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UNICORN User Manual - GE Healthcare Life Sciences

1. 0000 o a Oligosynthesis system aa ahi PC with UNICORN PC where UNICORN PC with UNICORN agua is NOT installed 2 Oligosynthesis system Figure 11 1 A network installation with 4 synthesis systems and 5 workstations PCs The synthesis systems physically connected to PCs 1 and 5 can be controlled locally Alternatively any of the PCs with UNICORN installed can be used to remotely control any of the synthesis systems via the network In this example PC 4 is connected to the network but it cannot be used to control any synthesis systems since it does not have UNICORN installed Note also that the server does not have UNICORN installed and is not involved in the control process per se Creating user groups and users of UNICORN This is performed by the network administrator 1 Log onto theNT domain controller with administrative rights 2 Enter the User Manager for the domain by selecting Start Programs A dministrative T ools Common 3 Create a group that all UNICORN users will be part of Select the User menu and the New Local or Global Group item 11 5 1 1 Network setup 11 6 In the dialogue that is displayed enter a suitable name for the group and click on OK Create the users that will run UNICORN Select the User menu and the New User item In the dialogue that is displayed enter user name maximum 20 characters and must beunique within the domain description and passwo
2. Figure C 1 Baseline box with Shortest baseline segment and Noise window The rectangular box is allowed to be tilted with a maximum slope corresponding to the Slope limit see Figure D 2 The box is not allowed to move up above the Max baseline level Max baseline level Slope limit i Figure C 2 Slope limit and Max baseline level C 4 Evaluation functions and instructions C 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 2 2 Selecting baseline points for Classic algorithm 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 see Section 10 1 5 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 necessarily pass through
3. Cancel Help Figure 6 9 Properties dialogue Y Axis tab 3 Select the appropriate curve and click on the Fixed button Enter a minimum and maximum range in the fields within the specified limits 4 Repeat steps 2 to 3 for other curves 5 Click on OK Valueson the y axis apply to the curve with the same colour as the axis markings Click on the legend to get the correct Y axis Changing the scale of x axis 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 Alternatively select the x units in the X Axis tab of the Curve Properties dialogue Y ou can also set the viewed portion of the total run 1 Select View Curve Properties or select Properties from the right mouse button menu The Properties dialogue is displayed 6 11 sorom a run 2 Select the X Axis tab 3 Select the appropriate base Time or Volume Note Switching between bases may alter the resolution since the sampling frequency is adjusted according to the flow rate Y Axis Curve Style and Colour Flow Scheme Logbook Run Data Groups Run Data Colour Curves Anis m Base oj A C Volume m Axis scale Total C Window fio r Figure 6 10 Properties dialogue X Axis tab 4 Select the appropriate Axis scale either Total or Window The Total option
4. Instruction Parameter Curve operation ADD Integration C File operation C Export C Chom C Other C Test Insert Replace ddi Delete Figure 9 38 Procedure editor dialogue 6 Therecorded procedure can be viewed in the Procedure Editor dialogue Restore the iconised dialogue if necessary 7 M oresteps can be added to the evaluation procedure by repeating steps 2 6 The new steps are added to the previous procedure Note New lines will be inserted into the procedure after the selected linein the currently listed procedure This can be used to insert new instructions between existing instructions 8 If required edit the evaluation procedure see Section 10 3 2 9 Select File Save or File Save as from the dialogue menu and enter a name for the procedure The evaluation procedure is saved within UNICORN and is specific to your user name If you have Edit global list s access you may also check the Global procedure option to make the procedure available to all users Global 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 10 Choose dialogue File Exit menu command Evaluating results 9 Note If you already have an existing procedure open for editing in the Procedure Editor dialogue see Section 10 3 2 and you follow the above procedure new instructions will be added to the curren
5. 3 Change the values as appropriate and save the file Caution Do not make any other changes in the UNICORN INI file since this may severely affect the function of UNICORN Logon and file handling 3 3 5 Logging off To log off from UNICORN click on the Logoff button or select the Logoff menu command Processes that are running when you log off will continue to run and may be left locked with a locking password or unlocked see Section 6 5 for more details If the M ethod editor module was active at the time of logoff it will be re opened when the same user logs on again UNICORN will still be open after a user has logged off and another user may log on We recommend that you always log off when you leave the computer to prevent other users from accidentally changing or deleting your files or disturbing your runs 3 6 Quitting UNICORN To quit UNICORN and close the program select File Quit program in the M ain menu Y ou will be prompted to save any unsaved data in the M ethod editor or Evaluation module If a run is proceeding when you quit do not shut down Windows NT or turn off the computer while the run is in progress Note You can not quit the program if you are performing a M ethodQ ueue run Ion and file handling Introductory material Evaluation System management Appendices Creating methods from method templates 4 4 Creating methods from method templates UNICORN is supplied with a set of method tem
6. The installation program is about to create the system table and needs some additional information Enter a name that you want to use for the system defined below If you want to connect a strategy to the system select a strategy in the drop down list box Only strategies that have been installed are visible System name System 1 System type Oligo X Seia OPII_310 System settings Control board CU 900 Control unit 1 IRQ 10 Address 100 Cancel Figure 12 15 System Table Settings dialogue After the installation is complete the computer must be restarted for the installed software to be properly configured Click on Finish to exit the UNICORN Setup dialogue and restart the computer 12 17 1 2 Installation 12 18 Unicorn Setup Setup has completed installing Unicom C No will restart my computer later Click Finish to exit Unicom setup Figure 12 16 UNICORN Setup dialogue 12 5 2 Installing selected software components after the initial installation If your UNICORN installation should be damaged for example 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 files and suggests the components to install Check some or all of Program files U
7. 1 0 1 2 0 1 Recycle ON flowrate LFlow 0 1000 cm h OFF flowrate LFlow 0 1000 cm h B 5 B Strategy for OligoPilot II B 1 3 Alarms amp Monitors Instruction Description Comment Tetrazole Percent tetrazole of the column volume 1 100 AmiditeC onc 0 01 0 5 M Base Id 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 used 1 100 mm in Lflow functions CondiAlarm Sets the alarm and warning limits for the signal from the monitor CV Sets the column volume for calculation 1 200 of CT flows and volume of Capping and Beaucage CV_Cap Column volume of capping CapA 0 1 10 CV and Y2 CapB CV_Thiolat Sets the column volume for thiolation 0 1 1 0 CV Cycle Start M arks thestart point for an integration and thus where from retention is calcu lated DelayVol The dead volume from port1 to the 0 1 10 ml column inlet Eq_AM Equivalents of amidites dependent on 1 10 eq the scale Eq_OX Equivalents of oxidation dependent on 1 10 eq the scale pAlarm_Det pAlarm_ACN 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 6 Strategy for OligoPilot II B C ycleStart Indicates the synthesis cycle start in order to calculate r
8. 7 Enter your name company and product serial number for the software Click on the Next button to continue User Information Type your name below You must also type the name of the company you work for and the product serial number Name Amersham Pharmacia Company Biotech Serial Figure 12 3 User information dialogue 12 8 Installation 1 2 Note The serial number can be found on the UNICORN License Agreement that is shipped with the software 8 Select the components to install The components User information and Strategy and template files contain sub components These sub components can be accessed by clicking on the Change button W hen all selections are made click on the Next button to continue Select Components x Select the components you want to install clear the components you do not want to install Components Program Files User Information Adviser 2296 K Strategy and Template files OK System Installation 126K Description This component includes the UNICORN aes application files anae Space Required 21112 K Space Available 26400 K cos Figure 12 4 Select Components dialogue Stand alone installation e Check all components N etwork installation Program Files must be checked on all stations both local and remote User Information contains global files for the UNICORN software procedures BufferPrep recipes report formats
9. 9 3 4 Points to watch In recording and editing evaluation procedures for automatic evaluation beware of the following potential pitfalls M akesure 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 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 e In calculating a baseline using the classic algorithm UNICORN suggests default values for the four control parameters see Section 10 1 2 based on the appearance of the curve To instruct UNICORN to use default values appropriate for the curve every time the procedure is run choose the D efault setting in the appropriate fields for the parameters For example 9 Evaluating results CALCULATE_BASELIN E 01 06 XXX XXX XXX XXX can be changed to CALCULATE_BASELIN E 01 06 DEFAULT DEFAULT DEFAULT DEFAULT Similarly for baselines calculated using the Morphological algorithm
10. CALCULATE_BASELINE_M ORPH 01 06 XXX XXX XXX XXX can be changed to CALCULATE_BASELINE_M ORPH 01 06 DEFAULT DEFAULT DEFAULT DEFAULT 9 3 5 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 and select the dialogue Control Run menu command or click on the Play button 9 3 6 Batch runs Itis 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 9 5 3 1 Select Procedures Batch run and the Open Procedure dialogue is displayed Select the evaluation procedure and click on OK The Batch Run dialogue which allows you to search for the result files and or chromatograms on which you wish to perform the batch run Evaluating results 9 Chromatogram selection Feds eretan l Result
11. Variables Questions Notes Reference Curves Evaluation Procedures Method Information Result Name Start Protocal Sequence Selected Evaluation Procedures will run at the end of the method Import Print Synthesis Data Rename Edi Figure 5 33 The Evaluation Procedures 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 See Section 10 3 2 for a description of how to edit evaluation procedures N ote 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 procedureis executed at the end of the run See Section 10 3 for more details 5 33 ons and editing methods Selecting procedures to run The Evaluation Procedures page lists all evaluation procedures associated with the method Click on the procedure s which areto be executed at the end of the run The procedures will be executed in the order they appear in the list Importing procedures To import an evaluation procedure 1 Select the Evaluation Procedures page and click on Import 2 Choose a procedure from the Sele
12. oe and editing methods 5 2 For example in a sequencecontaining the bases 5 AC 3 theDNA base C is assigned to a specific block in the method template called Add_DNA_C Consequently by clicking on the Create button in the Sequence page the C base in the sequence causes the block Add_DNA_C to be copied into the method The next base in the sequence DNA base A is assigned to the block name Add_DNA_A Thus the block cross referenced to base A 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 W 0 00 Bloc DNA_C_ 2 Base Time Set_mark DNA C v2 Block Detritylation Block Detrit_wash Base_Id _dC Block Coupling_recycle_DNA_C_v2 Block Oxidation_DNA Block Capping_DNA Oligonucleotide synthesis direction C Add_DNA_C E 0 00 Block Add DNA_A_ v2 A Add_DNA_A Add__DNA_A_v2 0 00 Base Time 0 00 Set_mark DNA A 2 H E 0 10 Block Detritylation Mi 0 20 Block Detrit_wash 0 20 Base_Id _dA 1 0 25 Block Coupling_recycle_DNA_A_v2 Mi 0 30 Block Oxidation DNA Mi 0 35 Block Capping DNA 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
13. Determines if the manual changes can be performed also from the instruments Determines which pump flow rate GradientPump or SamplePump will be used to calculate method volume 14 3 Curves System settings 1 4 PumpType Only for OligoPilot Determines the types of pump used for pumps A B and C PumpGain Only for O ligoPilot Sets the gain factor for the pump control signal N ormal value 1 0 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 stored DPI Curves Instructions x r Instructions m Cond Parameters C Alarms iei Time between samples 1 000 s e OFF 2 ON C Specials PressDet A Store ON Time between samples C Monitors Time between samples 1 000 s F000 z PressACN T Time between samples 1 000 s Curves Set Selected Parameter To Strategy Default Value Cancel Help Figure 14 4 Curves settings in System control Settings in the Curves group include StoreD etermines whether the curve data is stored in UNICORN Time between samples The Time between samples determines the frequency with which curve data is recorded in UNICORN this does not affect the reading frequency of the monitor itself Default values are the shortest possible time between samples Caution If a curve is set to Store OFF data from the monitor concerned cannot be displayed in the curves win
14. If you receive the error message Cannot connect to system ina network installation check e that the local computer to which the system is connected is turned on and logged on to the network that the computer from which you are trying to establish a connection is logged on to the network e that 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 F 2 3 Run data Connection in System control displays a No xX In System control if the Run data option Connection says N o 1 or No2 e Check that the UNICORN PC Control board is configured according to the settings made during the installation of the program i e the same Control unit number Address and IRQ must be set at the Control board see Chapter 13 4 e Thecommunication may also fail if the UNICORN PC Control board configurations conflict with other boards in the PC If this is the case select a free Address and afreeIRQ during UNICORN installation and at the Control Board see Chapter 13 4 If the Run data option Connection says N o 3 there is no contact with the OCI Troubleshooting In Main menu select Administration System Setup In the dialogue select the system with prob
15. Security features 1 0 results in the Failed folder This policy has the advantage that the Failed folder can conveniently be used to temporarily store methods and results from runs performed from the local station when the network is not running e Grant access to the Failed folder only to one or a few users who share the responsibility for retrieving results files and deleting old files from the Failed folder 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 folder should also have access to other users home folders to be able to copy or move result files to suitable destinations In granting access to the Failed folder it is sufficient to grant access to DRIVEs Unicorn Local Fil Failed since this is the path to the Failed folder on each local station 10 3 2 Local station failure If the local station fails during a run the run may continue but the results generated after the failure cannot be saved An autosave feature saves a temporary result file on the local station during runs at a pre set interval defined in the system definition see Section 14 1 When the local station is restarted the temporary result file will be transferred to the original result file destination or to the Failed folder if the original destination was on a network drive which is not currently available The result file will contain the results of the run
16. column files and the users file Click on the Change button to select sub components to install These files need only be installed once in a network For all following installations to the same network these files will be already be present on the server Note Theusers file should be checked for the first installation only If the users file 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 12 9 1 2 Installation 12 10 10 Adviser should be checked on all stations both local and remote Strategy and Template files should be checked for each new strategy to be installed usually when a new system is installed on a local computer Each strategy needs only to be installed once since they are stored on the server e System should be checked on all stations where a system is connected but not on demo or remote stations If global procedures report formats BufferPrep recipes columns or ausersfile already exist 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 report formats BufferPrep recipes columns and the users file will be deleted and replaced by the defaults Select the disk drive where the program is to be installed and click on the Next button to continue This should bea physical disk drive usually c on
17. e Direct measurement e Viewing peak table data Direct measurement 1 Doubleclick on the small XY icon within the chromatogram area at the top of the y axis The active XY box contains coloured text which shows the x axis co ordinate and y axis co ordinate both with their respective units The colour and units of the y axis information corresponds to a specific curve of the same colour 9 Evaluating results 4 322401 1 L 15 322401 1_UV1_280nm pu 3 55 min 10 602 mAT 1D 2b 3b ab 5b 6b 7b min Figure 9 28 Active bar linked to XY box for determining curve point co ordinates Click on the desired curve legend and the correct y axis is displayed A vertical cursor line can also be seen on the chromatogram which is the same colour as the selected y axis scale Use the mouse pointer to drag the line back and forth along the chromatogram Co ordinates corresponding to retention time and peak height will change accordingly in the active XY box Double click on the active XY box to close this function Viewing peak table data The retention time and amplitude of any peak can be directly viewed in a peak table after an integration if selected for in the Chromatogram Layout dialogue 9 1 9 Measuring HETP HETP height equivalent to a theoretical plate calculations allow you to check how well the column has been packed 1 Perform a run with injection of a non i
18. these can arise if a method is written for onesystem and saved for another All such instructions must be deleted or changed before a method can be run see Sections 5 4 3 and 5 4 4 Normal text 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 0 Text with a loop symbol When a block is called from within itself this will generatea potentially infiniteloop which might exceed the maximum number of calls allowed in a method Double click on a Block or Watch instruction to display or hide the instructions in the called block or click on the symbol for the block respectively Double clicking on the Main keyword at the beginning of the method will show or hide instructions in all blocks in the method E Main E Main 0 00 Base CV 100 Column_Volume ml Any 0 00 Base CY 100 Column_Volume ml Any 0 00 Message Fill your column with DNA T support Screen 0 00 Message Fill your column with DNA T support Screen 0 00 Message Press CONTINUE when ready Screen 0 00 Message Press CONTINUE when ready Screen 0 00 Pause 1 0 0 00 Pause 1 0 0 00 Base_Id _dT Double 0 00 Base_Id _dT om et H 0 00 Block Purge_G click START_parameters 0 00 Block Purge_C 0 00 Base Time ic 0 00 Block Purge_T_U 0 00 C 100 C ml 0 00 Block Purge_A 0 00 Scale 1 00 Weight_of_support g 10 Loading_of_support
19. 0 00 Base_ld _dT Ml 0 00 Block START_parameters 0 00 Block Purge_G 0 00 Block Purge_C MM 0 00 Block Purge_T_U MH 0 00 Block Purge_A 0 00 Block Purge_Tetrazole Ml 0 00 Block Purge_solvents_ox 0 00 Block Column_wash MH 0 00 Block Add_DNA_T MM 0 00 Block Add_ DNA Mi 0 00 Block Add DNA 0 00 Block Add_DNA_ Ml 0 00 Mi 0 00 Block Add_ DNA 0 00 Block Add_DNA_ MI 0 00 Block Add_DNA_G ME 0 00 Block Add_DNA_A Ml 0 00 Block Add_DNA_A Mi 0 00 Ml 0 00 Ml 0 00 Block Add_ DNA Ml 0 00 Block Add_ DNA M_0 00 Block Add DNA C 2 S fa amp gt 4 4 5 z gt gt Ll ge ee gt p zz ZE FP ae zz Pr ZE Figure 5 9 The Method editor block window Conditional W atch instructions areindicated by a green line showing the start and duration of the watch The example above has a W atch instruction to start thefraction collector which is active throughout the gradient elution block Loop instructions to repeat a group of instructions are also indicated in the block window If you click on the line representing a block in the block window the first instruction in the block will be highlighted in the text window 5 3 2 Calling blocks To execute the instructions contained within a block in a method the block must be called by the program When a block is called the instructions in the block are executed in the order that they are written until the block is finished or the End_Blo
20. A 2 Control capacity A 2 1 Stand alone installations A 2 2 Network installations 13 4 13 4 13 4 13 7 13 9 13 10 13 12 13 12 13 12 13 13 13 13 13 14 13 14 13 14 13 17 13 17 13 18 13 19 13 22 14 2 14 4 14 5 gt gt bP PPDP D NN Y NBR a Contents A 3 Data sampling A 3 B General strategy for Oligo Synthesis B 1 Method instructions B 1 B 1 1 Pump B 1 B 1 2 Flowpath B 4 B 1 3 Alarms amp M onitors B 6 B 1 4 Watch B 7 B 1 5 Other B 8 B 2 Manual control B 9 B 2 1 Pump B 9 B 2 2 Flowpath B 9 B 2 3 Alarms amp M onitors B 9 B 2 4 O ther B 10 B 3 System settings instructions B 10 B 3 1 Alarms B 10 B 3 2 Specials B 11 B 3 3 Monitors B 12 B 3 4 Curve B 13 B 3 5 M ethod variables B 13 C Evaluation functions and instructions C 1 Smoothing algorithms C 1 C 1 1 Moving Average C 1 C 1 2 Autoregressive C 1 C 1 3 M edian C 2 C 2 Baseline calculation theory C 2 C 2 1 Defining baseline segments C 3 C 2 2 Selecting baseline points for Classic algorithm C 5 C 2 3 Drawing the baseline C 5 C 2 4 Estimating the baseline parameters from the source curve for Classic algorithm C 5 C 2 5 M easuring the Slope limit using D ifferentiate and curve co ordinates for Classic algorithm C 6 C 3 Peak table column components C 7 C 4 Evaluation procedure C 12 C 4 1 Curve operations C 12 Contents C 4 2 Integration C 15 C 4 3 File O perations C 16 C 4 4 Export C 17 C 4 5 Chromatogram functions C 21 C 4 6 O t
21. Baseine OOOO l Wel All Chromatogram Poo y Browse All r Found chromatograms Baseline example 2 1 Baseline example 11 Figure 9 41 Batch run dialogue The search will take place in the displayed folder only To select another folder click on the Browse button and open the desired folder By default all chromatograms are searched for within the designated folder and result files as denoted by the asterisk As for result files you can select a specific result file using the Browse function or you can use wildcard characters to search for chromatograms with a specific name profile Y ou can use standard wildcard characters in the file name specification stands for any number of characters and for any single character For example iex will search files named iex iex will search all files with names beginning iex iex will search all files with names ending iex iex will search only 4 character names ending in iex User entered search filters up to a maximum of 10 will be saved in the drop down menus for both Result and Chromatogram selections M ore than one string can be used as a search delimiter by inserting a between strings Search filters are automatically saved and stored within user profiles 9 Evaluating results To return to the default setting to search for all chromatograms click on All Click on the Search button and a list of chromatograms will be displayed base
22. End_method 83 66 End_method E E ic E E Ej Ej rc E E Ej re Base le Time Volume Figure 5 40 Log Format dialogue displaying the cumulative time for a method in rectangle with arrow 5 46 Creating and editing methods 5 The concept of block time or volume versus accumulated time or volume is illustrated in the following table Accumulated Block Instruction time vol time vol Main 0 0 0 00 Block Purge_G 0 0 0 00 Base Volume 0 0 sete 1 1 1 00 End_block 1 0 0 00 Block Purge_C 1 0 0 00 Base Volume 1 0 Sante Se 2 1 1 00 End_block 2 0 0 00 Block Purge_A 2 0 0 00 Base Volume 2 0 me deo ne 3 1 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 9 4 Messages and set mark 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 e g when Watch instructions are used for conditional events or at the end of a gradient see below 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 Scre
