrated peaks Note that this value may differ in time and volume base if the flow rate is not constant throughout the method UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA C 7 C Evaluation functions and instructions Type of peak limits Identifies the criteria for peak start and peak en
275. value should be set large enough so that signal noise does not activate the conditions and small enough so that the condition Is activated close to the valley or peak As a general guideline set the value to 2 3 times the noise level and 5 10 of the smallest expected peak height Setting a value that is too high can prevent a new peak from being detected after a local minimum D_ Baseline D_ Baseline defines the permitted variation for the Stable baseline condition For this condition to be activated the signal may not vary by more than the D_Baseline value up or down over the time interval specified in the Stable baseline condition in the watch instruction The D_ Baseline setting affects only the Stable baseline condition 5 46 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Creating and editing methods 5 WATCH Watch Sabe bering inierra Stable EPEE riers a Sama Bare Ls Tat A m i oy ier bne rs Til 4 wt Af has Pp a Mast A Fi Le i To Tamal LE i codio mar cardin a ee Figure 5 41 Illustration of the D_Baseline setting The condition WATCH Stable_baseline is met if the signal does not deviate by more than D_Baseline from baseline during the time interval specified for the watch The baseline value is determined by the signal at the start of the watch If the condition is not met a new interval is started with a new baseline value defined by the signal level at the
276. vea default answer Example Instrument identification a OligoFilot Il Figure 5 29 Options for input field questions eeMultiple choice allows the user to choose one of a defined set of answers To allow a blank answer enter a space in one of the predefined answers Example Mark the type of support k E Frimersupport 30 HL a Primer Support Other gt Figure 5 30 Options for multiple choice questions eeNo answer does not require an answer This kind of question may be to display important information or to split a question over more than one line by setting all but the last line to No answer Example The information on this side will not effect the method Figure 5 31 Options for no answer questions 5 28 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Creating and editing methods 5 eeValue accepts only numerical answers Value questions may have specified maximum and minimum limits and may be defined to accept only integer values Question Type the weight of the support in the column V Mandatory _ Authorized V Chromatagram Answer eines un 2 Multiple choice Max 200 No Answer e Value _ Integer Figure 5 32 Options for value questions Press the Preview button to display the questions as they will appear when the method is run Alternative answers to multiple choice questions are not shown in this m
277. warning message is issued without interrupting the process Alarm and warning messages are displayed on all stations with a connection to the system concerned regardless of the activity currently being performed in UNICORN OS and regardless of the identity and access rights of the current user Alarms and warnings can however only be acknowledged from the control mode connection If allowed by the system strategy limits for certain monitor signals may also be set locally in a method overriding the global setting as long as the method is in operation Error messages from the monitors and pumps are reported if the respective Error settings are Enabled Important Alarms are not active unless the mode is set to Enabled Use the scroll bar in the Parameters panel to access this setting if the mode parameter is not displayed 13 2 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA System settings 1 3 The hysteresis setting for an alarm determines the extent to which the signal can oscillate around the warning limits without re activating the warning Figure 13 2 Warning limit Phytera as K i WARNING WAR NING Figure 13 2 The hysteresis setting defines the limits within which the signal may oscillate up or down from the threshold without re activating a warning After the signal has activated a warning the warning will not be repeated as long as the signal remains within a window d
278. where you want to insert text in the chromatogram and click the left mouse button once UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Presenting results 3 Inthe dialogue box that appears type the desired text and then click on OK Now the text can be viewed on the chromatogram The text is saved at the position where it is placed in the chromatogram window and is not linked to any curve Text cannot be moved in the window once it has been placed If you want to edit the inserted text 1 Select Edit Text Edit 2 Select the specific text that you want to edit and make the appropriate changes and click on Replace Click on OK If you want to delete inserted text 1 Select Edit Text Delete 2 Select the specific text that you want to delete and click on Delete and then OK 7 4 Comparing different runs The previous sections dealt with the manipulation of single curves within a chromatogram The following sections describe how to make comparisons between two or more curves or chromatograms from different runs and details how best to present them It is possible to eeview several chromatograms at the same time eeoverlay curves from different runs in one chromatogram eestack curves from different runs in one chromatogram eestretch curves to make comparisons easier eecreate mirror images 7 4 1 Comparing chromatograms from different runs Two functions can be used to import chromatograms from