23. Select Edit Rename and the relevant menu cascade option Chromatogram Curve or Peak Table 2 Select the appropriate object in the displayed dialogue and typein thenew name Click on OK Thenew namewill replace theold one rather than creating a new curve or chromatogram Note Theoriginal raw data curves cannot be renamed and are not therefore given as options 8 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 detail how best to present them It is possible to e view several chromatograms at the same time e overlay curves from different runs in one chromatogram e stack curves from different runs in one chromatogram e stretch curves to make comparisons easier create mirror images 8 4 1 Comparing chromatograms from different runs To import chromatograms from other result files into an already opened result file two functions can be used namely File Open to compare or File Open The former option is most useful in searching Presenting results 8 for many chromatogramsin a specific folder based on defined selection criteria The latter option is best used to import any individual chromatograms from result files in different folders The imported chromatograms are sequentially numbered 11 12 13 etc for identification purposes Up t
24. mi bar b do 1000 1800 2060 2500 3000 35b0 mi Draw Spline Spline Through assa ml Delete Delete all Point mode foro bar Draw straight to next point Zoom mode Cancel Help Figure 9 37 Create Curve chromatogram window 9 31 9 Evaluating results 5 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 6 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 contrast Spline through creates a curve that passes through all of the data points 7 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 button This may have to be repeated for several consecutive points to achieve the desired curve 8 Click on OK and save the curve Y ou can change the curve name from the default CreatedCurve and also the curve destination 9 2 4 Measuring salt concentrations in the fractions If you need to know the approximate condu
25. monitoring of the synthesis process The run status can be displayed as numerical display of run data from selected monitors e graphical display of curves from monitors e aflow scheme showing the current open flow path in the system alogbook recording the control events in the run Systems can be controlled either manually with interactive commands or through pre programmed methods 2 7 2 UNICORN concepts 2 8 System Control 1 OPII Method d default HI01 m01 File View Manual System Adviser Help Run Hold eaused pause Commie End alala per poe Block Volume Acc Time Block Time Run Status ii Volume Cond 0 00 min Run 38 02 14 10 03 17 Method HIO1 Result qingrid HIO1 res Manual Logbook For Help press F1 End Bock Controlled By default Figure 2 4 The system control screen By using M ethodQ ueue facilities several methods may be run in a pre defined automatic sequence involving one or more oligosynthesis systems With suitable oligosynthesis system equipment this allows unattended operation of quite complex multi step synthesis processes 2 5 2 System connections For controlling a synthesis process the operator establishes a connection between the computer and the oligosynthesis system in one of the system control windows in UNICORN Two kind of connections may be established e Control mode connections which permit full
26. simultaneous view mode connections to one system on different computers T his allows a running process to be monitored from several locations at the same time M ultiple logons with the same username are treated internally as separate users for the purpose of System control Note Do not confuse Windows NT network logon with UNICORN logon Y ou log on to the network to gain access to network resources shared drives printers and other networked equipment You log on to UNICORN to gain access to the oligosynthesis systems that are installed in the network The username and password for logging on to the network are entirely independent of the those for logging on to UNICORN 3 2 Toolbar Guide 3 2 The UNICORN Toolbar Guide dialogue is shown after start up and logon reminding you about the M ain menu toolbar buttons TheM ain menu toolbar buttons allow you to begin using UNICORN quickly for example to create a new method in the M ethod editor start an instant run open aresult filefor evaluation or execute manual instructions in System control is eile ic Logon and file handling 3 Toolbar Guide x Begin your work directly by using the toolbar buttons UNICORN Main Menu m Methods Launch the Evaluation module immediately start a new run PP EERE Control your system Figure 3 2 UNICORN Toolbar Guide dialogue The toolbar buttons are About UNICORN This gives you information about the UNICORN v
27. stored here lt home gt H ome folders for users in the system There may be any number of home folders in the system In network installations home folders may be created on the server disk as well as on local disks method Holds method files M ethodQ ueue folders and user created method folders result H olds result files scouting result folders and user created result folders failed H olds result files in the event of network communication failure strategy H olds sub folders for each system physically connected to the local computer system0 system1 system2 system3 System information for systems physically connected to the local computer H olds a copy of the strategy flow scheme and templates and files for the system settings and system audit trail folder system0 corresponds to installed System 1 Folders for systems that are not installed are empty server H olds the original file for system definitions system tab This file is copied to the local folder File organisation E fil H olds the original files for user definitions users30 mpm global procedures globproc gpl global report formats globproc grf BufferPrep recipes global rcp comp rcp and the column list columns cmn The user definition file Column list and BufferPrep recipes are copied to the local fil folder
28. unconditional calls 5 11 Index UNICORN access problems F 1 cannot quit or logoff F 3 in the network environment 11 1 installation 11 13 installation summary 12 1 installing in NT domain with TCP IP 11 9 installing on network with IPX SPX Novell server 11 10 installing software 12 6 migrating from an earlier version 12 1 operations after installation 13 1 problem re establishing system connection F 5 system administrator 11 2 Unicorn concepts 2 1 introduction 1 1 main menu windows 3 5 network installation 3 2 oligosynthesis systems i 1 1 quitting 3 15 security features 2 11 security system 1 1 starting 3 1 summary of functional features 1 1 user interface 2 2 units adjusting in page layout 8 44 Unlocked 6 23 user administration 13 9 creating anew home folder 13 11 defining new user 13 10 deleting 13 12 home folder selection 13 11 identification 13 9 password 13 11 selecting user accessible folders 13 11 viewing and changing user definitions 13 12 user groups and users creating on local computer 11 11 user groups and users creating on NT domain with TCP IP network 11 5 user groups and users creating on NT workstations connected to a Novell server 11 10 user interface 2 2 user setup information printing 13 14 username entry 3 1 Vv Var button 5 24 variable Index in run documentation 8 52 removing 5 26 renaming 5 26 start protocol items 6 3 variables 5 38 defining 5 24 editing in run setup 5 27 identify
29. utility Follow the procedure below to install UNICORN For a network installation follow this procedure on each computer in the network Y ou can quit the installation at any point by clicking on either the Installation 1 2 Cancel button or the Exit button If you do this however the installation will be incomplete and the software cannot be used Note Beforetheinstallation is performed on a local station be sure that you know which interrupt and address that the system s will use It is possible to install the UNICORN software without this information but the system s will not work Refer to chapter 13 3 for information about the system settings 1 For network installations log on to the network and check that you have access to the server disk and folder where UNICORN network components are to be installed 2 Insert the CD rom disk into the CD rom drive 3 Select Run from the taskbar Start button menu 4 Write the command d setup where a is the unit for your CD rom drive Click on OK 5 TheUNICORN setup program is launched and you will see the Welcome dialogue Click on Next to continue Welcome to the Unicorn Setup program This program will install Unicorn on your computer It is strongly recommended that you exit all Windows programs before running this Setup program Click Cancel to quit Setup and then close any programs you have running Click Next to continue with the Setup program WARNING This p
30. 1 8 8 Click on the X Axis tab in the Chromatogram Layout dialogue Curve Style and Colour Edit Texts Layout Library Header CurveNames Y Anis Axis Cuve PeakTable Base C Volume Gol i m Axis scale Auto r Min Mag C Fixed 0 10 min IV Adjust retention zero to injection number 1 v I Apply to all chromatograms Cancel Figure 8 7 Chromatogram Layout dialogue X axis tab Select the appropriate choice from the Base field either Time of retention Volume or Column volume Presenting results 8 Note Some calculated curves e g baselines exist in only one base and may seem to disappear when the base is changed In addition switching between Time and Volume base may alter the resolution since the sampling frequency is not adjusted according to the flow rate 3 Click on the Axis Scale Fixed option Type in the desired minimum and maximum values for the x axis 8 2 8 Viewing information about the run Y ou may wish to display header information at the top of a chromatogram detailing the variables questions and or notes H eader information cannot be displayed for imported chromatograms 1 In the Chromatogram Layout dialogue click on the Header tab 2 Check the options to be included in the header of the chromatogram window test20res 1 Variables Column_Volume 630 m cV 630 ml Weight_of_support 1 879 g test20_PressACN t Mo M WM M e M ee
31. 1 Select the destination folder in the Methods window or Results window 2 Without selecting afileicon bring up the right menu button menu and select Copy from external or select File Copy from external T he Copy from external dialogue is displayed 4 Select the wanted file s from the relevant source drive and folder Click on the Save button Jaon and file handling Copy from External Copy From 2 SASAS a sco1 m01 a sco2 m01 Figure 3 9 Copy from external dialogue in this example used to copy method files If result file s were selected these will be copied into the previously designated folder in the Results window If method file s were selected the Method System connection dialogue is displayed Method System Connection x Method files Systems Select a method file Double click on a system Help Figure 3 10 Method System connection dialogue Each copied method listed in the Method files field must in turn be connected to the same type of system same strategy for which the method was originally created listed in the Systems field Highlight a method and double click on a system Click on the OK button Logon and file handling 3 T he Method System connection dialogue is displayed again listing the remaining methods to be connected R epeat the process until all methods have been connected Note M ethod syntax errors may arise if a method created on one system is con
32. 10 these will be reformatted so that they can be used by the new system e Document your user setup access rights and folder structure so that they can be redone in the new version e Print out the system settings and calibration constants 12 1 12 Installation 12 2 2 Migration and post installation setup 1 2 Install UNICORN 3 10 Install strategies either from diskette supplied with the original system using the Have Disk option or by selecting from the standard systems supplied in the installation CD ROM Set up the users folders and access rights R ecalibrate the system Enter system settings Copy the methods and results you want using Copy from External If you are using multiple systems you might need to associate your copied files with a system in this process 12 3 System requirements For the hardware software and network requirements please refer to Appendix A 12 4 Hardware installation 12 2 In most cases an installation of your system will be performed by Amersham Biosciences authorised personnel If your system is not preinstalled then follow the steps below to install the 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 remove the power cable from the mains socket O pen the PC cover Refer to your PC documentation
33. 12 Select the program folder in which you want the UNICORN icon to be placed Y ou can either create a new folder or select one that already exists The folder will be placed in the programs menu under the Start button menu Select Program Folder Ea Setup will add program icons to the Program Folder listed below You may type a new folder name or select one from the existing Folders list Click Next to continue Program Folders Unico Existing Folders Administrative Tools Common tartu Figure 12 8 Select program Folder Installation 1 2 13 At this point the setup program is ready to start copy the files A dialogue will display all the selections that have been made If the settings are correct click on the Next button to start copying the files If you want to make any changes you can click on the Back button Start Copying Files x Setup has enough information to start copying the program files If you want to review or change any settings click Back If you are satisfied with the settings click Next to begin copying files Current Settings UNICORN 3 10 Software Setup User Information User name Amersham Pharmacia Company Biotech Component Selection Figure 12 9 Start Copying Files dialogue 14 If you have chosen to install systems the system installation dialogueis displayed after the file copy process Select a system for OligoPil
34. 2 3 Choosing the curve s you want to see 8 2 4 Changing curve names 8 2 5 Changing the colour and style of curves 8 2 6 Defining and positioning curve text 8 2 7 Changing and fixing the axes 8 2 8 Viewing information about the run 8 2 9 Saving and applying a layout 8 2 10 Viewing a grid in the chromatogram window 8 3 Other presentation possibilities 8 3 1 Showing part of a curve 8 3 2 Reducing noise and removing ghost peaks 8 3 3 Subtracting a blank run curve 8 3 4 Adding curves 8 3 5 Entering text in the chromatogram 8 3 6 Renaming chromatograms curves and peak tables 8 4 Comparing different runs 8 4 1 Comparing chromatograms from different runs 8 4 2 Comparing curves 8 4 3 Stacking and stretching curves 8 4 4 Mirror images of curves 8 5 Saving results 8 6 Printing active chromatograms 8 7 Printing reports Contents 7 5 7 5 7 6 pp P N LPPPPPPPPP P P OONNOUUOBHBW N N So e 8 10 8 13 8 14 8 17 8 17 8 18 8 18 8 18 8 22 8 27 8 31 8 32 8 32 8 33 Contents 8 7 1 Creating a new customised report format 8 7 2 Creating a new standard report format 8 7 3 Modifying an existing report format 8 8 Run documentation 8 9 Exiting Evaluation 9 Evaluating results 9 1 Integrating peaks 9 1 1 Baseline calculation for integration 9 1 2 Performing a basic integration 9 1 3 Optimising peak integration 9 1 4 O ptimising the baseline parameters using a morphological alg
35. 21 measuring peak asymmetry 9 25 measuring retention time and peak heights 9 23 optimising peak integration 9 4 peak table column components C 7 purity 9 26 reject peaks 9 5 skimming 9 7 splitting a peak 9 21 viewing peak table data 9 24 peak table renaming 8 18 Peak_Frac Parameters setting 14 4 Permissions button 11 6 PID feedback tuning D 1 tuning parameters D 1 Point list 9 19 Point mode button 9 19 pressure curve selecting units 6 13 print active chromatograms 8 32 audit trails 13 17 can not print screen on printer F 5 printer setup 3 14 report 8 46 8 49 reports 8 33 user setup information 13 14 Index Print_Chromatogram 5 32 procedure instructions editing the parameters 9 36 inserting new instructions 9 36 removing instructions 9 36 saving the edited procedure 9 36 viewing the parameters 9 35 procedures defining and viewing 5 33 deleting 5 35 editing 5 35 importing 5 34 placing on the menu and running 9 41 recording 9 33 removing 9 37 renaming 5 35 9 37 selecting to run 5 34 PumpGain setting 14 5 PumpT ype setting 14 5 Q questions defining for answer type 5 28 deleting 5 31 display at start of run 5 38 display in start protocol 6 3 editing 5 31 Input field 5 28 inserting 5 30 multiple choice 5 29 no answer 5 29 types mandatory authorised chromatogram 5 27 value 5 30 quit problem quitting UNICORN F 3 quitting Unicorn 3 15 R ratio value in peak skimming 9 7 ratios between UV curves 9 26 re
36. 5 Entering text in the chromatogram Basic annotations can be added to the chromatogram 1 Place the mouse pointer in the curves view of the chromatogram window and select Add text from the right mouse button menu Alternatively select the Edit Text Add command The mouse pointer is replaced with an ABC pointer 2 Position the pointer where you want to insert text in the chromatogram and click the left mouse button once 3 Inthe dialogue that appears type the desired text and then click on OK N ow 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 The text cannot be moved within the window once it has been placed If you want to edit or delete an inserted text 1 Open the Chromatogram Layout dialogue and select the Edit Texts tab Alternatively select the Edit Text Edit command and the Edit Texts tab is displayed automatically 8 Presenting results 2 Select the specific text that you want to edit and make the appropriate changes in the Selected text field Click on Change text 3 Select the specific text that you want to delete and click on Delete text 4 Click on OK to close the dialogue and apply the changes 8 3 6 Renaming chromatograms curves and peak tables Sometimes it may be desired to change the name of a chromatogram curve or peak table To do this close the Chromatogram Layout dialogue and then 1
37. 52 03 AM Connection change User default at computer MARIA connected to system Explorer 4 28 1399 osasi AM New System OPII r Global 4 28 1999 11 10 55 AM Logoff default 4 28 1999 11 11 00 AM Connection change User at computer MARIA disconnected from system Explorer 4 28 1999 11 11 23 AM Logon default User West 999 11 11 36 AM Connection change User default at computer MARIA connected to system Explorer 28 1999 11 12 10 AM Connection change User default at computer MARIA disconnected from system Explorer ea 999 11 12 16 AM Connection change User default at computer MARIA connected to system OPII M Backup 28 1999 6 55 30 PM Logoff default bet 999 6 55 32 PM Connection change User at computer MARIA disconnected from system OPII Logon Logoff 4 29 1999 8 54 48 AM Logon default eat a 8 54 57 AM Connection change User default at computer MARIA connected to system OPII PM Defi t 9 48 22 AM Connection change User default at computer MARIA disconnected from system OPII moguin ne ea 9 54 17 AM Connection change User default at computer MARIA connected to system OPII I MethodQueue m Renew log file Renew every 1 day Close Help Administration 1 3 Figure 13 10 Global audit trails User Backup Log on off Define system MethodQueue Sequence Displays all user creation deletion and re definition operations Displays backup operations for global audit trail files Displays all logon and logoff att
38. 8 the overall method time or volume may vary according to watch events during the run Log format The difference between block time and accumulated time can be viewed in M ethod editor by viewing the Log Format To view the accumulated time for a method select View Log Format or ry click on the Log Format toolbar button The Log Format dialogue is displayed The Log Format dialogue displays the cumulative time or volume for the current method Log Format x E Main 0 00 Base CY 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 Pause 1 0 0 00 Base_Id _dT W 0 00 Block START_parameters 0 00 Block Purge_G_ 2 W 0 00 Block Purge_C_ 2 W 0 00 Block Purge_T_U_ 2 W 0 00 Block Purge_A_ 2 A A A E 0 00 Block Purge_Tetrazole Ml 0 00 Block Purge_solvents_ox E m 0 00 Block Column_wash 1 Base CY 6 30 Column_Yolume ml Any H E 26 33 Block Add DNA T_ 2 Message Fill your column with DNA T support Screen H 51 33 Block Add_DNA_T_v2 Message Press CONTINUE when ready Screen ll 74 66 Block Add_DNA_G_v2 Pause 1 0 H 78 66 Block Final_detritylation Base_ld _dT Block START_parameters Block Purge_G_ 2 Block Purge_C_ 2 Block Purge_T_U_V2 Block Purge_A_ 2 Block Purge_Tetrazole Block Purge_solvents_ox Block Column_wash Block Add_DNA_T_v2 Block Add_DNA_T_v2 Block Add__DNA_G_v 2 Block Final_detritylation
39. Attachments You have the option of attaching result files method files and or log files to the report 1 To makean attachment first select the appropriate tab Result method System log or Global log Result Method System log Global log Baseline example 1 res r System information IV Computer amp operating system information gt ARTA hardware mformation I Integrity check lt Back Next gt Cancel Help 2 Click on the Add button and browse in the displayed dialogue to find the file that you want to attach 3 Select a file and click on Open The selected file is added to the tab in the Attachments dialogue System information can also be included in the report by checking the appropriate option boxes By default the following options are checked Computer amp operating system information This summarises information about the computer and operating system e g type of processor processor speed RAM memory hard disk capacity and printer Integrity check When UNICORN is installed a checksum calculation is performed on the stationary files dll and exe for the system An integrity check involves a new checksum calculation being performed for the same files in their 13 21 1 3 Administration 13 22 folders and comparing the new calculated value with the checksum value obtained during installation The results of the comparison are presented in the report and any devi
40. C _Column_wash C _column_wash fe00 Cv 0 00 9938 00 Detrit_peak_start Detrit_flow 300 emh 0 00 2000 00 zl Figure 5 7 Run setup with the Variables page displayed To access a given pagein the Run setup click on the relevant tab Pages in the Run setup editor are described in Section 5 6 5 3 Method blocks 5 8 Viewing a method as a long list of individual text instructions would be confusing and inconvenient Text instructions are therefore conveniently grouped into blocks of instructions that define a specific functional use For example one block might contain the instructions necessary for equilibrating a column and another block contain instructions for adding a single defined nucleotide in the sequence etc By using such blocks it is easier to build up a total method for a run 5 3 1 Viewing blocks It is possible for one block to contain one or several other blocks This is most evident for example in the T ext instruction window Unexpanded blocks in a method Expanded block ina method containing textinstructionsand blocks Creating and editing methods 5 In the text instruction window In the text instruction window the method is shown asa list of blocks denoted by the blue square symbols Beside each block is also a symbol which you can click on to expand the view of the instructions within the block N ote that a block can also contain other blocks as denoted by the blue square sy
41. Calibration Displays monitor calibration operations Viewing older audit trails W hen you choose Administration Audit trail the audit trail dialogue 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 If you have saved log files on diskette these drives can be selected from the pull down list Files are named by date and serial number Choose Current log file to return to the current audit trail after viewing older files Administration 1 3 Open log file xi Drive Log drive x Log files oK Cancel Help Figure 13 12 Open Log File dialogue Printing audit trails Click on Print in the audit trail dialogue menu prints the audit trail file as currently displayed in the window 13 4 2 Renewing audit trail files The audit trail file is renewed at regular intervals between 1 and 30 days To set the interval choose 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 This will not affect the automatic setting e g if the audit trail is set to
42. Cancel Help Figure 8 22 Open curves dialogue Select File Open The Open Curves dialogue is displayed Select the folder in which to search for curves Click on the result file of choice and where appropriate the specific chromatogram containing the desired curve The chosen chromatogram and curves contained therein will be listed Select the desired curve and click on the Select button T he selected curve will now be displayed in the Selected curves list If you want to choose more curves from other chromatograms repeat steps 3 4 When all the desired curves have been selected click on OK Restore the chromatogram window and open the Chromatogram Layout dialogue 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 9 2 6 Alternative C Copying curves into one chromatogram Curves can be copied between chromatograms present in the Evaluation desktop For effective comparison of curves it is suitable to transfer all relevant curves to a single chromatogram This is best achieved by creating a new chromatogram using File New Chromatogram and copying curves into it from other chromatograms or copying an existing chromatogram using Edit Copy Chromatogram and importing more curves into it or Presenting results 8 copying curves into the Temporary chromatogram see Section 9 1 2 You can perform evaluation
43. Exports the source curve to the file Source curve Left CURVE_W_ defined in Export to Filein WKS limit Right limit KS format In the part of source curve Every lt n gt sample limited by Left limit and Right limit Export to file Every lt n gt samples are exported EXPORT_ Exports an evaluation log in ASCII Export to file EVAL_LOG format to the file defined in Export _ ASCII to file EXPORT_ Exports an evaluation login WKS Export to file EVAL_LOG format to the file defined in Export _WKS to file C 17 Cora functions and instructions EXPORT_ Exports an evaluation log as XLS Export to file EVAL_LOG format to the file defined in Export _ XLS to file EXPORT_ Exports a method to the file defined M ain Blocks Export METHOD_ lin Export to filein ASCII format If to file ASCII all parameters are OFF then no method is exported If M ain is ON then the main method is included and if Blocksis ON then all blocks are included in the exported file EXPORT_ Exports a method to the file defined M ain Blocks Export METHOD_ lin Export to filein WKS format If to file WKS all parameters are OFF then no method is exported If M ain is ON then the main method is included and if Blocksis ON then all blocks are included in the exported file EXPORT_ Exports a method to the file defined M ain Blocks Export METHOD_ lin Export to filein XLS format If all to file XLS parameters are O
44. Figure 6 11 The flow scheme for a run Viewing multiple flow schemes If there is more than one flow scheme picture for the system there may beup to five pictures per system then these can be selected and viewed in the right mouse button menu 6 2 5 Logbook All actions including method start and end base instruction method instructions and manual interventions such as Pause or Hold 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 The log is saved in the result file Performing a run 6 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 Pause 1 0 0 00 Base_Id _dT 0 00 Block START_parameters 0 00 Block Purge_G_V2 0 00 Block Purge_C_ 2 0 00 Block Purge_T_U_ 2 0 00 Block Purge_A_V2 0 00 Block Purge_Tetrazole CSCO CoC Coca 0 00 Block Purge_solvents_ox 0 00 Block Column_wash Block Add_DNA_T_v2 0 00 Block Add_DNA_T_v2 0 00 Block Add_DNA_G_v2 aaa 0 00 Block Add_DNA_A_v2 0 00 Block Add_DNA_A_v2 0 00 Block Add_DNA_G_v2 0 00 Block Add_DNA_C_v2 0 00 Block Add_DNA_G_v2 0 00 Block Add_DNA_A_v2 0 00 Block Final_detritylation 0 00 End_method Le LLLLLEELELELLELELEL 8 SCS CoC Coc
45. Items I Method editor I Run methods I Evaluation I Manual interaction IV User setup Levels I Pause M Audit trail System setup T Calibrate Tune I Delete move Home only IV System settings M Delete move T Edit MethodQueue M Copy file s T Run MethodQueue I Confirm IV Edit global list s I Unlock locked system VV Maintenance Quit program coc ee Figure 13 3 Suggested authorisation profile for the system administrator Development staff Developers need to be able to edit and run methods and M ethodQ ueues issue manual instructions configure system parameters calibrate monitors and evaluate data T hey may also copy move or delete files 13 7 1 3 Administration Define Access Levels Berane m a a a e m m e a AIAI A I 4 Figure 13 4 Suggested authorisation profile for development staff Process supervisors Supervisors may pause a method and issue manual instructions as well as start methods and M ethodQ ueues Supervisors are also allowed to calibrate monitors configure system parameters unlock running processes evaluate run data and quit UNICORN Define Access Levels Berane a M al BE al al al M M AAAA A I 4 Figure 13 5 Suggested authorisation profile for process supervisors 13 8 Administration 1 3 Process operators Process operators are allowed to run and pause methods and M ethodQ ueues but may not perform any other oper
46. Select File Export and then the appropriate cascade 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 a spreadsheet application software 9 3 10 Exporting results Data can be exported to ASCII text and Lotus 1 2 3 wks or xls spreadsheet formats Select the format that best matches your other application software Peak tables are exported as text strings in the ASCII format but as numerical values in the Lotus 1 2 3 wks or xls format When exporting peak tables all possible columns in the table are exported 9 Evaluating results Export Documentation x Export documentation parts tes Iv Info M Start notes I System settings M Run notes I Scouting IV Evaluation notes IV Variables T Questions I Log book IV Evaluation procedures T Result name I Calibration IV Column parameters V Reference curves T Buffer prep recipe M Start protocol Cancel Help Figure 9 42 Export Documentation dialogue Curves are exported as two series of numerical co ordinates referring to time volume and signal respectively M ultiple curves 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 option T
47. UNICORN click on the lt Windows M gt keys on the keyboard Note Minimizing a module window to the taskbar does not close the module Once opened UNICORN modules remain active until you quit the program A minimised System control module may thus be actively in control of a running process 2 2 3 On line help A comprehensive on line help utility is included in UNICORN software Entry to the general help utility can be accessed from the Help menu Dialogue or window specific help topics can be obtained by clicking on the Help button in the dialogue or by pressing lt F1 gt o0n the keyboard In the dialogues for method instructions procedure instructions and system settings pressing lt F1 gt when an instruction is highlighted will display an information box with short help on the function and use of the selected instruction 2 3 Files and folders UNICORN M ain menu interface divides user files into two categories for methods and results see Figure 2 1 Only folders to which the current user has access are shown in the M ain menu windows Method window and Results window Files may be displayed in several viewing options see Chapter 3 for more details 2 3 1 Method files M ethod files contain instructions for controlling a run and are shown in the Methods window of the M ain menu The Methods window also displays icons for M ethodQ ueues which allow several methods to berun in an automatic pre programmed sequence on the same
48. UNICORN 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 on to the network e A 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 e A system that is locked by a user can be unlocked on any computer in the network not possible during M ethodQ ueue runs by any user with sufficient access rights see above e A UNICORN user may connect to a system with up to eight different remote workstations in the network Each successive multiple instance of the user automatically establishes the same System control connections as the first instance when the logon is performed M ultiple instances are however treated by UNICORN as separate users although they are not distinguished in the System control workspace 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 modules independently of each other once the logon is performed See Section 2 6 2 6 6 Calibrating monitors 6 24 Certain system monitors need to be calibrated regularly for correct results According to the ro
49. User information 12 8 User set up 13 10 Variable name definition 5 25 Variables page in Documentation 8 52 Welcome 12 7 Windows 5 7 X Axis tab in Properties dialogue 6 12 Y Axis tab in Properties dialogue 6 11 Divide 9 26 Draw a frame around the pages 8 44 Draw spline 9 32 Draw straight to next point 9 32 E edit chromatogram layout 8 4 editing text inserted in a chromatogram 8 17 existing procedure 9 35 method instructions 5 6 method variables 4 7 M ethodQ ueues 7 4 methods overview 5 1 parameters in procedure instruction 9 36 procedures 5 35 system definitions 13 4 text instructions 4 12 5 6 Index error cannot connect to system F 2 connections not available F 2 couldn t create result file F 3 M ethod System Connection dialogue keeps appearing F 3 No x F 2 strategy file error F 1 evaluating results 9 1 Evaluation exit 8 55 functions and instructions C 1 problems F 5 evaluation overview of evaluation facilities 2 9 Evaluation button 3 4 Evaluation log adding Evaluation log object to report 8 41 evaluation module overview 2 3 evaluation procedures 5 32 abort problem F 5 automated evaluation procedures 9 33 chromatogram functions C 21 curve operations C 12 display after run 6 4 editing 9 35 export C 17 file operations C 16 integration C 15 miscellaneous C 22 potential problems 9 37 reporting 9 40 running 9 38 evaluation procedures display at start of run 5 39 Execute button 6 19 exiting
50. a slight instability leading to oscillations in the actual flow can have values between 0 and infinity where smaller values have greater effect and a value of infinity has no effect The value infinity is set as 9999 in UNICORN e In certain cases the D parameter can reduce the oscillations introduced by a Pl regulator D can have values between 0 and infinity where larger 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 OligoProcess System is configured by default with the D parameter set to zero D 1 D Feedback tuning setpoint Stationary 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 Tuning a feedback control system in practice is largely a matter of trial and error The following recommendations summarize the Ziegler Nichols 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 D 2 I M ake sure that the flow path is open Select System Tune in System control Select Flow for Feedback Control O pen the valves and start the pump with the column in line Set P 0 05 9999 and D 0 Set the flow rate in Setpoint Press New parameters and
51. adapter to use by clicking on Select from list If your adapter is shown in the displayed list select it and click on OK Alternatively click on Have disk and insert the disk with the latest version of your driver Enter the path to it in the dialogue and click on OK 11 11 1 1 Network setup 11 12 10 12 13 14 15 16 17 18 19 20 21 22 23 24 Click on Next M ake sure your selected adapter is shown in the list and click on Next Select NWLink IPX SPX Compatible Transport as the protocol to use Deselect TCP IP Click on Next Select all default the services and click on Next Select Client Service for NetWare and click on OK Click on Next Click on Next again to install the selected components Insert the Windows NT CD ROM disk and enter the path to it in the dialogue Example D Click on Continue A dialogue is displayed displaying where the files will be read Click on Continue The bindings are displayed Click on Next to continue Click on Next to start the network Select the Workgroup radio button and enter a name for the workgroup Thenetwork administrator should supply this name click on Next to continue Click on Finish Remove the CD ROM disk Select the option to restart your computer now The computer is restarted Log on with administrator rights The Select NetWare Logon dialogue is displayed The network administrator must supply the Defau
52. and results destined for network drives cannot be saved in their correct folders In this event the run continues under control from the local station R esults are saved on the local station with the original result file name in the folder DRIVE Unicorn Local Fil Failed from which they can be retrieved after the run is complete To retrieve results from the Failed folder 1 Start UNICORN if it is not already started on the local station where the synthesis process was run 2 Logon asa user with access to the Failed folder 3 You can now open the result file in the evaluation module and moveit to a suitable location on the network server when network communication is re established so that it is accessible from remote stations Note Result files are saved directly under the Failed folder 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 folder Access to the Failed folder The system administrator may choose one of two policies concerning access to the Failed folder e Grant access to the Failed folder 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 folder on the individual users N ote that with this policy any user will be able to examine copy move and delete the other users
53. and stores the result in the target curve position Target curve position Shift factor C 13 Cora functions and instructions C 14 SMOOTH_A Smooths source curve with an Source curve R autoregressive filter and stores the Target curve result in target curve position The position Filter parameter decides the strength _ Filter of the filter SMOOTH_ Smooths the source curve with a Source curve MA moving average filter and storesthe Target curve result in Resulting Curve The Filter position width parameter decides how many _ Filter width samples wide the filter is SMOOTH_ Smooths the source curve with a Source curve MEDIAN median filter and stores the resultin Target curve target curve position The Filter width position parameter decides how many samples Filter width wide the filter is SUB Subtracts two curves to gain a third First source curve curve which is the difference of the Second source two curves The two source curves curve must have the same y axis unit and Target curve not be fraction or injection curves position TDIV Divides two curves to gain a third First source curve curve which is the quotient of thetwo curves The two source curves can have any y axis unit The threshold values are used to avoid division of numbers close to zero At those points where source curve 1 has amplitude less than Threshold1 or source curve 2 has amplitude less than T hreshold2 the re
54. are installed and one of the protocols TCP IP or NW Link IPX SPX User rights The requirement in the above list regarding user right is satisfied if a default installation of the network is performed The default is for everyone to have the user right Access this computer from network H owever if this is nor the case then the user rights should be set by a person with administrative privileges on the N T workstation using the following steps 1 Select Start Programs Administrative Tools Common User Manager 2 Select Policies User rights from the dialogue menu 3 Select Access this computer from network from the drop down list 4 Click on the Add button 5 Select the group or person s who will get this right It should bea group within which the person logged on to the local UNICORN station isa member or that person is specifically selected 6 Click on the OK button Network setup 1 1 11 3 Installation guide 11 3 1 TCP IP NT domain This is an example of how to setup the network so that UNICORN may run across the network when the network protocol is TCP IP and there isan NT domain controller The setup of the NT domain controller is not described here a competent network administrator is generally needed to setup an NT domain server Oligosynthesis system Fefe PC with UNICORN Network sever PC with UNICORN Oligosynthesis system
55. be controlled with manual instructions issued from the Manual menu The available instruction options are dependent on the strategy and only instructions for the components defined for the system are displayed To save the results of a manual run issue the instruction Record_on in the Other instruction group at the beginning of therun UNICORN will prompt for a result file name at the beginning of the run The Manual menu opens a dialogue similar to the text instruction box in the M ethod editor see Section 5 4 Instructions p Parameters C Pump FlowRate 10 1000 Insert Flowpath WasteDut 0 000 H omh Dalis Recycle Alarms Mon ACN Pump Te Detrit_Pump c Valvel OFF ON C Other Valve2 Figure 6 15 The Manual instruction dialogue Thenameof the connected system is displayed in the title bar of System control Available instructions are determined by the strategy and selected optional components for the connected system Instructions for the O ligoPilot strategy is listed in Appendix B Performing a run 6 M anual instructions are entered in the same way as method instructions from the dialogue in the M ethod editor T he Insert button places the current instruction in the list at the bottom left of the dialogue Clicking on Execute executes all instructions in thelist at the same time or executes the currently marked instruction if the list is empty N ote that although al
56. clicking on an object with the right mouse button T his generates a popup menu that allows you to the highlighted section 3 Youcan also modify the contents of the highlighted report section by choosing the Properties command From the popup menu click on Properties make the desired choices on the resulting dialogue and press OK 4 Once the desired formatting choices have been made clicking on the Preview button will display the page layout Press the Print button to print out the report Also you can save the modified format under a new name by selecting File Save As Standard format Choosing the Standard format button generates a second Edit Standard Report Format dialogue with tabs representing format options for the major sections of the report The options within the various tabs are identical to those available when creating a new standard format see Section 9 7 2 Presenting results 8 Create Standard Report Format x Evaluation Log Sequence Contents Header Method Documentation Chromatogram The selected items will be included in the report r Select Items Select all Clear Figure 8 45 Standard format dialogue Once the desired formatting choices have been made clicking on the Preview button will display the page layout Press the Print button to print out the report Also you can save the modified format under a new name by clicking on the Save As button 8 8 Run doc
57. column 1 4 p1 0 p2 1 5 2 6 1 valve6posl G to column 1 5 p1 0 p2 1 5 2 6 1 ACN_T U to column 1 7 p1 0 p2 1 5 2 6 1 T U to column 1 6 p1 0 p2 1 5 2 6 1 ACN_A to column 2 1 p1 1 p2 1 5 2 6 1 A to column 2 2 p1 1 p2 1 5 2 6 1 ACN_C to column 2 4 p1 1 p2 1 5 2 6 1 c to column 2 3 p1 1 p2 1 5 2 6 1 ACN_G to column 2 4 p1 1 p2 1 5 2 6 1 G to column 2 5 p1 1 p2 1 5 2 6 1 ACN_T U to column 2 7 p1 1 p2 1 5 2 6 1 T U to column 2 6 p1 1 p2 1 5 2 6 1 ACN_X to column 1 1 p1 0 p2 1 5 2 6 1 X to column 1 8 p1 0 p2 1 5 2 6 1 ACN_Y to column 2 1 p1 1 p2 1 5 2 6 1 Y to column 2 8 p1 1 p2 1 5 2 6 1 Solvent Beaucage 3 7 p1 0 5 2 6 1 ACN_3 1 to column 3 1 p1 0 5 2 6 1 Cap_A to column 3 2 p1 0 5 2 6 1 Cap_B to column 3 3 p1 0 5 2 6 1 ACN_3 4 to column 3 4 p1 0 5 2 6 1 OX to column 3 5 p1 0 5 2 6 1 Extra_3 6 to column 3 6 p1 0 5 2 6 1 Tetrazole to column 3 8 p1 0 5 2 6 1 Every amidite and solvent setting can also be set to waste This means setting value 6 from position 1 to position 2 B 4 Strategy for OligoPilot II B Waste ut Waste ACN valve 6 1 Waste Detrit valve 6 2 Waste A valve 6 3 Waste_C valve 6 4 Waste G valve 6 5 Waste_T valve 6 6 Waste_X valve 6 7 Waste_Y valve 6 8 ACN Pump to waste to column Detrit Pump to waste to column Valve 1 1 8 2 1 8 3 1 8 4 1 8 5 1 3 6 1 3 Port
58. connections 2 8 control unit number selection 13 3 Controlled by status bar message 6 17 copying blocks 5 14 copying files and folders 3 9 copying files from external 3 11 copying files to external 3 10 Correlated baseline 9 1 creating user groups and users 11 11 cumulative time in blocks and methods 5 46 cursor vertical 6 9 curve adding curves 8 17 adding points 9 31 calculating a baseline 8 16 changing and fixing the axes 8 7 changing colours and styles 6 10 changing curve names 8 5 changing the colour and style 8 6 comparing curves 8 22 comparing curves by mirror image 8 31 components of a curve name 8 5 copying curves into one chromatogram 8 26 creating 9 30 cut 8 12 defining and positioning curve text 8 7 deleting a point 9 32 export 9 41 importing a blank run curve 8 15 importing curves 8 22 importing individual curves 8 25 mirror 8 25 move 8 29 moving a curve using the Shift function 8 30 normalise 8 29 optimising presentation in Evaluation 8 10 overlay 8 25 presentation options for comparing imported curves 8 24 reading coordinates using vertical cursor line 9 24 renaming 8 18 selecting curves displayed on screen 6 8 selecting part of curve for integration 9 6 settings 14 5 showing part of a curve 8 10 size 8 29 smoothing 8 13 stack 8 25 stacking and stretching curves 8 27 stacking and stretching curves using the normalise function 8 28 Index stretching and shrinking a curve using multiply 8 30 subtracting a bl
59. control instructions B 9 manual instructions 6 18 M anual menu 6 18 M argins adjusting in page layout 8 44 M ax baseline level adjustment 9 18 maximising resolution A 3 Index M edian 8 14 median algorithm C 2 menu manual 6 18 messages during run 5 47 method adjusting method object in report 8 39 base 2 5 blocks 5 8 controlling block and method length 5 45 creating anew method 4 1 creating from method templates 4 1 cumulative time 5 46 default notes in template 5 32 display of instructions at start of run 5 38 display of method information at start of run 5 40 display of notes at start of run 5 39 editing operations summary 5 7 editing the variables 4 7 execution in M ethodQ ueues 7 5 information 5 36 instructions 2 5 instructions entering editing and deleting 5 17 log format 5 46 naming 4 10 notes 4 9 notes entry 5 32 open file 3 5 overview creating and editing 5 1 pausing 5 48 problems F 3 red instructions F 4 running file 3 6 saving 4 10 5 40 saving method as a template 5 41 starting a method 6 1 starting a method from the M ain menu 6 2 starting a method from the System control 6 2 templates 2 7 text instructions display in start protocol 6 3 using selected unconditional method instructions 5 43 5 49 variables 5 23 viewing notes 5 31 method editor overview 2 2 method file copying from external 3 11 copying to external 3 10 method files overview 2 3 Index method information display 6 4 method instruction
60. control of the connected system e View mode connections from which the progress of the synthesis can be monitored but the system cannot be controlled A system can be started from a computer in for example the laboratory Control of the system can be released without affecting the run and the control of the system can be later taken from another computer station for example in the office 2 6 Evaluation UNICORN concepts 2 Each oligosynthesis system can have only onecontrol modeconnection at any onetime but it can have several view mode connections In a network installation the same or different users may establish simultaneous view mode connections to one system on different computers T his allows a running process to be monitored from several locations at the same time The evaluation module chapter 9 and 10 provides extensive facilities for presentation and evaluation of synthesis results Essential features of evaluation include e Trityl data This is stored in the result file and can be printed in a report as a table e Curve manipulation A wide range of operations can be performed on curves such as addition and subtraction of two curves differentiation integration normalization and scaling The original raw data curves are always kept unmodified in the result file e Curve comparisons Curves from different result files can easily be compared in the evaluation module e Evaluation procedures O perations per
61. corresponding instructions in the text 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 5 6 2 Questions Questions provide a means for entering structured run specific information at the start of a run M ethod templates supplied with UNICORN aredefined with a set of questions for sample column and eluent identification To define questions which will be shown when the method is started open the Questions page in Run setup Note For questions to be shown in the start protocol the Questions option must be checked in the Run set up Start Protocol page 5 27 oe and editing methods Questions may have the following status Mandatory these questions must be answered before a method is started Authorised answers to these questions must be acknowledged by a user with Confirm Unlock authorisation see Section 14 2 The user s password must be given to acknowledge the answers Chromatogram these questions will be printed with the answers on the same page as the chromatogram if Diagram header is chosen in an evaluation report see Section 9 5 Variables Questions Notes Reference Curves Evaluation Procedures Method Information Result Name Start Protocol Sequence No 2 Type the weight of the support in the column Insert Input field No
62. curve determines the min and max values for the x axis Select help curve s m Y axis scale 01 test20_Cond 02 test20_PressDet 03 test20_Press4CN Minimum jo Maximum jo Unit s gt j New unit Cancel Help Figure 9 35 Create Curve dialogue If you want to create new unit click on the New unit button and enter the new unit name and number of decimal places the values will receive Evaluating results 9 Create New Unit xi Name Number of decimals fo Cancel Help Figure 9 36 Create new unit dialogue Click on OK to return to the Create curve dialogue and again on OK when you have made your selections there W ith Point Mode selected you can usethe left button to insert new curve points on the chromatogram The co ordinates of each new point are automatically entered into the Point list The co ordinates of the mouse cursor are displayed beneath the curves thus allowing you to precisely position a new data point Theco ordinates for the new curve are always displayed Selecting Zoom mode allows you to use the mouse to select an area of the view and zoom in The right mouse button menu allows you to undo the last zoom step with Undo zoom or to reset the default zoom scale with Reset zoom In Zoom mode you can also drag the vertical cursor line and the co ordinates presented reflect where the line bisects the curve of the same colour Create Curve SEE Point jst