279. work M ethod and result files stored on network drives will of course not be accessible For runs performed in this stand alone mode where the result file is directed to a network drive the results will be saved in the FAILED directory on the local station see Section 9 3 Several simultaneous connections can be established to one system but only one may bein control mode i e able to actively control the system The other connections are in view mode and can monitor the system activity but cannot issue commands except Pause if the user has Pause access UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Performing a run 6 6 5 2 Connection modes The possible connection states of a System control window are indicated by the connection mode button and the status text on the control bar as summarized below C onnection State Action mode Not a Click on the connection mode connected button or choose System Connect to establish a connection Control Controlled by Click on the connection mode mode lt user gt button to leave the system but retain the connection with the System control window Y ou may leave the system locked or unlocked View mode Controlled by The indicated user has a control lt other user gt mode connection Clicking on the connection mode button has no effect View mode _ Locked by The indicated user has left the lt other user gt system in a locked state Cl
280. work installation these definitions must be set up for each local computer in the network but the actual set up operations can be performed on any computer Rights of access to system are controlled at the user administration level Sections 12 2 and 12 3 To manage system definitions choose Administration System from the M ain menu To use this menu command you must have Audit trail System setup authorization see above System Systems oy STEMI El Figure 12 1 System definition dialogue box 12 1 1 Defining and editing systems To define a new system click on New in the System dialogue box To edit an existing system choose the system in the System dialogue box and click on Edit Change parameters as required UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Administration 1 2 Edit system Definition System name OligaPilot 2 Strategy name Information System title bar colour Colour Sampe Fipe server name ROOM_E32 Control unit number 1 4 E Autosave interval minutes Cancel Figure 12 2 Dialogue box for defining and editing systems 1 Enter the system name In the System name field The system name can be set only when defining a new connection and cannot later be edited since users access rights are linked directly to the system name Names can be up to 30 characters long 2 Select a strategy for the system from the pull down list in the Strategy
281. y selected Y axis scale to calculate the noise range as the difference between the max and min values Add an extra 20 and insert this value as the Noise window value M easuring the Slope limit using Differentiate and curve co ordinates To measure the slope at any point on the curve UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA C 5 C Evaluation functions and instructions 1 Select Operations Differentiate A box will appear 2 Select the desired source curve check that a First order calculation iS selected and click on OK The differentiated curve will appear in the active chromatogram 3 Measure the Y axis values on the differentiated curve using the XY curve co ordinates function Remember to select the appropriate Y axis scale Any Y axis value Is interpreted as the UV curve slope at the selected retention point If the differentiated curve Is very noisy it can be filtered using a light M oving average filter in the Operations Smooth function 4 Determine the highest slope value of the baseline non peak part of the curve Add an extra 10 and insert this value as the Slope limit E i 7 Al a Pai wa Figure C 3 Measurement of the Slope limit after differentiation C 6 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Evaluation functions and instructions C C 3 Peak table column components C A F Figure C 4 Diagram ill