63. e Access level and user administration e Network 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 T hese activities are described in the present chapter System administration duties may also include routine monitor calibration Section 6 6 Access level and user administration concerns authorisation 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 are considered in Chapter 12 After installation the following operations should be performed in UNICORN by the administrator before other users can use the program 1 Set up system definitions for the synthesis systems Section 14 1 2 Define access levels for the installation Section 14 2 3 Define new users with home folders and access profiles Section 14 3 Note These operations can be performed on any station in a network installation It is however important that the administrator is logged on to the network on the station being used so that the changes will apply globally throughout t
64. e M e M eeo l aobo ff _ Figure 8 8 Chromatogram with header information displayed 8 2 9 Saving and applying a layout All configurations that you make in the Chromatogram Layout dialogue can be saved as a layout It is possible to apply saved layouts to other chromatograms All saved layouts are user specific 8 9 8 Presenting results To save a layout 1 Open the Chromatogram Layout dialogue and make the appropriate layout configuration within the various tabs N ote that you can return to the chromatogram window by clicking on OK to see the applied affects of a given configuration and return again to the Chromatogram Layout dialogue to perform further changes 2 Select the Layout Library tab and click on Save current layout as 3 Enter aname for the layout in the displayed dialogue If you want the current layout to be the new default layout check the Save as default option 4 Click on OK to save the layout The new name is added to the Saved layouts list To apply a layout 1 Select the Layout Library tab 2 Select a layout from the Saved layout list and click on the Apply selected layout button The layout is automatically applied to the active chromatogram window If the same layout is to be applied to all chromatograms on the Evaluation workspace select check the Apply to all chromatograms option 8 2 10 Viewing a grid in the chromatogram window Y ou can display a grid in the chromatogram window 1 In th
65. even though some versions of the N ovell N etware driver have known problems A valid network connection A 2 Control capacity A 2 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 A few basic facts about a network installation Synthesis systems must be locally linked to a workstation and then the workstation is linked to the network i e the synthesis systems are not directly linked up to the network Each local workstation can be connected to four separate synthesis systems A network can support up to 90synthesis systems which are connected locally to the workstations in the network A workstation can locally or remotely actively control up to four synthesis systems This is achieved using the four possible System control windows in UNICORN available on each workstation Each synthesis system may be controlled by only one active System Control window and be viewed by seven different other System control windowsin UNICORN Technical specifications A A 3 Data sampling Data from synthesis system monitors are stored temporarily in data buffers in the local system controller Data are transferred from the buffers to disk storage by UNICORN whenever a chromatogram is closed i e when the N ew_Chromatogram instruction is issued or the result file is closed Data a
66. for your system Check your system components under Administration System Setup Components in M ain menu e The Copy function was used instead of Copy from external when importing a method from a diskette e Thewrong system may have been selected in the Save As dialogue in M ethod editor e You may also have templates not intended for your system which often happens for custom designed systems There are several actions that you can take e Check that the method has been connected to the correct system either in the System Method Connection dialogue when using the Copy from external dialogue or in the Save As dialogue in M ethod editor e For custom designed systems go to the M ethod editor select the red instruction and either delete it or replace it with a corresponding instruction if available from the Instruction box Repeat this for all red instructions before saving the method Troubleshooting F 3 7 I ve logged out of Windows NT and then logged in again but can not get system connection in UNICORN only for local systems not remote If you shut down Windows NT using the command Start Shutdown Close all programs and log in as a different user you will not beable to obtain a system control connection in UNICORN thenext time you or another user logs on This is because the aforementioned shutdown procedure automatically shuts down a number of processes including those needed for system connection that are only st
67. from Pump P6000 to the column or waste Flow_Reag Starts the flow with reagent con nected to valve x x to the column or waste LFlow_ACN Starts a linear flow of acetonitrile to the column or waste and is dependent on column diameter LFlow_Det Starts a linear flow of detrit solution to the column or waste and is depend ent on column diameter LFlow_Reag Starts a linear flow of reagent to the column or waste and is dependent on column diameter PFlow_ACN Pressure controlled flow of ace tonitrile from Pump P6000 to the col umn or waste B 2 Strategy for OligoPilot II 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 3 B Strategy for OligoPilot II B 1 2 Flowpath Instruction Description Comment Amidite M eans ACN_A to column 1 1 p1 0 p2 1 5 2 6 1 valve pos A to column 1 2 p1 0 p2 1 5 2 6 1 portlpos ACN_C to column 1 4 p1 0 p2 1 5 2 6 1 port2posl C to column 1 3 p1 0 p2 1 5 2 6 1 valve5pos2 ACN_G to
68. if you are not sure how to do this Locate an empty full length expansion slot Takethe expansion card out of theanti static bag H andle the card by its edges and avoid touching the electronic components as far as possible because 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 Installation 1 2 electricity from your body by touching an earthed metal surface e g a water tap or radiator before handling the card Two setings that are made on the expansion cards are for interrupt IRQ and address These settings must be entered in the UNICORN software installation so that UNICORN can find the systems when started To see which settings the card has look at the card and compare it with the tables below The IRQ isset by a jumper and the address is set with a dip switch W rite down the settings on a paper for future use When you are installing the UNICORN software and the setup program asks for the card settings enter the correct values for IRQ and address If for some reason the default settings can not be used on a specific computer for examplethereis a conflict with another device that it already installed the settings have to be changed CU 900 0 ligosystems Alternative RQs for this card are3 4 5 7 10 11 12 and 15 The manufacturing default setting is IRQ10 T headdress is made up of tw
69. in a baseline that reaches too high up in the peaks of the curve see figure below This situation may arise when for example a wider peak is not recognised dueto it containing a cluster of smaller peaks The default Structure width is therefore set to a default value according to the largest width of the identified narrower peaks EE Baseline example 2 1 UUU mapp104 1_UV1_215nm_Morphological_default mapp104 1_UV1_215nm_Morphological_4xdetault Bl Figure 9 10 Chromatogram displaying a curve with two baselines A default morphological algorithm settings and B morphological algorithm with increased structure width value 9 Evaluating results 9 10 Conversely a too large Structure width value means that narrower peaks especially in fluctuating curves may not be properly followed This may arise when an artifact in a curve is identified by the morphological algorithm as the widest peak and hence used to set the default Structure width value Minimum distance between points The Minimum distance between points is a measure of the distance between the data points used to generate a baseline The largest number of data points is produced at the slopes of the curves and so by increasing the Minimum distance between points value fewer points will be collected on the slopes mwa AX aw 20 i 100 i 200 i 300 i 40 0 f 50 0 i min Figure 9 11 A curve in a chroma
70. is performed can be undone using Edit Undo Presenting results 8 8 2 1 The chromatogram window The chromatogram window is divided into three main views for header information run curves and peak tables The displayed areas for the views can be adjusted by dragging the borders with the mouse cursor between the views Viewing the curves The first time a chromatogram window is opened and viewed a default layout is applied to display all of the original curves The default layout can be changed by the user see Section 9 2 9 test20res 1 of Figure 8 2 Displayed chromatogram in a newly opened result file AJ LOU NE Each curve is automatically assigned a default colour and style with default information about each curve displayed in the key above the curves T his information includes the result file name chromatogram name and curve name Each curve has a correspondingly coloured y axis To choose the appropriate y axis scale click on the y axis until the desired scale is displayed or simply click on the name of the curve of interest Optimising the workspace Chromatograms can be minimised in the desktop by clicking on the minimize button in a chromatogram window Icons can be neatly arranged in the workspace by selecting Window Arrange icons To restore a window click on the restore button for the iconised window or select the chromatogram name from the Window menu Y ou
71. level While holding the SET button on the pump you can observe in the UNICORN Calibration dialogue that an internal M easured value is assigned Click on the R ead 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 M easured value and M easured 1 fields 6 7 Maintenance Some strategies support the possibility to view system information for the components in a synthesis unit and to define warnings on the components for maintenance purposes Note OligoPilot II systems do not support these Maintenance functions 6 26 Performing a run 6 6 7 1 Viewing system component information To view the maintenance functions for a maintenance supported oligosynthesis system 1 Select System Maintenance T he Maintenance manager dialogue is displayed The Info tab is shown by default and UNICORN takes a few moments to scan the connected oligosynthesis system for the components present Maintenance manager Figure 6 18 Maintenance manager dialogue Info tab When UNICORN has completed its scan all system components are displayed Maintenance manager H 0 Valve 7 Figure 6 19 Maintenance manager dialogue Info tab with system components displayed 6 27 sorom a ru
72. not logged on will apply locally and will be lost the next time you log on to the network to use UN ICORN 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 folder on the local station see Section 11 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 6 5 2 Connection modes The possible connection states of a System control module are indicated by the connection mode button and the status text on the status bar as summarised below Button Connection Text State A ction mode Not connected none Click on the connection mode button or choose System Connect to establish a connection Control mode Controlled Click on the connection by lt user gt mode button to leave the system but retain the connection with the System control module You may leave the sys tem locked or unlocked 6 21 sorom a run 6 22 Controlled by lt other user gt View mode View mode Locked by lt other user gt z System is available View mode Theindicated user has a control mode connec tion Clicking on the connection mode but ton has no effect The indicated user has left the system in a locked state Click on the connection mode b
73. 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 customised methods either by adjusting variable values or changing method instructions For your first run you are recommended to select the method template Fix Column Recycling Coupling Click on a template to display information about the particular template in the Method notes field Choosea specific column to be used O nly columns for the selected technique are displayed If you do not find your specific column it can beadded to the list see Section 5 9 Relevant column data are automatically copied into the method thus reducing the need to edit the method Creating methods from method templates 4 If Any is selected you can use any column but must enter the column volume in the method on the Variables page It is recommended that a specific column is selected 5 Click on OK once you have made your selections The method template will now be opened in Run setup view as an untitled method 4 2 Saving and running a test sequence method Several of the 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 Notel To view what the various symbols mean for the bases please see the table in Section 4 3 Note2 The synthesis reaction proceeds in the 3 5 direction so
74. or different systems 2 3 2 UNICORN concepts 2 4 Methods 2 4 2 3 2 Result files Result files are created by UNICORN when a method is run and contain A copy of the method used in the run e Run data from the monitors in the oligosynthesis system e g UV absorbance flow rate conductivity etc e Saved results from evaluation of the run data see Chapter 10 e Run documentation including information on for example the run log calibration settings scouting parameters text method etc Oligosynthesis runs are programmed as methods in UNICORN This section gives a brief overview of the concepts and principles of methods See Chapters 4 and 5 for a description of how to program methods and Chapter 6 for how methods are used to control oligosynthesis systems 2 4 1 Method structure Blocks Methods in UNICORN aredivided 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 e Detritylation e Detrit wash e Coupling e Oxidation thiolation e Capping M ethod templates supplied with UNICORN contain all the blocks that are likely to be used in a specific method When 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
75. result file containing the blank run curve The curves in the first chromatogram are displayed cA Defaut Select curves Name Size Type Mo 4 Chromatogram Selected curves FPLC Directory example fies User Folder aii 5 iid 1992 Scouting Folder 372 CJ SMART Manager example files User Folder 3n Svalable i Baseline example 1 1173KB Result File ae test20_Cond i Baseline example 2 985KB Result Fle aye test20_PressDet ait y test20_PressACN L Baseline example 3 929KB Result File 3 8 id 18501 1180KB Result File 3n a id 18502 959KB Result File 3h iti Peak skim example 998KB Result File 127 w 1164KB Resul File sz m 5 Select gt gt Remove o tore in chromatogram 1 Figure 8 14 Open Curves dialogue with a result file selected Select the curve corresponding to the blank run curve and click on the Select button The selected curve will now be displayed in the Selected curves list To remove a curve from the list select it and click on Remove If there is more than one chromatogram in the result file and the blank run curve resides in another chromatogram select the appropriate chromatogram from the drop down list The curves for that chromatogram are displayed from which you should make the appropriate selection To import the curve click on OK Note For more detailed information about how to import curves chromatograms and other results
76. s logon password A locked system can also be unlocked with the logon password for a user with Unlock locked systems authorisation This authorisation should be restricted to a small number of users to prevent indiscriminate unlocking of locked systems 6 5 4 Disconnecting a system To disconnect an oligonucleotide synthesis system from a System control module choose System Disconnect If you are disconnecting a control mode connection you will be asked if you wish to leave control of the system under a password protected lock Logging off or quitting UNICORN automatically disconnects all connected systems displaying the Leave control dialogue for each system Systems which are disconnected in this way will bere connected automatically when you log on to UNICORN again N ote however that you may 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 6 23 Ox arun Note You can disconnect a system during a run and the run will continue It isnot recommended to do this without locking the system since this can leave a run on the system with no responsible user Y ou cannot however disconnect from M ethodQ ueue runs 6 5 5 Network considerations In a network installation an oligonucleotide synthesis system can be controlled from any computer in the network provided that the user has sufficient access rightsin UNICORN
77. see Section 9 4 Alternative B Calculating a baseline If there is no blank run curve you can instead create a baseline with Integrate Calculate baseline see Section 10 1 1 Subtracting the blank run curve Select Operations Subtract to subtract the blank run curve or the baseline away from the sample curve Click on OK All resulting curves from the subtract operation receive the SUB suffix Presenting results 8 8 3 4 Adding curves In some runs several sequential chromatograms may have been created for example when the instruction New chromatogram has been used in the method thus creating different chromatograms during the run In order to view and evaluate the resultant curve of all the chromatogram parts the curves must be added together T he common situation is when you have a number of chromatograms within the same result file and you want to add the curves In some circumstances curves may need to be imported from other result files To add curves 1 Select and view the first chromatogram in the sequence 2 Select Operations Add Add the first curve in the sequence to the second curve in the sequence from the appropriate chromatograms 3 Add the result of the previous step to the next curve in the sequence 4 Repeat this process until all curves have been added together The final curve should be the cumulative curve for the whole run All curves created using the Add operation receive the ADD suffix 8 3
78. strategy template files on a diskette or another source click the Have Disk button to locate the strategy template files Strategy and Template Installation x Select the strategy you want to install and press the INSTALL button If your strategy template is located on a floppy or on another source press the HAVE DISK button to locate it When all strategies templates are installed press NEXT to continue Options I Install strategy IV Install template Strategy template Spade xl inal Have Disk f aup Cancel Figure 12 12 Strategy amp Template Installation dialogue If the options for both strategy and template are checked the template files are installed automatically together with the strategy If the strategy and template files are located on different diskettes the setup program will ask for the template disk when it is needed When installing strategies you will be prompted for a name for the strategy that is being installed Y ou can either keep the default name or enter another name The name for a strategy can not be more than 8 characters 12 15 12 Installation Enter Information Figure 12 13 Enter Information dialogue If you are installing template files separately you must enter the appropriate destination for the correct files It is not possible to detect which template files correspond to which strategy A list with all strategies installed on the statio
79. system is installed Also 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 Note For strategies where you can select the system components only thesettings for the selected components will be displayed Assigning a new value to the actual system setting is performed in the System control module and Configuration access is required to make such changes see Section 14 2 1 To access thesettings click on the appropriate System control icon on the Windows NT taskbar and select System Settings The Instructions dialogue for the current system is displayed OPII Alarms Instructions Instructions j Cond1 Alarm Parameters Te ane a High alarm 0 399900 Husem CondlAlarm High alarm 999900 000 uS cm Low alarm 0 000 uS cm Specials High wam 999900 000 uS cm Low wam 0 000 uS cem Low alarm a 0 999900 c Hysteresis 1000 000 uS cm 0 000 WEED Mode Enabled ZyuS em p lam_Det p High alarm 20 000 bar High wam 10 999900 Low alarm 0 000 bar 999900 000 tel usvem Cina High wam 20 000 bar z Loman m1 ANN har Low wam 0 g 999900 Set Selected Parameter To Strategy Default Value 0 000 F uS em coe eeo Figure 14 1 Instructions dialogue in System control The illustration shows the Alarms group of settings 1 4 System settings 14 1 Alarms 2 Theinstructions are classified into Ins
80. the M ain menu Enter the old password and the new password in the appropriate fields Passwords are not displayed explicitly in this dialogue 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 Administration User setup select the user and enter a new password in the Password field see Section 14 3 1 Change passwords regularly and avoid obvious passwords like secret and open_sesame 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 13 4 2 13 3 3 Viewing and changing user definitions To view the setup for any user click on the user name in the User setup dialogue To change the user definitions make changes in the dialogue fields as appropriate and click on OK 13 3 4 Deleting users To delete a user select the user from the username list and click on Delete 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 folder or method and result files Administration 1 3 13 3 5 Defining new home folders To define an existing folder as a home fold
81. the computer where you are installing UNICORN not a network disk drive Select Drive x The Unicorn software have to be installed to a folder named Unicorn located in the root directory of the selected drive Select the fixed drive that you want to install Unicom 3 00 04 to and push the Next button to continue Available disk drives on the system D Destination Path C Unicom p Cancel Figure 12 5 Select Drive dialogue Choose whether the setup type is stand alone or network For a stand alone installation the network options settings are ignored A stand alone installation can be either a local station or a demo station If you want to install a demo station make sure that Demo system is checked Installation 1 2 A network installation can be either a local station or a remote only system To get a remote only system i e a computer to which no systems are physically connected check Remote only system For a network installation you will also have to select a server disk where the server files are to be located W hen you perform a network installation the necessary parts of UNICORN software will be copied automatically to the network server disk Note If you perform a stand alone installation and later want to connect the system to a network you must remove the current installation and install the software with the appropriate settings Setup Type x Select the type of in
82. the method andata To Authorized brTametoareny Figure 5 30 Options for no answer questions 5 29 B ieira and editing methods 5 30 Value 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 M Mandatory P Authorized Chromatogram Answer Value C Input field Mec jas C No Answer Max 20q Multiple choice Value T Integer Figure 5 31 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 mode From preview mode press Edit to return to question editing mode Inserting a question To insert a new question after an existing question 1 2 Click on the existing question in the questions list Enter the text status type and answer for the new question as required The Answer pane depends on the type of question e Input field questions Enter a default answer if required e Multiple choice questions Click on the input field under Alternatives enter the answer and click on Add Delete Repeat 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 e Value questi
83. the network server if the computer is logged on to the network F 1 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 13 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 on to the network beforeUNICORN is started so that the strategy file on the server disk is accessible F 2 UNICORN access problems F 2 1 Unable to access certain UNICORN functions UNICORN 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 Administration User setup in the main menu F 1 Troubleshooting F 2 F 2 2 Connections are not available Check the connection between the PC and the chromatography system Check that the power to the chromatography system is turned on If the connection appears to be correct and the power isturned on switch off the chromatography system and quit UNICORN 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
84. the new values will apply N ote the response Increase the value of P until the actual flow rate oscillates with a constant period and amplitude Note 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 oscillation is satisfactory note the P value PO and the oscillation period in seconds T0 Calculate suggested PID values for the required regulator type from the table below Feedback tuning D 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 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 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 D 3 D Feedback tuning D 4 File organisation E E File organisation This appendix documents the file structure ina UNICORN installation UNICORN strategy system sy
85. the specific Periodicity parameter has been reached Pe check Figure 6 24 Warning dialogue for a lamp check MethodQueues 7 7 MethodQueues M ethodQ ueues provide a means for linking several methods together on the same or different systems For example if a system wash procedure is programmed in a separate method it can be linked in a M ethodQ ueue to a series of different process methods ensuring that the same wash procedure is used before every process Alternatively the product of a separation on one system might form the starting material for a separation on the next allowing fully automated multi step processing Specific user authorisation is required to edit and run M ethodQ ueues separate from that required for editing and running methods 7 1 Setting up a MethodQueue 7 1 1 Defining a MethodQueue To create a new M ethodQ ueue 1 2 In M ain menu select File New MethodQueue or MethodQueue New T he MethodQueue Editor dialogue is displayed MethodQueue Editor Untitled x w System2 System System4 MethodQueue Start Immediate Start End of MethodQueue Setup Save Save As Close Help Figure 7 1 MethodQueue Editor dialogue To add a method to the M ethodQ ueue list select the End of MethodQueue instruction in the list Click on theappropriate System button The Load MethodQueue dialogue is displayed Note Thenumber of available
86. 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 5 6 7 Result Name Use the Result N ame page to specify how the result files will be named for the results of a run and where the result file will be saved Creating and editing methods 5 Variables Questions Notes Reference Curves Evaluation Procedures Method Information Result Name Start Protocol Sequence Result name Serial number is added to all resultnames C Method name Date C Name Result directory Home Browse Scouting subdirectory Figure 5 36 The Result Name page in Run setup The result file nameis constructed by adding a 2 digit serial number to one of the base options listed below The serial number is incremented automatically each time the method is run e the method name plus a 2 digit serial number e thedateof therun in an 8 digit format determined by the country setting in Windows NT plus a 2 digit serial number a freely specified name within the file naming restrictions in the operating system plus a 2 digit serial number If the result file folder already contains files with the same file name base the serial number is incremented automatically By default result files are stored in the home folder of the user who starts the run To change the folder where the resu
87. 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 use the Text instruction editor and click on the New block toolbar button or select Block New New block x Name r Base Same as main C Time Volume Column volume 010 al Lenath 0 00 w r Call Erom Mn StS C SsS At p 00 wy Cancel Help Figure 5 11 New block dialogue 5 11 B ieira and editing methods 5 12 Alternatively you can enter the Block instruction in the Instruction box enter a name for the block in the parameter field and click on Insert 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 Block names must be unique within the method The case of letters is retained but is not significant the names Add_DNA_W and add_dna_w are treated as identical Base Choosea basefor theblock If you choose SameAsMain thenew block will inherit the base from the M ain block in the method The corresponding Base instruction will be inserted in the block at breakpoint 0 If y
88. will show the curves as far as they have come in the run The Window option allows you to set the portion of the total window to be displayed either in minutes or ml depending on the selected base 5 Click on OK The zoom function To zoom in on a selected region of the curve window 1 Press down and hold the left mouse button and drag a rectangle out on the screen to encompass the area to be viewed 2 Release the mouse button The display is now zoomed in on the selected area 3 Repeat the process for further magnification of selected areas 6 12 Next Layout Colour Settings Performing a run 6 Y ou can reduce the scale of the zoom in function in two ways either e reverse each zoom in action a step at a time by displaying the right mouse button menu and selecting Undo Zoom or reverse all of the zoom in actions to the default scale setting by displaying the right mouse button menu and selecting Reset Zoom Viewing hatch marks Y ou can display a grid in the curve window by selecting Hatch from the right mouse button menu Selecting curve pressure units If the Pressure curve is displayed in the curves window you can set the displayed units 1 Click on the pressure curve with the right mouse button to display the menu Select Set Unit and then the appropriate unit either MPa bar or psi from the menu cascade The selected unit is now displayed on the y axis Alternatively 1 Select Properties
89. 00 Pause 1 0 0 00 Base_Id _dT 0 00 Block START_parameters START_parameters 3 ml 1 00 200 00 Base Time __ z CY 6 30 C ml Weight_of_support 1 00 g 0 10 100 00 0 00 0 00 0 00 0 00 0 00 0 00 Scale 1 00 Weight_of_suppoit g 10 Loading_of_support umol g _ Loading_of_support 1 ColDiam 20 Col_Diam mm z Delay ol 2 00 ml End_block UNICORN concepts 2 Main Column_VYolume 3 ml 0 10 999999 00 START_parameters wy umol g 10 00 250 00 Col_Diam mm 1 00 100 00 Hii Figure 2 3 Relationship between variables in text instructions and in the Variables page of run set up 2 4 2 Method templates M ethod templates are basic methods which provide convenient starting points for developing customised methods see Chapter 4 for more details M ethod templates for most synthesis techniques are supplied with UNICORN installations for OligoPilot and OligoProcess N ew methods are created by selecting a suitable system technique and template and column The method can then if necessary be modified on the Variables page or in the Text instructions Fully adequate customised methods for many applications can be created simply by adjusting the values of method variables in a suitable template 2 5 System control 2 5 1 Control facilities The system control module allows independent control of up to four oligosynthesis systems from one computer with continuous real time
90. 1 TCP IP NT domain 11 5 11 3 2 IPX SPX Novell server 11 10 12 Installation 12 1 Installation summary 12 1 12 2 Migrating from UNICORN OS 1 10 to UNICORN 3 10 12 1 12 2 1 Before migration 12 1 12 2 2 Migration and post installation setup 12 2 12 3 System requirements 12 2 12 4 Hardware installation 12 2 12 5 Software installation 12 6 12 5 1 Installing UNICORN for the first time 12 6 12 5 2 Installing selected software components after the initial installation 12 18 13 Administration 13 1 System definitions 13 2 13 1 1 Defining new systems 13 2 13 1 2 Editing system definitions 13 4 Contents Appendices 13 1 3 Deleting system definitions 13 2 Access levels 13 2 1 Defining access levels 13 2 2 Access level examples 13 3 User administration 13 3 1 Defining new users 13 3 2 Changing user passwords 13 3 3 Viewing and changing user definitions 13 3 4 Deleting users 13 3 5 Defining new home folders 13 3 6 Deleting home folders 13 3 7 Printing user setup information 13 4 Audit trails 13 4 1 Examining audit trails 13 4 2 Renewing audit trail files 13 4 3 Backing up audit trail files 13 5 Report Generator Wizard 13 5 1 Generating a report from the main menu 13 5 2 Generating a report from System Control 14 System settings 14 1 Alarms 14 2 Specials 14 3 Curves A Technical specifications A 1 System requirements A 1 1 Hardware requirements A 1 2 Software requirements A 1 3 Network requirements
91. 20 mm 1 00 100 00 CV_Column_wash E CV_column_wash 2 00 CY 0 00 999999 00 Detrit_peak_start Detrit_flow 300 em h 0 00 2000 00 x Figure 4 5 The Variables page in Run setup 4 7 4 Creating methods from method templates 4 8 Work through the variable list adjusting the values to suit your synthesis To change a variable value simply type the required valuein the field Remember that the values you enter here will be default values suggested each time the method is run If the whole variable list does not fit on one screen a scroll bar will be shown to theright of thelist 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 Typical blocks are illustrated with the list below taken from a method created for OligoPilot II with the Fix Column Recycling Coupling template The list is organised according to the blocks in the method with mentioned variable parameters identified in italics O ther method templates have different structures and variables e Start_parameters These variables together 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
92. 3 Type the loading of the support Rae Input field Input fiel Delete No 5 Mark the type of support Multiple choice Delete all No 6 Enter Column volume Input field No 7 Instrument identification Input field Text The information on this side will not effect the method Te Mendeton E Aunoa fe Ghrometogren Answer C Input field Multiple choice Figure 5 27 The Questions page in Run setup Questions may be defined to accept four types of answers e Input field accepts any alphanumerical input as the answer Input field questions may have a default answer Example r Question Instrument identification I Mandatory I Authorized V Chromatogram Answer p Default text Input field jo ligoPilot 11 C Multiple choice C No Answer C Value Figure 5 28 Options for input field questions 5 28 Creating and editing methods 5 e Multiple 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 pre defined answers Example Mark the type of support PrimerSupport 30 HL Primer Support Other Figure 5 29 Options for multiple choice questions No 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
93. B 7 instructions groups 14 2 instructions in methods 2 5 instructions manual alarms and monitors B 9 miscellaneous B 10 instructions system settings alarms B 10 curve B 13 monitors B 12 specials B 11 instructions conditional 5 10 Integrate_and_Print 5 32 integrating peaks 9 1 integration C 15 common problems 9 12 including negative peaks 9 8 optimising 9 4 performing 9 2 selecting part of a curve for integration 9 6 integration problem noise detected as peaks 9 11 9 15 peak limits too high up on the peaks 9 13 peaks missing 9 16 introduction to Unicorn 1 1 IPX SPX installing on the computer 11 11 IRQ setting 12 3 Index K Keyboard setting 14 4 L layout creating 6 7 deleting 6 7 saving and applying 8 9 selecting 6 7 leaving system 6 22 length entering for a block 5 12 license agreement 12 8 linear flow rates 5 49 local station 11 3 local station failure 10 3 Locked 6 23 Locked by 6 18 Locked by status bar message 6 18 locking system 6 22 log format for cumulative time 5 46 logbook autoscroll 6 15 logbook for a run 6 14 logging off 3 15 logging on 3 1 Logoff button 3 15 logoff problem logging off F 3 logon problem logging on F 1 Logon Logoff button 3 3 loop symbol with text 5 18 M ain menu for starting a method 6 2 main menu functions overview 2 2 managing system connections 6 20 manual assumed skills of user ii bold typeface entries ii contents overview i manual control 6 16 manual
94. C is cross referenced to the block name Add_DNA_C which is then copied into the method Similarly for the following base A this is cross referenced to the block named Add_DNA_A 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 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 Creating and editing methods 5 For example you may decide that the DNA sequence 5 ACTGGT 3 should havea column wash step after the addition of each base in the sequence By cross referencing a base not used in the current sequence eg A to the column wash block in the method template this can be directly incorporated into the sequence to signal a column wash Thus in the sequence 5 AAACATAGAGAT 3 the appearance of A does not mean that a thiolated DN A 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 a new method by clic
95. Click on the View Windows toolbar button or choose the View Windows menu command to open a dialogue for choosing which windows to display M Flow scheme IV Instruction box Cancel Help Figure 5 6 The Windows dialogue for selecting which windows to display in the Method editor M ethod editing operations which can be performed in the various windows are summarised in the table below Window O perations See Section Text window Display and hide block 5 4 instructions Select current instruction M ove instructions within a breakpoint Flow scheme window For information only The 5 4 5 flow scheme picture is static and is therefore not updated according to system status or changes in the method Instruction box Specify breakpoints instruc 5 4 2 tions parameters and varia 5 4 4 bles Insert change and delete instructions 5 7 oe and editing methods 5 5 2 1 Run setup The Run setup is a series of pages for defining the method properties To access the Run setup pages press the Run setup toolbar button or select View Run Setup from the menu Variables Questions Notes Reference Curves Evaluation Procedures Method Information Result Name Start Protocol Sequence Main 4 Column Volume mi 0 10 999999 00 START_parameters cv 6 30 mi 1 00 200 00 Weight_of_support fico fg 0 10 100 00 Loading_of_support fio umoa 10 00 250 00 Col_Diam o frm 1 00 100 00
96. Connect As field Give the password and click on OK Install UNICORN see Chapter 13 select Network version Reboot thePC and log on as one of the domain users that will run UNICORN 11 9 1 1 Network setup 11 10 10 Connect the shared folder again this is necessary since each user has his own connected network drives Repeat steps 1 2 3 5 and 6 above and make sure the Reconnect at Logon option is checked 11 Start UNICORN and set up system definitions and user profiles see Chapter 13 11 3 2 IPX SPX Novell server This is an example of how to set up the network so that UNICORN can run across the network when thenetwork protocol is IPX SPX and uses a N ovell server The general network organisation is described in the following picture 9 G008 tala Oligosynthesis system z FN a 9 E508 PC with UNICORN PC with UNICORN Network sever Oligosynthesis system 0500 Oligosynthesis system 0508 PC with UNICORN PC where UNICORN PC with UNICORN BBRA is NOT installed SS Oligosynthesis system Figure 11 2 General network organisation based on a N ovell network controller The setup of the Novell server is not described here a competent network administrator is normally needed to setup a N ovell server Creating user group and users of UNICORN on the Novell server This is performed by the network administrator
97. Evaluation 8 55 export data or curves 9 41 results 9 41 F failed folder access 10 2 feedback control aims D 1 feedback tuning D 1 file copying from external 3 11 copying to external 3 10 deleting 3 13 Index file organisation E 1 file organisation in local and remote computers E 1 file organisation in network installations E 1 file organisation in network server E 1 file organisation in stand alone installations E 1 filtering files displayed 3 7 finding 3 8 moving and copying 3 9 presenting 3 6 renaming 3 13 sorting order 3 7 files method files overview 2 3 opening and running 3 5 result files overview 2 4 filter files displayed 3 7 Filter type 8 13 filtering peaks from view 9 4 finding files 3 8 First H eader 8 44 Fixed button 6 11 Fixed y axis 8 8 flow rate tuning D 2 flow scheme adjusting display 6 14 flow scheme display 6 14 flow scheme window 5 23 flowpath instructions B 4 folder creating a new folder 3 5 defining new home folder 13 13 deleting 3 13 moving and copying 3 9 renaming 3 13 user access 13 11 footer selecting contents 8 45 Frac Parameters setting 14 4 FractionN umbering M ode setting 14 4 G generate report 13 22 generating report from the main menu 13 19 ghost peaks removal 8 13 global audit trails 13 14 gradient tuning D 3 grid in chromatogram window 8 10 Index H hardware installation 12 2 requirements A 1 hatch marks viewing 6 13 help on line 2 3 HETP C 11 calcu
98. FF then no method is exported If M ain is ON then the main method is included and if Blocks is ON then all blocks are included in the exported file EXPORT_ Exports multiple curves previously Export to file M ULTI_ defined with CURVES EXPORT_SEL_CURVES instruc ASCII tions in ASCII format to the file defined in Export to file EXPORT_ Exports multiple curves previously Export to file M ULTI_ defined with CURVES EXPORT_SEL_CURVES instruc WKS tions in WKS format to the file defined in Export to file EXPORT_ Exports multiple curves previously Export to file M ULTI_ defined with CURVES X EXPORT_SEL_CURVES instruc LS tions in XLS format to the file defined in Export to file EXPORT_ Exports the peak table in Peak table Peak table source PEAKTABL sourceto thefile defined in Exportto Export to file E_ASCII filein ASCII format Evaluation functions and instructions C EXPORT_ Exports the peak tablein Peak table Peak table source PEAKTABL sourceto thefile defined inExportto Export to file E_WKS file in WKS format EXPORT_ Exports the peak table in Peak table Peak table source PEAKTABL sourceto thefiledefined inExportto Export to file E_XLS file in XLS format EXPORT_S Selects a curve for subsequent export Source curve EL_ using the EX PORT_M ULTI Left cut limit CURVES CURVES instruction Thecurveis Right cut limit cut according to the right and left cut Export every li
99. ON OFF C0000 9 OFF ON JON OFF C4000 10 JON OFF JON OFF C8000 11 OFF OFF JON OFF CC000 12 ON JON OFF OFF D0000 13 OFF JON OFF OFF D4000 14 ON OFF OFF OFF D8000 15 OFF OFF OFF OFF DC000 6 Closethe cover on your PC 7 Connect the 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 12 5 Software installation 12 6 UNICORN will normally be installed by your Amersham Biosciences representative Follow the instructions below if you need to install the program yourself If the system is connected to the network and installed to support remote control make sure that the same version of UNICORN is installed on all stations in the network 12 5 1 Installing UNICORN for the first time Theinstallation procedure described below assumes that the operating system Windows NT is correctly installed on your computer R efer to the operating system documentation for details For network installations the network must be correctly set up UNICORN is supplied on a CD rom Files on the CD rom are compressed and cannot simply be copied onto the hard disk The installation procedure described below creates the required folder structure on the hard disk and decompresses the files Do not attempt to decompress the distribution files using any other file decompression
100. 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 Calibration dialogue that an internal M easured 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 M easured value and M easured 1 fields 6 25 sorom a run ACN solvent pump System Control 1 OPII Calibration r Calibration procedure Set pressure to zero and press button bar 0 0000 Read value 1 1 Reference value 1 ae 2 Measured value r TEE 3 Reference value 2 gow 0 0 20 0 q nevie bar Measured 1 Close Help Figure 6 17 Dialogue box for pressure calibration of the ACN pump Select PressAC N in theM onitor pull down list to calibratethe 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 value2 box OntheACN pump press and hold the SET button and manually set the pressure to the desired
101. Queue authorisation is required to define a new M ethodQ ueue 7 1 2 MethodQueue folders and icons M ethodQ ueues are saved in a separate M ethodQ ueue folder within the folder that you specified during the save The M ethodQ ueue folder is represented by a special icon in the Methods window of the main UNICORN menu M ethodQ ueue folder Double click on the M ethodQ ueue folder icon to open it A M ethodQ ueue folder contains the M ethodQ ueuedefinition and copies of all methods included in the M ethodQ ueue M ethodQ ueue definition in a M ethodQ ueue folder It is important to realise that the M ethodQ ueue works with copies of the original method files If changes are made in the original method these will not affect the method in the M ethodQ ueue To implement changes in a M ethodQ ueue method edit the method in the M ethodQ ueue folder Alternatively edit the original method then use the M ethodQ ueue editor to update the M ethodQ ueue replacing the old method with the changed version It is a good idea to make sure that M ethodQ ueue definitions always contain updated methods to avoid confusion between different versions of method files 7 2 Editing MethodQueues 7 4 To edit an existing M ethodQ ueue open the M ethodQ ueue icon with the right mouse button menu command Edit The MethodQueue Editor dialogue is displayed for the selected M ethodQ ueue Edit MethodQueue authorisation is required to edit a M ethodQ ue
102. Section 9 4 2 Alternative A and more besides If the curves have been stacked with the Open to compare curves operation and you want to changethe stack offset the 8 Presenting results easiest way is to repeat the operation with another offset The curves can also be stacked and stretched individually as described below The operations presented below all require the curves to be present in one chromatogram see Section 9 4 2 xy fi Figure 8 24 Stacking curves Alternative A Stacking and stretching curves using the normalise function The simplest method to align curves with respect to the x axis or the y axis for easier visualisation is to use the normalise function To select the curve to be moved within a chromatogram 1 Select Operations Normalise The Normalise dialogue is displayed 2 Select the curve to be normalised and a reference curve to be normalised against For example if you want to stack curves select the curve at the bottom of the stack to be normalised against and the curve to be moved as normalised Click on OK 3 TheNormalise window is displayed A box surrounds the curve selected to be normalised Presenting results 8 Select curve to normalise Select help curve to normalise against 01 test20_Cond 02 test20_PressDet 03 test20_PressA4CN 02 test20_PressDet 03 test20_Press4CN gt Cancel Help Figure 8 25 Normali
103. Setup Method dialogue is used to define the settings for the meth od object Setup Method x Selected items will be included M Fonts Title Text ain Method Block I Start on new page MV Expand main Cancel Help Figure 8 36 Setup Method dialogue 1 Select the items to be included Main Method This is the method on which the run was based Blocks These are the blocks that were used in the method 8 Presenting results 2 Select as appropriate for the inserted method to Start on new page and or to Expand main to show the expanded view of the method 3 If appropriate change the characteristics of the Fonts by clicking on the buttons Title and or Text 4 When you have made your selections click on OK in the Setup Method dialogue The method is now inserted into the report Documentation The Setup Documentation dialogue is used to define the settings for the documentation object Setup Documentation x Selected items will be included Select all Clear tk Title Text d I Start on new page Cancel Help Figure 8 37 Setup Documentation dialogue 1 Select theitemsto beincluded from the Evaluation Documentation menu To clear the current selection click on Clear To select everything click on Select all 2 If required select Start on new page 3 If appropriate change the characteristics of the Fonts by clicking on the button
104. Size Type Prev Folder Z test20 OPII 312KB Method File Explorer Technique Anion_Exchange Cation_Exchange RPC K 4 HIC Size_Exclusion Affinity Chromatofocusing Method name MENTI Figure 4 7 Save as dialogue for saving a method 1 If required select another folder than the default home folder in which to save the method 2 Enter a Method name for the method M ethod names may be up to 256 characters long The method name must be unique for the chosen system within the folder see steps 2 and 3 3 If you have more than one system connected to the computer choose the System for which the method is intended The method can only berun on the system for which it is saved Remember that different systems may have different configurations and control capabilities Note Each method is written for a specific strategy The function of the method cannot be guaranteed on systems having other strategies 4 Choose the Technique for which the method was written 5 Click on OK Creating methods from method templates 4 Note The method templates are written for standard strategies If you receive a syntax error message when the method is saved oneor moreinstructionsin the method areinvalid These may becalls to blocks which are not defined or instructions which are invalid in your customised strategy this can also arise if a method is written for one system and saved for another Invalid instructions