282. y the original view of the chromatogram by clicking on the Unzoom command button in the top right corner of the active window Fixed scale axes Another way to display only part of a curve is to fix the minimum and maximum values of the y and or x axes in the Chromatogram Layout box See Section 7 2 7 Cutting curves T he Operations Cut curve function allows a region of the curve between two values on the x axis to be cut and stored as a new curve This is done in the following way 1 Select Operations Cut curve 2 A dialogue box will open in which you must select the curve to be operated on Click on OK 01 96041 04 1_ Cond 02 96041 04 1 _PressDet 03 96041 04 1 _PressA4ch 04 96041 704 1_CondP Figure 7 14 Select Curves to Operate On dialogue box 3 Theselected curve will now be shown in a new window which also contains two vertical cursor lines To facilitate the cutting process it is possible to use the zoom function within the window 7 14 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Presenting results Gil Sea Lea ad ih ia ai a E s a a a s s a m pe lke m P ee SSS _ e m r rE e F F p _ _ i Figure 7 15 Defining the limits of the curve to be cut using the cursor lines To make the cut ee drag the two cursor lines to define the left and right limits of the cut
283. yed in groups of three bases beginning from the 5 end 5 TCCATGGTAC 3 ES EJ 5 TCC ATG GTAC 3 Ea EJ Figure 4 4 An ungrouped top and grouped bottom sequence in the sequence editor field of the Sequence page 6 To save the sequence you have created click on the Save As button and type in a name for you sequence The name can be up to 256 characters in length Click on OK The name of the saved sequence will now be displayed in the Sequence page containing the specific sequence N ote that saved sequences are personal to the current user i e users logged in under a specific username will not see the saved sequences of another user 7 Placea check mark in those boxes beside the Optional method steps that you want to be included in your method UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA 4 5 4 Creating methods from method templates 8 Create the method for the sequence you have entered by clicking on the Create button The Create button serves four main purposes ee to check the sequence for invalid combinations ignoring the 3 base e g it is not possible to include both base A DNA and base a RNA in the same sequence since they both take up the same reagent bottle position on the instrument ee to generate a method based on the sequence and cross reference list see Section 5 1 1 ee update the method variables based on the generated method ee display the S
284. ystem administrator for UNICORN OS using the Administration User Setup command in the M ain menu Both the system and user definitions are stored on the network server and apply globally throughout the network Changes to system and or user setup can be made from any station in the network UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Installation 7 7 11 Installation This chapter describes how to install UNICORN OS hardware and software 11 1 Installation summary The following installation procedures are required before UNICORN OS systems can be used 1 Set up the network environment for network installations only Chapter 10 2 Install UNICORN OS hardware and software this chapter 3 Set up system definitions for the attached synthesis systems Section 12 1 4 Define access levels for the installation Section 12 2 5 Define users with home directories and access profiles Section 12 3 6 Check the system settings for the attached systems Chapter 13 11 2 System requirements 11 2 1 Hardware requirements Computer type Operating system CPU RAM minimum Hard disc recomm Floppy disc drive Communication N etwork support Printer or plotter M ouse M onitor IBM compatible PC Compaq with at least one free full length expansion slot IBM OS 2 Warp registered trademark of IBM Inc version 3 0 or later Pentium 75 M Hz or higher 24 M
285. yzed Alternatively x axis values 8 4 UNICORN for Oligo Synthesis version 1 10 User M anual 18 1118 52 Edition AA Evaluating results 8 for the Left limit and Right limit may be typed in Click on OK to return to the main dialogue box The baseline will be calculated from the whole curve but calculation of the areas beneath the peaks is only performed on the selected section of the curve The default peak window includes the entire curve HITS Ld ond Figure 8 4 Peak Window chromatogram window 4 Ifyou want to include negative peaks in the integration select check Accept negative peaks in the Integrate Peak integrate dialogue box or In Integrate Calculate baseline T he negative peaks will be reported as negative areas in the peak table By default negative peaks are not included in the integration 5 Inthe event that the automatic baseline calculation is not satisfactory it is possible to edit the baseline manually by inserting and deleting baseline points This is done with the Integrate Edit baseline function see Section 8 1 4 6 The lIntegrate Edit peak table function see Section 8 1 5 allows you to manually adjust the peak start and end points split and join peaks 8 1 4 Manually editing a baseline Once a baseline has been calculated it is possible to add or remove baseline points on it and then draw a new baseline from the new set of data points The edited baseline curve can then be used in a new peak i

UNICORN® OS User Manual - GE Healthcare Life Sciences

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UNICORN® OS User Manual - GE Healthcare Life Sciences