105. System buttons is dependent on the number of systems that were selected during installation of UNICORN 7 1 MethodQueues 7 2 Load MethodQueue x o Default Name Prev Folder 312KB Method File Figure 7 2 Load MethodQueue dialogue Usethe dialogue to locate and select the required method Click on OK or double click on the method item The system type for the selected method e g Basic 10 is assigned to the selected System button and the System name on the button is replaced with the name of the system A lineis also inserted into the M ethodQ ueue list before the previously selected End of MethodQueue instruction The new line contains the name of the selected method MethodQueue Editor Untitled x End of MethodQueue Figure 7 3 MethodQueue Editing dialogue with a method selected for a Basic 10XT system To add more method steps click on the Insert button and repeat steps 3 4 N ote that you must use a new System button to add methods written for a different system type For example if you have designated System 1 to methods for the Basic 10 system then only methods written for Basic 10 can be added with this button If you want to add to the list methods MethodQueues 7 written for for example Explorer 100 then you must use another free System button such as System 2 In this case System 2 will be assigned and renamed Explorer 100 and only methods writ
106. The network administrator creates the users that will be running UNICORN on theN ovell server The procedure for this is not described here Network setup 1 1 Sharing a folder This is performed by the network administrator The network administrator creates shared folder on the N ovell server which UNICORN will use for shared data The procedure for this is not described here Creating user group and users of UNICORN on the local computers 1 Logon to the client computer with administrator rights 2 Enter the User Manager with Start Programs A dministrativeT ools Common User M anager 3 Create the users that will run UNICORN Select the User menu and the New User item 4 In the dialogue that is displayed enter the user name the UNICORN users created by the network administrator description and password enter the password the user has in the N ovell network M ake sure that the Password Never Expires option is checked and that the User Must Change Password at Next Logon option is not checked 5 Click on OK in the New User dialogue and the user is created 6 When completed close User Manager Installing IPX SPX on the computer 1 Open the control panel with Start Settings C ontrol Panel 2 Doubleclick on the Network icon 3 A question will bedisplayed asking if you want to install Windows NT Networking Click on Yes 4 Inthenext dialogue select Wired to the network and click on Next 5 Specify the network
107. Threshold Result curve name fe je PressACN 0303 DI Cancel Help Figure 9 29 Divide dialogue 4 Theresulting curve can then befiltered by Operations Smooth see Section 9 3 2 It is suggested that you smooth using the median filter to remove noise that appears as spikes or occurs in a small area of the curve The ratio can be used to check peak purity If the peak is pure the absorbance spectra are the same over the whole peak and therefore the ratios should remain constant If the absorbance ratio is not the same over the whole peak then the peak is probably not pure Co eluting peaks A B amp Ratio 3 A270nm A255 nm Ratio A270nm A255 nm 209090909009000 oni w minnow y Z 2 3 Time units gt Figure 9 30 Simulated chromatogram of two co eluting components with differing absorbance spectra and a small difference in retention time The resulting ratio can also be used for peak identification as different compounds have a specific ratio between absorbencies at different wavelengths 9 27 9 Evaluating results Separated peaks Ratio 4 gt B esoscsesoocses o DKW D ISS ma o c Time units Figure 9 31 Simulated chromatogram of two components with differences in their absorbance spectra 9 2 2 Finding the slope values for Peak Fractionation or Watch instructions Peak fractionation parameters are set in the M ethod editor with the instruction Peak_FracP
108. UNICORN version 3 10 for oligonucleotide synthesis gt yg 0 9a 6 g f A 18 1134 71 Edition AA User Manual Amersham e Biosciences BioPilot BioProcess FPLC FPLCdirector SMART UNICORN and AKTA are trademarks of Amersham Biosciences Limited or its subsidiaries Amersham and Amersham Biosciences are trademarks of Amersham plc Compaq is a trademark of Compaq Computer Corporation M icrosoft and Windows are trademarks of Microsoft Corp Novell and NetWare are registered trademarks of Novell Inc All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group which supplies them A copy of these terms and conditions is available on request Amersham Biosciences AB 1999 All rights reserved Amersham Biosciences UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Biosciences AB SE 751 84 Uppsala Sweden Amersham Biosciences Inc 800 Centennial Avenue P O Box 1327 Piscataway NJ 08855 USA Amersham Biosciences Europe GmbH M unzinger Strasse 9 D 79111 Freiburg Germany Preface Preface About this manual This manual provides a full reference to UNICORN version 3 10 from Amersham Biosciences AB UNICORN isa complete package for control and supervision of oligosynthesis systems suitable for use with Amersham Biosciences O ligoPilot II and OligoProcess systems UNICORN consists of
109. User M anual Amount As above M olecular size As above Sigma a 5 p Ci Xyna Sigma n 3 A peak where n is the number of data points x is the volume or time value y is the amplitude value Xymax S the volume or time value at the maximum amplitude value A peak iS the area of the peak The peak width for a Gaussian peak is 4 x Sigma Resolution Vpo Vp X 1 177 Writ W R h2 where Vp 1 retention volume for peak 1 Vp retention volume for peak 2 Wh peak width at half height for peak 1 for Gaussian peaks Wh2 peak width at half height for peak 2 for Gaussian peaks C 9 Cora functions and instructions The peak resolution is calculated with one of the following three algorithms 1 VR2 VRDA W p2 W p1 2 2 VR2 Vr1 Sigma Sigma x 2 3 VR2 E VpR1 2 X W p2 W p3 2 354 where V7 Wp1 Sigma and Wy are the retention width sigma and width at half height of the previous peak and Vp gt W p2 Sigma and W p2 are the retention width sigma and width at half height of the current peak respectively The UNICORN INI variable EVAL ResolutionAlg determines which of the three algorithms is actually used If this variable has the value 1 2 or 3 then the algorithm used corresponds to the numbered list above If the variable has the value 0 or if the varible is not defined or has a value other than 0 1 2 or 3 then the default 3 algorithm is used To ch
110. _amid and CT5 Cap e CV_column_wash The number of column volumes CV to wash the column is set here If zero is entered no wash will take place e Detrit_peak_start The flow rate of the detritylation solution is set here e Detrit_wash The pressure of the detritylation wash and the number of column volumes of detritylation solution to be used are set here e DNA_Parameters These 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 e DNA Recycle The amidite recycling flow rate and time used are set here Creating methods from method templates 4 e Oxidation 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 e Capping 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 Click on the x axis button in the graphical display to change a base for the graphical display Changing the display base will not affect the base in the method 4 5 Method notes al Click o
111. a Bae C Time C Volume Close Help Figure 6 12 The logbook panel for a running method The logbook window can autoscroll to display the latest entries if you select the Autoscroll function from the right mouse button menu or else check the Autoscroll option in the Properties dialogue 6 2 6 Synthesis Data The Synthesis Data window can be accessed under View Synthesis Data from within the System Control module a summary Synthesis Data view is also available in the Evaluation module For the current run Synthesis Data displays a spreadsheet with summary information for each base in the chosen sequence Units time or volume are identical to those in the chromatogram Synthesis Data x 17 54 2003 80 371 00 100 00 17 02 185700 365 00 100 00 17 02 1786 20 344 00 96 20 EA EA 4 5 50 544 10 290 00 100 00 Ba EA EA 841 70 280 00 100 00 7 Le Figure 6 13 The Synthesis Data window within System Control 6 15 i arun 6 3 Manual control 6 3 1 The toolbar The toolbar at the top of the System control workspace contains a set of buttons for starting and stopping the run accessing documentation and locking the system Run Hold C Pause I Continue End alale Figure 6 14 The toolbar in the System control workspace Run Hold Pause Continue End 6 16 Starts a run when the system isin End state and a method is loaded Suspends execution of a method
112. a compromise between noise removal and preservation of peak shape The easiest way to find the optimum smoothing effect is to start with a low parameter value e g the default value and increase it until the best result is achieved A useful strategy is to increment the parameter value by the default value for each try 5 Click on OK The formulae for the filters are described in Appendix D 1 8 3 3 Subtracting a blank run curve This is a frequently used function in presentations especially if the curves have a drifting baseline or ghost peaks Presenting results 8 Note If the ghost peaks come from impurities in the eluents all equilibration of the columns should be the same from run to run If for example the equilibration volume with buffer A is larger before a blank run curve than before a synthesis your ghost peaks might be higher in the blank run curve Figure 8 12 UV curve with baseline prior to subtraction of the baseline Figure 8 13 UV curve after subtraction of the baseline Alternative A Importing a blank run curve If a blank run curve was done this may have been stored in another result file To access the blank run curve 1 Ensure that the destination chromatogram has been opened and is the active window on the workspace 8 Presenting results 8 16 5 Select File Open and then from the select Curves from the menu cascade The Open Curves dialogue is displayed Locate and double click on the
113. a split select the desired peak in the list or mark in the curve and press the Split Peak button Note The peaks will be renumbered according to the split Refer to the description below about adjustment of the drop lines 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 chromatogram or in the peak table 9 Evaluating results 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 The original intervening drop line is removed and all peaks are 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 Peak start Figure 9 26 A drop line at the start and end of a peak W here there are two peaks beside one another the end of thefirst peak will be at the same point as the beginning of the next peak Thus there will bea drop line below and above the line at the same point End of first peak start of second peak Figure 9 27 A drop line between two peaks Evaluating results 9 It is possible to move the drop lines for a selected peak and thus affect the area beneath the peak 1 Click on the peak of interest on the curve or in the peak table and two vertical cursor bars become superimposed on the left and right drop lines that delimit the s
114. able was based is displayed at the bottom of the panel 3 Doubleclick on the desired peak table in thelist or click on OK A chromatogram window is displayed containing the selected peak table with corresponding curve and baseline The various editing features are described below Asan aid it is possible to use the zoom function 4 Onceyou have completed your changes click on OK and verify the destination of the new edited peak table Evaluating results 9 Q Edit Peak Table olx test20_Cond test20_Gond 01 BASEM 224 6225 54 627 1042 46 a 1896 37 2327 58 2764 03 3202 36 3640 99 4036 70 fest ene fresno mi Peak start m1 Peak end m1 area uS cm ml 24581 7831 23883 5104 gt A Delete peak _Spitpeak_ Join lett Join right OK Heb Figure 9 25 Edit Peak Table chromatogram window Deleting a peak in the peak table To deletea peak from thetable 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 delimiting drop lines to the left and right of the peak It is possibleto split the peak into two new peaks by inserting a drop line The drop line is always inserted at the middle point between two existing drop lines The area under each new peak will not be the same if the symmetry of the original peak was not perfect To make
115. age layout Page Layout which allows you to set the page Margins and the Units cm or inch Y ou can check the option to have the Same header on all pages and also you can check the option to Draw a frame around the pages Page Setup First Header Footer Margins Page Setup x Top cm Bottom 2 54 cm Unit o cm Left L 1 5 cm 9S Right 1 5 cm r Settings M Same header on all pages T Draw a frame around the pages Figure 8 39 Page Setup dialogue Page Setup tab First Header which allows you to select the components to beincluded in the header for the first page Note If you have not selected the setting Same header on all pages on the Page Setup tab then a fourth tab option Header is visible in the Page Setup dialogue The Header tab allows you to select items to includein the headers of the individual pages except for the first page Page Setup Eg Page Setup First Header Footer Header Presenting results 8 The selected items will be included in the report Select Items Current user Alignment Left amp ti Select all C Middle C Right Method file name _ Gear plau age number Layout name T Line over T Line under Report title Report Tite Figure 8 40 Page Setup dialogue First Header tab 1 6 7 Select the items to be included in the header Click on Select All or Clear to faci
116. an constitute a serious security risk All user passwords are visible in the User setup dialogue For security reasons make sure that access to this function is restricted 5 Select ahome folder from the Home folder drop down list Y ou may choose any folder from this list even if you created a home folder in the New user dialogue 6 Inthe Access field select other folders to which the user will have access from the folders list Up to 20 folders can be set up to be shared Selecting a folder here will give the user access to all files and folders therein Folders that are not selected in this list will not be visible in the methods or results panel of the M ain menu Note All users should be given access to the Failed folder on each local station in a network installation This will ensure that users Can access results saved in the Failed folder in the event of anetwork communication error 13 11 1 3 Administration 13 12 7 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 drop down list Y ou can continue to define new users as long as the User setup dialogue is open Click on OK to close the dialogue If you close the dialogue by clicking on Cancel all changes made since you opened the dialogue will be lost 13 3 2 Changing user passwords Every user can change his or her own password with the Administration Change Password command in
117. ance 11 2 minimum requirements for running UNICORN 11 4 requirements A 2 security implications 11 2 setup 11 1 setup introduction 11 1 sharing a folder NT domain with TCP IP 11 6 sharing a folder NT workstations connected to a N ovell server 11 11 supported network cards A 2 UNICORN in thenetwork environment 11 1 network installation 2 10 network creating user groups and users NT domain with TCP IP 11 5 network creating user groups and users NT workstations connected to a N ovell server 11 10 N ew M ethod button 3 4 new method creation 4 1 N ew unit button 9 30 noise window 9 11 noise window adjustment 9 15 9 16 noise reducing 8 13 normalise function 8 28 notes 4 9 5 31 display in start protocol 6 3 entering 5 32 in run documentation 8 52 0 oligo system type selection 13 2 oligosynthesis systems used with Unicorn i 1 1 on line help 2 3 open chromatogram layout dialogue 8 4 method file 3 5 Index result file 8 1 overlay curves 8 25 P parameter setting changing 14 2 restoring default value 14 2 viewing 14 2 password 12 11 13 12 password entry 3 1 13 11 peak adjusting peak start and end points 9 22 adjusting the peak limits 9 20 changing peak labels 9 4 deleting a peak in the peak table 9 21 direct measurement of retention time and peak heights 9 23 excluding peaks from integration 9 5 filtering peaks from view 9 4 identification 9 26 identification names for peaks 9 23 integrating 9 1 joining a peak 9
118. ancelled the M ethodQ ueue is paused Select MethodQueue Display Running in the M ain menu and Restart or end the run in the displayed dialogue Performing a run 6 6 2 Monitoring a run BI The System control workspace displays the status of the current system On the Windows NT taskbar there may be up to four System control modules available that can be connected to different systems Separate systems may be controlled and displayed independently of each other Each System control workspace displays up to four windows for monitoring different aspects of the run Click on the View Windows toolbar button or choose View Windows from the menu to select which windows to display M Rundata Teves M Curves MV Flow scheme I Logbook Cancel Help Figure 6 3 Dialogue for choosing windows to display in the System control workspace 6 2 1 General window techniques Windows in the System control are always displayed over the full width of the workspace The boundaries between the displayed windows can be moved by selecting and dragging up or down to change the size of a specific window Any window can be maximized to the full view or restored to its original size by selecting the Maximize or Restore toggle command respectively in the associated right mouse button menu To hide a window from view select Hide in the relevant right mouse button menu 6 2 2 Run data The run data window displays the current values fo
119. ange the peak resolution algorithm edit the UNICORN IN I file by 1 Minimize UNICORN and locate the file UNICORN INI within C UNICORN BIN 2 Open the file and locate the following line EVAL ResolutionAlg If the line does not exist then add it before the Begin line 3 Choose the desired value for the algorithm 4 Save the file Note Donotmakeany changesintheUNICORN IN I filebetween thelines Begin and End as this may severely affect the functionality of UNICORN 10 peak height Pou Evaluation functions and instructions C Capacity factor where Vp retention volume V total liquid volume REE Kay where Vp retention volume Vo void volume Vc column volume SAsymmetry Asymmetry width B width A where A and B are the partial peak widths measured at 10 of the peak height with A representing the leading part of the peak and B the tailing part of the peak HETP HETP L N N 5 54 x Vp wy where N no of theoretical plates L bed height in cm Vp peak retention volume or time Wp peak width at half height expressed in the same units as Vp Plates meter is the number of theoretical plates per meter N x 100 L GC functions and instructions C 4 Evaluation procedure C 4 1 Curve operations Instruction Description Parameters ADD Adds two curves to gain a third curve which is the sum of the two curves The two source curve
120. ank run curve 8 14 subtracting the blank run curve 8 16 system settings instructions B 13 curve comparisons 2 9 curve manipulation 2 9 cut curve 8 12 D data buffers A 3 buffers capacity A 3 export 9 41 minimising loss in the event of communication failure A 3 security 10 2 transfer A 3 date and time incorrect F 5 delete instruction 5 20 Delete button 6 19 deleting blocks 5 13 files or folders 3 13 text in a chromatogram 8 17 Demo system 12 10 dialogue Batch run 9 39 Calibration page in Documentation 8 53 choosing windows displayed in System control workspace 6 5 Chromatogram Layout X axis tab 8 8 Chromatogram Layout Curve tab 8 4 Chromatogram Layout Y axis tab 8 7 Condition 7 3 Contents tab in Create Standard Report Format 8 49 Copy curve 8 27 Copy from external 3 12 Copy to external 3 11 Create Curve 9 30 Create new unit 9 31 Create N ew User 13 10 Create Standard Report Format 8 48 Curve N ame 8 5 Curve Style and Colour tab in Properties dialogue 6 10 Curves tab in Properties dialogue 6 9 Delete Folder 13 13 Differentiate 9 29 Divide 9 27 Enter Information 12 16 Index Evaluation log page in Documentation 8 54 Export Curve 9 43 Export Documentation 9 42 Filter 3 8 Find file 3 9 First H eader tab in Page Setup 8 45 Footer tab in Page Setup 8 46 Generate Report 8 34 Import Block 5 16 Import procedures 5 34 Instant Run 3 4 Instructions Alarms group 14 1 Integrate 9 2 Leave Control 6 23 Li
121. arameters StartSlope and EndSlope values are set The procedures for finding suitable slope values for a particular run are described below It is also possible to set up conditional Watch instructions which allow the progress of a run to be determined by the events during the run eg start collecting fractions when the first peak emerges The slope of the curve may be set as a condition used to satisfy a Watch condition in the method during therun It is thereforeimportant to use accurate slope values for the specific W atch instruction parameter ope Figure 9 32 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 Evaluating results 9 Evaluation module 1 Ensurethat you have selected Time as the x axis scale for retention in the Chromatogram Layout dialogue X Axis tab 2 Select Operations Differentiate Select the desired UV curve check that a First order calculation is selected and click on OK The differentiated curve will appear in the active chromatogram Differentiate x Source chromatogram Target chromatogram 7 02 test20_PressDet 03 test20_Press4CN oa gt Curve name gt Condi 01 Ad First order Secondorder C Inverse Cancel Hep Figure 9 33 Differentiate dialogue 3 M easure the y axis values on the differentiated curve by clicking on the XY ico
122. 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 be run The method remains open in the M ethod editor when it has been saved so that you can continue editing if you wish Once the method has been saved choosing File Save saves the current state of the method under the same name 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 4 7 Starting a run This section briefly summarises how to start arun with a method The method must benamed and saved before it can be started See Chapter 6 for more details of how to run a method Note If you 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 whileit is running therun will not beaffected It isthe version of the method that is saved on disk at the time when the method is started that controls the run 1 Establish a control mode connection to the system where the method is to be run See Section 6 5 for details You cannot start a method without a control mode connection to the appropriate system 2 Choose File Run from System control for the required connection and select the method to run Alternatively click on the method in the Methods window of the M ain menu and select Run from the right mouse button me
123. area or e type the desired left and right limit values in the boxes marked Left limit and Right limit Presenting results 8 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 curve will not begin at zero unless designated as one of the limits The original curve is not changed Click on OK A new dialogue is displayed Select whether to save the new cut curve in the Source chromatogram i e the current active chromatogram or in a New chromatogram If you select the latter option you can change the name of the chromatogram Click on OK If the destination of the cut curve was the source chromatogram the cut curve is automatically displayed in the source chromatogram If the destination of the cut curve was a new chromatogram this will be represented as a new open chromatogram window 8 3 2 Reducing noise and removing ghost peaks Sometimes the chromatograms may contain curves with a noisy baseline Thenoise can be caused by several things e g a dirty flow cell air bubbles electrical noise dirty buffers etc The amount of noise can usually be reduced by taking proper precautions eg filtration of buffers and instrument maintenance Smoothing a curve The smoothing function allows background noise to be reduced or removed from any selected curve The type of smoothing function you should choose depends upon the type of noise encounte
124. arted when the computer is booted up In other words you must restart the computer in order to obtain a system connection in UNICORN F 3 8 Print screen does not send a copy of the screen to the printer Print screen only makes a copy of the screen to the clipboard and not to the default printer If you wish to make a print out of the view on the screen press the lt Print Scrn gt key and paste the image from the clipboard into an appropriate program such as M icrosoft Paint and then print out the image F 4 Evaluation problems F 4 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 4 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 compatible 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 5 Troubleshooting F 6 Index Index A About UNICORN button 3 3 access 13 11 authorisation level items 13 4 defining levels 13 4 examples of levels 13 7 security 10 1 ActionlfPC Fail setting 14 4 adding curves 8 17 adding blocks 5 11 administration overview 13 1 user 13 9 alarm activation 14 3 alarms display 14 2 introductio
125. 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 Text instruction editor and the following sections present the components of a method and how to edit and create blocks The text instruction editor is used for entering and editing method T instructions Click on the Text instructions toolbar button or choose View Text Instructions to display the text instruction editor Method Editor UNTITLED AA E File Block View Adviser Help oasa alal feo 8 E Main Base CV 100 Column_Volume ml Any Message Fill your column with DNA T support Screen age Press CONTINUE when ready Screen o ts_ox Block Column_wash Block Add_DNA_ Block Add_DNA_1 Block Add_DNA_ Block Add_DNA J Block Add_DNA_A Block Add_DNA_ Block Add_DNA Block Add_DNA_G Block Add_DNA_G Block Add_DNA B Breakpoint Thetiuctions C Pump 0 00 aNd Flowpath EA C AlarmetMon Column Insert any E Base Volume 101 389999 fioo jm C Watch Other Ready I ESE Figure 5 5 The Method editor in text instruction mode showing the Block window top text instruction window centre and instruction box bottom 5 6 Creating and editing methods 5 Up to four windows can be displayed together with the instruction box
126. at is currently active in the Evaluation window All chromatograms This inserts all chromatograms contained within the Evaluation window 1 2 etc This inserts the specific chromatogram corresponding to the selected number name 2 Check the appropriate Settings for the inserted chromatogram s Y ou can select to view and print the chromatograms with Thick lines view the chromatogram s in Landscape orientation insert the chromatogram s so that they Start on new page and or show a chromatogram on a Full page 3 To define the layout for the chromatogram s click on the Define layout button The Report Chromatogram Layout dialogue is displayed M ake the appropriate selections in the various tabs for the report Presenting results 8 Notel Selections made in this layout only affect the report and not the view of the chromatograms in the Evaluation window Note2 Appropriate variables can be selected for the chromatogram header for example if you wish to mark the chromatogram with the sample ID when using the autosampler Click on OK when you have made your selections to return to the Setup Chromatogram dialogue 4 If appropriate change the characteristics of the Fonts by clicking on the buttons Chromatogram Peak table and or Header text 5 When you have made your selections for the chromatogram s click on OK in the Setup Chromatogram dialogue The chromatogram s are now inserted into the report M ethod The
127. ation Check the System installation option if you are installing a new or an additional system on a stand alone computer or a local station in a network or if you want to change the settings for an existing system Systems are not installed on demo or remote only systems Strategy files Check the Strategy files option to reinstall system strategies or to install additional strategies 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 Template files Check the Template files option to re install template files or to install template files for a new strategy N ormally templates are installed together with strategies If you are not installing a strategy at the same time a dialogue will be shown displaying all strategies that are installed on the system Y ou must select the strategy to which the method template files correspond Templates are installed on the network server in a network installation Adviser Check the Adviser option to reinstall the Adviser This will not affect any existing method or result files in the system T he Adviser is always installed locally even in network installations 12 19 12 Installation 12 20 Administration 1 3 13 Administration There are three main aspects of administration of a UNICORN installation System administration
128. ation Help Figure 4 2 Sequence page in Run setup containing the 20 base sequence pre defined in the method template 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 window of the M ain menu To run the test20 method follow the instructions detailed in Section 4 7 and Chapter 6 Y ou can change the method variables prior to the commencement of the run 4 3 Creating a sequence and method 4 4 As described in Section 4 2 some of the method templates contain a partially built method for a pre defined 20 base sequence These templates are Fix Column Recycling Coupling Fix Column Flowthrough Coupling Adj Column Recycling Coupling Adj Column Flowthrough Coupling Creating methods from method templates 4 By replacing the pre defined sequence in these method templates with a sequence of your choice you can quickly and easily create a ready to run method 1 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 pageto display the pre defined sequence Select the predefined sequence in the sequence editor and delete it using the lt Delete gt key o
129. ations Access level name Operator kd Rename Items I Method editor Run methods I Evaluation Manual interaction I User setup Levels VV Pause TI Audit trail System setup T Calibrate Tune I Delete move Home only T System settings I Delete move T Edit MethodQueue T Copy file s T Confirm T Edit global list s I Unlock locked system I Maintenance T Quit program Cancel Help Figure 13 6 Suggested authorisation profile for process operators 13 3 User administration All UNICORN 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 functions Caution As part of the installation procedure new users should be created with passwords and restricted access rights as required The default user should be deleted or redefined to prevent unauthorised access to the system M aintenance of user authorisation information is the responsibility of the system administrator In a newly installed system log on as user default To define edit or delete users choose Administration User setup in the M ain menu To use this menu command you must have authorisation see above All user administration is performed from this dialogue 13 9 1 3 Administration User Setup default c Default c Failed Figure 13 7 The User
130. ations are included Generate Report The Generate R eport dialogue displays a tree structure of all drives that are reached from the computer performing the report generation By default the report is saved in the folder Unicorn R eports although you can change this folder to another one of your choice Generate report Select a directory to store the report and press the Finish button to create it 3 TEMP C Report_98011401 Report_98020801 H E Server Settings H E WINNT4 amp E amp gary on pl1500 H ine whe ee ILCON ri E Print Preview lt Back Cancel Help Y ou will also notice that the Print and Preview buttons are enabled in this dialogue Preview opens the report in N otePad while Print prints out the report without any preview 13 5 2 Generating a report from System Control If you activate the Generate Report Wizard by clicking on the Report button in an error message dialogue in System control the following dialogues are displayed Generate Report Wizard See description above Administration 1 3 Description All of the problems errors that are currently listed in the System control error message dialogue are recorded here H elp text is then shown specific to the selected error in the error message dialogue By selecting a different error in the dialogue the appropriate text is shown All problems with the help texts are included in the report You also have the possibility t
131. aved before performing a run Enter anamefor 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 The method templates are constructed from blocks representing the stages in a typical synthesis Each block has a set of method variables displayed on theV ariables pagein the run setup Y ou set default values for the variables in the M ethod editor and can change these values for a particular run in the start protocol before the run is started Variables Questions Notes Reference Curves Evaluation Procedures Method Information Result Name Start Protocol Sequence Main 4 Column Volume ml 0 10 999999 00 START_parameters wv 6 30 mi 1 00 200 00 Weight_of_suppott fico g 01010000 Loading_of_support 10 umol g 10 00 250 00 Col_Diam
132. ayed in either the source chromatogram or in a new chromatogram that you created 8 Select the curves that you want to view in the Chromatogram Layout dialogue Curves can also be scaled individually or all with the same scale using the All with this unit function in the Chromatogram Layout dialogue see Section 9 2 6 If you stacked the curves and you want to change the stack offset the easiest way is to import the curves again with another offset value Theindividual curves can also be moved see Section 9 4 3 9 If desired select the Store in new chromatogram box and give the chromatogram a new name Alternative B Importing curves using Open Using the File Open Curves function individual curves may be imported into the active chromatogram 8 Presenting results 8 26 1 Ensure that the destination chromatogram for the imported curve s is active on the screen CA ADefauk Select curves Name Size Type Chromatogram Selected curves EJ FPLC Directory example fies User Folder Test20_PressDet id1932 Scouting Folder test20_PressACN jez SMART Manager example files User Folder waj Baseline example 1 1173KB Result File Hi Baseline example 2 985KB Result File uy Baseline example 3 929KB Result File w id 18501 1160KB Result File a ig 18502 959KB Result File E Peak skim example 998KB Result File 4 gt uyiesi20ee 1164KB Result File Store in chromatogram Temporary oK
133. ayout dialogue Chromatogram layout 1 x Curve Style and Colour Edit Texts Layout Library Header Curve Names Y Axis XAsis Curve Peak Table r Curve name appearance Curve legend position C Left Alignment M ChromatogramName C Bight Alignment Curve Name Top Alignment T Apply to all chromatograms Cancel Figure 8 4 Curve Names dialogue 8 5 8 Presenting results 8 6 2 Check the appropriate option boxes for the Curve name appearance 3 Select the appropriate Curve legend position option It is usually sufficient to select the Curve name option if only one chromatogram is being evaluated H owever confusion may arise when more than one chromatogram is shown so more complete names may be necessary 8 2 5 Changing the colour and style of curves All curves within a chromatogram are represented by a default colour and line style Curves imported into the chromatogram or newly created curves are automatically assigned a colour and line style To reassign thecolour and or style of a specific curve do the following 1 In the Chromatogram Layout dialogue select the Curve Style and Colour tab 2 To change the colour and or line style of a curve select the curve of interest from the list 3 Select the desired colour and or style Chromatogram layout 1 x Header CuveNames Y Axis Axis Curve Peak Table Curve Style and Colour Edit Texts Layout Library Select cu
134. being installed is using CU 900 or UNICORN control board AT For each system you will have to set the correct settings for the control unit interrupt and address R efer to chapter 13 3 for the correct settings The Default button gives the default resource settings for the selected system type If you want to remove a system click on the Clear button to clear all fields AII fields must contain a value before you can continue Click on OK to save the changes or Cancel to abort the dialogue In both cases you are returned to the System Installation dialogue 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 12 If you installed more than one system makea note of which system is connected to which control unit This information is useful to have when you set up thesystem table or if you must in the future reinstall UNICORN Note If you want to define systems later or change the settings for an already defined system run thesetup program onceagain with only the System Installation option checked in the Component Selection dialogue 12 14 Installation 1 2 15 If you have chosen to install strategy and or template files the Strategy amp Template Installation dialogue is displayed If the correct strategy is visiblein thelist mark that strategy and click on the Install button If you have the
135. but continues to pump liquid at the current flow rate and eluent concentration settings 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 Continue is pressed Behaviour on Pause is strategy dependent Pause suspends execution of a method and stops all pumps so that the system comes to a standstill In O ligoPilot and OligoProcess valves remain in the position they werein beforethe pause Accumulated time and volumeis 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 Continue is pressed Resumes execution of a paused or held method Terminates method execution and puts the system into End state Performing a run 6 These commands can also be located under the System control Manual menu The available buttons in System control are dependent on the control status of the connection Status Available buttons End Run Running Hold Pause End M anual Run Pause End Hold Pause Continue End M ethod pause Hold Continue End M anual pause Run Continue End Other buttons on the toolbar are Opens a dialogue for choosing which wl window panels to display Clicking on this button is equivalent to choosing the menu command View Window O pens thedocu
136. button from within any of the tabs Saving the report format 1 Click on the Save As button 2 Inthe displayed dialogue enter a name for the format 3 Ifyou want the report format to be saved globally check the Save as global format option if you have Edit global lists authorisation 4 If you want the format to be used as the default format check the Save as default report format option 5 Click on OK Note If you selected the Save as default report format option the format name is changed to DEFAULT 8 49 8 Presenting results 8 7 3 Modifying an existing report format Another way of creating a new report format is to edit an existing format 1 Select File Report or click on the Report toolbar button and the z Generate Report dialogue is displayed 2 Select the report format of interest and click on the Edit button Select either Standard format or Customised format and click OK Customised format Choosing the Customised format button opens the Customised Report window As these options are dealt with for creating a new customised report see Section 9 7 1 they will be treated here only briefly To add formatting to the page layout 1 Add new objects as described in Section 9 7 1 Alternatively you can Cut Copy Paste or Delete objects using the right mouse button menu 2 Edit existing and new objects by moving them to new positions resizing them or changing their properties Change properties by
137. by the network administrator 1 2 10 11 12 13 14 15 Open the control panel with Start Settings C ontrol Panel Double click on the Network icon A question will bedisplayed asking if you want to install Windows NT Networking Click on Yes In the next dialogue select Wired to the network and click on Next Specify the network adapter to use by clicking on Select from list If your adapter is shown in the displayed list select it and click on OK Alternatively click on Have disk and insert the disk with the latest version of your driver Enter the path to it in the dialogue and click on OK Click on Next M ake sure your selected adapter is shown in the list and click on Next Select TCP IP as the protocol to use Click on Next Select all default the services and click on Next Configure the TCP IP protocol Information specific to the network will have to be entered This information should be supplied by thenetwork administrator Click on Next when ready Click on Next again to install the selected components Insert the Windows NT CD ROM disk and enter the path to it in the dialogue Example D Click on Continue A dialogue is displayed displaying where the files will be read Click on Continue The bindings are displayed Click on Next to continue 11 7 1 1 Network setup 11 8 16 17 18 19 20 21 22 23 Click on Next to start the network On
138. can also maximise a chromatogram window to fit in the whole Evaluation desktop by clicking on the maximize button To view several chromatogram windows side by side select Window Tile Alternatively Window Cascade will stack all of the open windows like a deck of cards 8 3 8 Presenting results 8 4 8 2 2 Opening the Chromatogram Layout dialogue M ost of the changes that you are likely to make regarding chromatogram presentation are made in the Chromatogram Layout dialogue This is opened in one of two ways Place the mouse cursor in the chromatogram window and select Properties from the right mouse button menu options N ote that the view from which you activate the Properties command determines the tab that is displayed in the Chromatogram Layout dialogue Select Edit Chromatogram Layout Note You can apply any changes made in the Chromatogram Layout dialogue to all open chromatograms by checking the Apply to all chromatograms option Chromatogram layout 1 x Curve Style and Colour Edit Texts Layout Library Header Curve Names YAris X Axis Curve Peak Table I Apply to all chromatograms Cancel Figure 8 3 Chromatogram Layout dialogue Curve tab Thelayout of the three views for header curves and peak table can be modified in the various tabs that are displayed in the Chromatogram Layout dialogue Y ou can work freely in the Chromatogram Layout dialogue and all of the configurations ar
139. change the home folder assignment for this user Select the user in the User setup dialogue and assign a different home folder e a folder to which several users share access To delete such a folder remove the access rights from each user first 13 3 7 Printing user setup information Y ou can print the settings for selected users 1 In the User Setup dialogue click on the Print button The Print dialogue is displayed 2 Select individual users for which you want to print information or click on the Select All button 3 Check the boxes for the Print Items that you want to include 4 Click on OK to print The audit trail accessed under Administration Audit Trail in the M ain menu provides a full record of UNICORN 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 13 4 1 Examining audit trails In the audit trail window select whether you wish to view the Global or System trail Global audit trails Check the items you want to display in the Global field All items are recorded in the audit trail the check boxes in the Global field 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 1999042800 xl heats ae 55 AM Logon default a Global C System
140. cified chromatogram File name Chro CHROM from the specified file matogram name RENAME_ Renames the specified chromatogram From chromato CHROM If is used as From then the gram name To current default chromatogram is chromatogram used name RESTORE_ Resets the destination for the subse DESTINA quent curve and peak table TION operations to the default _CHROM chromatogram Used in pair with the SET_DESTINATION_CHROM instruction SET_ DESTI O pens the named chromatogram as Chromatogram NATION destination for the subsequent curve name _CHROM and peak operations Used in pair with the RESTORE_DESTINATION _CHROM instruction C 21 Cora functions and instructions C 22 C 4 6 Other BASE Sets the x axis base in which the fol lowing 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 X axis base COMMENT Inserts a comment below the marked instruction Comment text ENDLOOP M arks the end of a LOOP statement LOOP The instructions between this state ment and the EN DLOOP 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 n Number of loops REPORT Prints a report with the
141. ck instruction is executed Any settings madein a block are valid throughout the method until the settings are changed Main 0 00 Message Fill your column with DNA T support Screen 0 00 Message Press CONTINUE when ready Screen 0 00 Pause 1 0 0 00 Base_Id _dT 0 00 Block START_parameters START_parameters 0 00 Base Time 0 00 CY 6 30 CV ml 0 00 Scale 1 00 Weight_of_support g 10 Loading_of_support umol g 0 00 ColDiam 20 Col_Diam mm 0 00 DelayVol 2 00 ml 0 00 End_block Purge_G_ 2 0 00 Block Purge_G_ 2 0 00 Base Volume 0 00 Flow_Reag 10 00 ml min 0 00 Amidite G Waste 0 50 End_block Figure 5 10 Illustration of the flow of process control through method blocks 5 10 Creating and editing methods 5 Calls may be of two types e Unconditional calls are made with the Block instruction e Conditional calls are made with a Watch instruction which makes it possible to call a specified block or an instruction when a particular monitor 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 W atch instruction is issued determines when the watch begins not when the block is activated The block will in fact never be activated if
142. click on the Peak window button in the Integrate dialogue A chromatogram window will open containing the curve and two vertical cursor lines These lines can be dragged to define a region between them that will be analysed Alternatively x axis values for the Left limit and Right limit may be typed in Click on OK to return to the main dialogue 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 Q Peak window _ ol x test20_Cond lusiem J L LL Lai 1500 2000 2500 Left limit 1345 03542 Right limit 2478 12356 Figure 9 6 Peak Window chromatogram window 9 6 Evaluating results 9 Peak skimming Thearea under a peak can be calculated either using drop lines or peak skimming Drop lines are vertical marks that split two peaks at the valley This is most commonly used for peaks of relatively similar size In some circumstances for example when a peak has a shoulder use of a drop line will cause too much area of the first peak to be lost to the peak that forms the shoulder Thus the skim peak function can be used when the smaller peak is skimmed off with a straight line starting at the valley between the peaks and ending at the point on the other side of the smaller peak where the slope of the skim line is equal to the slope of the curve In doing
143. cording a procedure 9 33 red bullet beside text 5 18 reference curves display in start protocol 6 4 reference curves display at start of run 5 39 reject peaks 9 5 remote station 11 3 renaming files or folders 3 13 Index report adding and deleting pages 8 35 adding objects 8 35 adjusting available viewing options 8 43 adjusting chromatogram object settings 8 37 adjusting documentation object 8 40 adjusting Evaluation log object 8 41 adjusting free text object 8 36 adjusting method object 8 39 adjusting page layout 8 44 adjusting settings for quantitate and molecular size object 8 41 creating a customised report format 8 34 creating a new standard report format 8 47 customising the format 8 50 defining placement and or sizing of object 8 41 defining the layout for the chromatogram 8 38 generate 13 22 generate from System Control 13 22 generating from the main menu 13 19 modifying an existing report format 8 50 moving and resizing objects 8 41 previewing and printing 8 49 previewing contents 8 48 printing 8 46 printing report 8 33 saving report format 8 46 saving the report format 8 49 selecting a format 8 34 selecting standard report options 8 48 standard format 8 50 report generator AKTA hardware information 13 21 attachments 13 21 integrity check 13 21 problem description 13 20 reproducibility information 13 20 systems list 13 19 report Generator Wizard 13 18 reports overview 2 9 resolution C 9 maximising A 3 measurem
144. ct list You can also choose to import a procedure from another method Select a method to show the procedures stored in the method If you have chosen a method click on Evaluation 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 remains open until you click on Close so that you can continue to add procedures from thesameor different method files c Default Select Name Print_Synthesis_Data Prev Folder oo 312KB Method File mpar Close Help Procedure List Figure 5 34 The Import procedures dialogue 5 34 Creating and editing methods 5 Deleting procedures To remove one or more procedures from the method 1 Select the Evaluation Procedures page and select the procedure s to be deleted 2 Click on Delete and confirm the deletion 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 Evaluation Procedures 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 remains open until you click on Close so that you can rename more than one procedure without closing the dialogue Editing procedures To edit a procedure in a method 1 Select a procedure on the Evaluation Procedures pag
145. ctivity or concentration of salt in your fractions it is possible to calculate these from the conductivity curve Note Theconductivity signal isnot linear above 0 3 M but you will still gain a relatively good idea of the salt concentrations above 3M A conductivity curve usually given the name Cond is stored in a chromatogram within a result file This curve represents the real conductivity data in mS cm and should be used for calculations Another curve Cond is also present and is the same as Cond but re scaled to display percentage values To make the calculations 1 Select Operations Fraction histogram Select the Cond curvein the left list and the fractions curve should already be selected in the middle list If you have earlier pooled fractions it is possible to select the desired fraction curve Click on OK 2 Select the appropriate fraction curve in the Chromatogram Layout dialogue In the active chromatogram you will see the fraction Evaluating results 9 marks the fraction histogram of the conductivity curve and any other selected curves Double click on the XY box to display the vertical cursor line Click on the desired curve legend and the corresponding y axis is displayed 3 Usethe mouse to drag the vertical bar back and forth along the x axis For a given fraction its conductivity is displayed in the active XY icon box 9 3 Automated evaluation procedures An evaluation procedure is a recorded sequenc
146. curves that have been created during a run such as UV conductivity fraction marks etc 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 created and saved during an evaluation session The default name for the first chromatogram in a result file is 1 8 1 2 Temporary chromatogram The Temporary chromatogram is essentially an empty chromatogram and is specific to the Evaluation module Thus curves can be copied into Temporary using Edit Copy Curve 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 It can also be used to keep blank run curves or curves to compare when opening different result files Information contained within the Temporary chromatogram is automatically saved from one evaluation session to the next but is not saved within the result files Click on the window restore button or select Windows Temporary The contents of the temporary chromatogram can be removed by selecting Edit Clear temporary chromatogram 8 2 Basic presentation of chromatograms 8 2 This section gives directions on how to access result files and optimise the presentation of a chromatogram and its curves via the so called Chromatogram Layout dialogue The last evaluation operation that
147. d and ONOFF EvalProc the corresponding parts will be ONOFF Method included in the exported file Info ONOFF Method N otes ON OFF StartN otes ONOFF RunN otes ONOFF EvalN otes ONOFF Sys Settings ONOFF Calibra tion ONOFF LogBook ONOFF Result N ame ONOFF Column Info ONOFF BufferPrep NAME Export to file EXPORT_ Exports the documentation in the cur ON OFF Variables DOC_ASCII rent result filein ASCII format to the ONOFF Scouting ONOFF Start Proto col ONOFF Questions ONOFF RefCurves ONOFF EvalProc ONOFF M ethod Info ONOFF M ethod N otes ONOFF StartN otes ONOFF RunN otes ONOFF EvalN otes ONOFF Sys Settings ONOFF Calibra tion ONOFF LogBook ONOFF Result N ame ONOFF Column Parameters N AM E Export to file Evaluation functions and instructions C C 4 5 Chromatogram functions Instruction Description Parameters COPY _ Creates a copy of the specified From chromato CHROM chromatogram If is used as gram name To source then the current default chromatogram chromatogram is used If isused name as destination then a default name will be created for the copy CREATE_ Creates a new chromatogram with Name NEW_ the given name If is used for the CHROM chromatogram name a default name will be generated and used DELETE_ Deletes the named chromatogram If Chromatogram CHROM trying to delete the current default name chromatogram a run time error will be caused OPEN _ Opens the spe
148. d on the designated search criteria Select the chromatograms that you want to import If you click on the Select All button all of the displayed chromatograms are selected If you want to clear the list of displayed chromatograms click on Clear Click on Run to perform the batch run Any created curves and peak tables will be saved in each result file automatically To view the results of a batch run on a specific result chromatogram open this in the Evaluation workspace Note If you include the currently opened result file in the batch run it will stop temporarily and you may exclude this result file from the batch run If you keep the current result file in the batch run the result file will be saved and then re opened to ensure that the contents of the Evaluation workspace reflects the results of the evaluation procedure The subsequent result files in the batch run will always be processed automatically 9 3 7 Evaluation procedures and reports The creation of evaluation procedures combined with batch runs isa very useful tool to produce printed documentation simultaneously for many result files This removes the necessity to open import result files onto the Evaluation workspace 1 Begin recording a new procedure by selecting Procedures Record on Select File Report and choose a report format see Section 9 5 3 Select Print in the Generate Report dialogue as the final instruction Stop the record function by selec
149. detected as peaks Sometimes you get too many peaks after the peak integration usually because noise on the baseline is erroneously detected as peaks The cause of this problem is that the Noise window parameter is too low Increase the Noise window parameter in the baseline calculation This may in some cases result in peak limits too high up on the peak slopes see example below 9 Evaluating results 9 16 noisepeak DefaultNoisewindow joj 322401 DetaultNoiseWindow_UV1_280nm 322401 DefaultNoiseWindow_UV1_280nm 01 BASEM3 Figure 9 18 A Noise detected as peaks B Peak integration after increase of Noise Window Another possibility isto use the Reject peaks function in the Integrate dialogue to reduce the number of peaks based on an appropriate criterion e g the number of peaks or the minimum peak height Peaks missing In cases where obvious peaks are not detected in the peak integration a probable cause is that the Noise window parameter is too high E Aa Figure 9 19 Peak integration with too high Noise window Evaluating results 9 Decrease the Noise window parameter until the peaks are detected Figure 9 20 Correct peak integration after decreasing Noise window Another possible cause of missing peaks is that an improper reject criterion has been used Check the criteria us
150. ding on the strategy and select the desired instruction from the displayed list For short help on the purpose of each instruction click on the instruction and press 1 gt c Enter values for instruction parameters in the Parameters field If a scroll bar appears on the right hand side of the Parameters field additional parameters are required Figure 5 17 The Method editor Instruction box 5 19 B ieira and editing methods 5 20 4 Click on Insert The new instruction will be inserted in the block either a at the 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 as the last instruction at the breakpoint if there are several instructions at thesame breakpoint as the new instruction and none of these is highlighted Note Instructions that are placed at the same breakpoint are executed simultaneously with the exception of Block instructions which are executed in the sequence in which they are written 5 4 3 Deleting instructions To remove an instruction 1 Select the instruction in the text instruction window 2 Press Delete in the Instruction Box or press on the lt Delete gt key or click on the right mouse button and select Delete An instruction that has been deleted can only be recovered by re inserting t
151. ditor UNTITLED FE File Block View Adviser Help 0 00 Message Fill your column with DNA T support Screen 0 00 Message Press CONTINUE when ready Screen 0 00 Pause 1 0 0 00 Base_ld _dT Block START_parameters Block Purge_G Block Purge_C Block Purge_T_U Block Purge_A Block Purge_Tetrazole Block Purge_solvents_ox Block Column_wash Block Add_DNA_T Block Add_DNA_T Block Add_DNA_G Block Add_DNA_A Block Add_DNA_A ET eri BEEBE ERE essessesssseeses 8888888888888888 5 z Block Add_DNA_C Block Ad d z p3 gt e em as E Po A 3 gt 2 l gt 5 Block Add_DNA_T Block Add__DNA Block Add_DNA_G r Parameters Column w Insert far E C AlamstMon Ea a a Volume 10 1 999999 ke 100 lace Loop_end wa m _Berloce_ C Watch Other age New_chromatogram Pause 2 e Dete Figure 5 14 The text instruction window top with the instruction box bottom Instructions are displayed in the text instruction window as follows Blue square beside text Valid call instructions i e Block and Watch instructions to other blocks in the method Bold text Valid instructions 5 17 oe and editing methods Red bullet beside text Instructions with invalid syntax These may be a calls to blocks which are not defined in the method or b instructions which apply to a different system strategy
152. 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 at log on time on the local station A local station can be used to control the synthesis system directly connected to the PC without logging on to the network The station must however have been logged on 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 the network drives will of coursenot be accessible For runs performed in this stand alone mode where the result file is directed to a network drive the results will besaved in the Failed folder on the local station Note Itisstill required that the user log on to the local Windows NT station using a valid user ID 11 3 1 1 Network setup 11 2 Requirements 11 4 The following are minimum network requirements for running UNICORN ina network installation e Windows NT workstation version 4 0 e 3Com Etherlink III PCI network card or a Compaq N etFlex 3 network card e A valid network connection e Theuser right Access this computer from network e Protocols and services such that named pipes are usable over the network Protocols and services such that folders can be connected to a drive unit The two last points are satisfied if the services Server and Workstation
153. dow during a process run and will not be recorded in any way 14 5 1 4 System settings Introductory material M ethods and runs Evaluation System management Technical specifications A A Technical specifications A 1 System requirements A 1 1 Hardware requirements e Compaq PC Pentium 11 333 M Hz or later minimum Pentium 90 M Hz e 64MbRAM minimum 32 M b for one system 128 Mb RAM minimum 64 M b for two or more systems e 1Gb of available hard disk space NTFS file system minimum 150 M b e Colour monitor 1024x768 pixels minimum 800x600 small fonts 64k colours e 11SA slot per connected system e CD ROM drive e 1 44 Mb 3 5 diskette drive Mouse Supported printers HP Desk et 660C HP Desk et 690C HP Desk et 870C xi HP Desk et 895 C HP Desk et 2500 C HP Laser et 4M HP Laser et 5M P H P Laser et 4000 N A 1 2 Software requirements Microsoft Windows NT Workstation 4 0 with Service Pack 4 or later A 1 A Technical specifications A 1 3 Network requirements These are the recommended network requirements for running UNICORN ina network installation Supported N etwork cards 3COM Etherlink III Compag N etelligent 10 100 TX Embedded UTP Controller Compaq Integrated N etFlex 3 Controller AMD PCNET PCI Ethernet Adapter Integrated N ovell N etWare version 4 50 189 or later or M icrosoft Windows NT Server 4 0 The UNICORN software works on earlier versions as well
154. e Chromatogram Layout dialogue select the Curve Style and Colour tab 2 Check the Grid option To remove the grid uncheck the Grid option 8 3 Other presentation possibilities The Evaluation module allows you to perform operations on the curves to optimise the presentation 8 3 1 Showing part of a curve This section deals with the selection of just part of a curvefor purposes of closer examination of details and for presentation This can be done in three different ways Presenting results 8 magnification using the zoom function fixing the axes cutting the curves The zoom function In the active chromatogram window it is possible to zoom in ona designated area of the chromatogram This is the easiest and quickest way to enlarge different parts of a curve 1 Place the mouse pointer in any corner of the intended area to be magnified Press and hold the left mouse button A magnifying glass icon will replace the mouse pointer arrow on the screen Drag out a box from the point of origin to cover the area to be magnified R elease the mouse button The selected region is now displayed in the entire chromatogram window together with appropriate scales for the y and x axes RQ 3500 3000 2500 2000 1500 1000 JANNA d sdo 1000 1800 2060 Figure 8 9 Illustration of the chr
155. e and click on Edit 2 Edit the procedure as described in Section 10 3 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 35 oe and editing methods 5 36 5 6 5 Method Information Variables Questions Notes Reference Curves Evaluation Procedures Method Information Result Name Start Protocol Sequence Method name UNTITLED Method created by default Date of creation 55 10 1997 2 41 09 AM Target system SYSTEM1 Method last modified by default Date of last modification 5 10 1997 2 41 09 AM Strategy name Unknown Strategy date Unknown Strategy size Unknown Figure 5 35 The Method Information page in Run setup TheM ethod Information page displays information about the method such as method name target system for creation and date of last change information about the strategy for which the method was created estimated eluent consumption and duration in time of the method These figures for the latter two are based on values for methods with variable length parameters and will be changed if the values are changed The M ethod Information page is for information only and cannot be edited 5 6 6 Sequence T he Sequence page contains
156. e applied when you click on the OK button If instead you want to close the dialogue without applying the changes you have made click on the Cancel button The main features of the Chromatogram Layout dialogue regarding chromatograms are described in the sections below Features regarding peak tables are described in chapter 10 Presenting results 8 8 2 3 Choosing the curve s you want to see In the Curve tab of the Chromatogram Layout dialogue is a list of all curves contained within the chromatogram numbered from 01 onwards Select the curves you want to seein the chromatogram Click on OK to return to the active chromatogram window 8 2 4 Changing curve names By default names are sequentially built up from three components e result name chromatogram name e curve name For example a curve with the name 9139401 1_UV1_280 is derived from the result named 9139401 The chromatogram name is a number automatically given during a run e g 1 The curve name corresponds to the curve type e g UV 1 for UV detection of an eluted component If two or more curves of the same type were created within a result file they will be numbered accordingly e g UV1 UV2 etc For systems using a variable wavelength detector the wavelength for the UV curve is also given e g 280 If you do not want to display the entire names of the curves in both the dialogues and chromatogram windows 1 Click on the Curve Names tab in the Chromatogram L
157. e in the Methods window of main menu Copying files to external Copying files to external may be useful when you want to store all results documentation etc in a common project folder on the network or want to back up the files in a special place To copy a method or result fileto external 1 Select thefileto be copied in either the Methods window or Results window 2 Select Copy to external from the right mouse button menu or select File Copy to external T he Copy to external dialogue is displayed 3 Select the destination drive and folder and click on the Save button Logon and file handling 3 Copy To External 21 x Select files Figure 3 8 Copy to external dialogue Note If you select the 3 Floppy Drive a as the destination drive the files will be automatically compressed into a zip file thus allowing approximately 5 10 times the storage capacity M oreover if the zipped file is greater than the storage capacity of the disk the file saving is automatically spanned across several disks Files are automatically decompressed when using the Copy from external operation see below The zip function does not work if you select the Copy function Copy files from external M ethod and result files can be copied from external If the selected files have been compressed using the Copy to external function then these will be automatically decompressed To copy a method or result file from external
158. e 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 A procedure can be recorded and run using the Procedures menu commands or from the commands available in the Procedure Editor dialogue The Procedure Editor dialogue also allows you to view and edit the instructions within a procedure The evaluation module is locked during a batch run 9 3 1 Recording a procedure 1 Open the appropriate results file in the Evaluation module 2 To begin recording a procedure select Procedures Record on The Procedure Editor dialogue is displayed in Record mode 3 Minimize the Procedure Editor dialogue 4 Perform the evaluation steps that the procedure is to contain These steps are recorded as you perform them 5 To stop the recording either e select Procedures Record off or e restore the iconised Procedure Editor dialogue and click on the stop button or select the dialogue Control End Record menu command 9 Evaluating results Procedure Editor Untitled BBE File Control Help feo ee e
159. e source data point The width of the window is determined by the parameter value expressed as number of data points When 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 Thefilter algorithm only accepts odd integer parameter values between 1 and 151 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 curve For each subsequent data point the previous processed pointis 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 C 1 Cora functions and instructions t S _ P tha Sn p 1 th where tn current processed point t 1 previous processed point Sn Current source point p smoothing parameter value Increasing the parameter value increases the smoothing effect The filter algorithm accepts integer parameter values between 1 and 25 C 1 3 Median For each data point in the source curve the processed 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 poin
160. e 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 M ethod templates supplied with UNICORN include procedures named Integrate_and_Print which integrates the first UV curve in the chromatogram and prints out the results Print_Chromatogram which prints the chromatogram from the run with the scouting variables printed at the top User defined procedures are created in the evaluation moduleand may be saved in method files see Section 10 3 Procedures saved with one method file can be imported to another Creating and editing methods 5 Note1 A procedurein a method will not be updated when a Note 2 procedure with the same nameis changed in Evaluation The same applies to report formats saved in a procedure If you use an 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 printer currently set up on the local station not on the remote station If however you execute the procedure interactively from the evaluation module on the remote station theresults will be printed on the printer set up on the remote station where you are working
161. e_flow 6 00 bar 0 00 20 00 0 00 Detrit_Pump _Column 0 00 _LFlow_Det 300 Detrit_flow cm h Figure 5 25 Relationship between variables in text instructions and in the Variables page of Run setup Default variable values can be changed either by editing theinstruction in the Instruction box or by changing the value in the Variables page of Run setup Changes made in the text instruction are automatically updated in the Variables page and vice versa Figure 5 26 5 25 oe and editing methods 5 6 Run setup 5 26 as Caution 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 breakpoint or gradient length 5 5 3 Removing a variable To convert a variable to a fixed value 1 Inthe text instruction window select the instruction where you want to remove the variable T he 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 Click on Clear to delete the variable name and click on OK 5 5 4 Renaming a variable To change the name of an existing variable 1 In the Text 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
162. eak integration UNICORN allows you to identify and measure a number of curve characteristics including peak areas retention times and peak widths 9 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 Using the Calculate baseline instruction for automatic calculation of the baseline which gives in most cases a very accurate measurement Baseline calculation can be performed using the Morphological algorithm or classical algorithm see Sections 10 1 3 to 10 1 5 Calculate baseline is the most common alternative and it is strongly recommended that you read the information contained within Appendix D 2 which describes the principles of baseline creation A blank run curve with the same chromatographic conditions as the corresponding sample can be used as the baseline for peak integration Another approach that may improve the peak integration if a blank run is available is to first subtract the blank run from the source curve see Section 9 3 3 and then perform peak integration on the resulting curve using the Calculate baseline option In addition to blank runs it is possible to select any curve present in the current chromatogram e g an edited baseline see Section 10 1 5 as baseline Using a Zero basel
163. eak_start H 0 00 Block Detrit_peak_end 0 30 Flow_Det 0 00 ml min 0 40 End_block W 0 20 Block Detrit_wash 0 20 Base_Id _dT E W 0 25 Block Coupling_recycle_DNA_T_v2 E m 0 30 Block Oxidation DNA Il 0 35 Block Capping DNA W 0 00 Block Add_DNA_T_v2 W 0 00 Block Add_DNA_G_v2 W 0 00 Block Add_DNA_A_v2 W 0 00 Block Add_DNA_A_v2 W 0 00 Block Add_DNA_G_v2 WE 0 00 Block Final_detritylation 0 00 End_method Ge A A A B A E Figure 5 41 A method containing the Set_mark instruction see arrows to show the start of detritylation 5 9 5 Pausing a method A method can be programmed to pause at critical points There are two instructions for this purpose Hold suspends execution of the method but continues to pump eluent at the current flow rate and concentration settings Creating and editing methods 5 Pause suspends execution of the method and stops the pumps so that the system comes to a standstill In O ligoPilot II 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 Continue button in the System control toolbar see Section 6 2 N ote N ever select Pause or Continue during a system H old Vol_amid Vol_Ox Vol_Thio Vol_Cap as this will interrupt the adding of reagent 5 9 6 Linear
164. ed for Reject peaks in the Integrate dialogue as well as Filter peaks in the Chromatogram layout function Baseline on top of peaks In rare cases the top of a broad flat peak will be incorporated as a baseline segment ky Figure 9 21 Baseline segment on top of peak 9 Evaluating results This is one of the very few situations where it is useful to change the Max baseline level M easure the height of the flat plateau of the peak using the XY icon on the chromatogram see Section 10 1 8 Insert a valuesomewhat lower than the plateau height as the Max baseline level in the baseline calculation Figure 9 22 Correct baseline after decreasing Max baseline level If there are several rather short segments that erroneously have been incorporated in the baseline an alternative remedy is to increase the Shortest baseline segment setting 9 1 6 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 integration 1 Select Integrate Edit baseline 2 Choose the desired baseline from the displayed dialogue and click on OK A window will appear displaying the baseline and the curve from which it was calculated Additionally blue crosses are displayed the baseline points and their co ordinates in the Point list Evaluating re
165. ed in the evaluation log 1 Select Operations Shift The Shift dialogue is displayed Select the curve to be shifted Select the axis along which the shift is to be made i e along the x axis Shift retention or the y axis Shift amplitude Enter the shift value and click on OK Shift Eg Source chromatogram Target chromatogram 1 hid 1 01 test20_Cond 02 test20_PressDet O3 test20_PressACN Curve name gt PressACN 03 SHFT I Shift retention jo ml Shift type Figure 8 26 Shift dialogue Alternative C Stretching and shrinking a curve using multiply Curves can be stretched or shrunk in thex or y plane using the multiply function This function is similar to Normalise Size except that each curve is repositioned with precise numbers instead of by eye and the instruction logged in the evaluation log 1 2 Click 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 Presenting results 8 Source chromatogram Target chromatogram 01 test20_Cond 02 test20_PressDet 10 zj Curve name PressACN 03 MUL Multiply type T Multiply retention jo I Multiply amplitude fo Cancel Help Figure 8 27 Multiply dialogue 8 4 4 Mirror images of curves A very useful way
166. ed 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 icon and select the options to be included The detritylation table alone can be viewed separately under View Synthesis Data This technique is useful if you would like to export Synthesis Data to an external spreadsheet such as M icrosoft Excel Use the cursor to highlight the desired sections of the table and then Ctrl C to copy the selection to the clipboard for export Synthesis Data x 371 00 365 00 344 00 290 00 280 00 250 00 218 00 225 00 AOLO FIL FLO A AI 4 Efficiency Total Yield 54 Average Efficiency 96 6 Close Help Figure 8 34 The Synthesis Data dialogue Chromatogram The Setup Chromatogram dialogue is used to define the settings for the chromatogram object 8 Presenting results Setup Chromatogram x Selected chromatogram s Current chromatogram Settings __ Fonts Tl Thick lines Chromat A Piei Sromatogram I Start on new page Peak table T Eull page Header text Define Layout Cancel Help Figure 8 35 Setup Chromatogram dialogue 1 Select which chromatogram s to insert from the Selected chromatogram s drop down list Current chromatogram This inserts the chromatogram th
167. een problem with fit of method editor window on screen F 4 Search button 8 20 search filters 8 20 security access 10 1 backup 3 14 connection 10 1 data 10 2 features overview 10 1 in network 11 2 recommendations for control stations 10 3 security features 2 11 security system in Unicorn 1 1 Select All button 8 21 set mark during run 5 47 setting ActionlfPCFail 14 4 Frac Parameters 14 4 FractionN umbering M ode 14 4 hysteresis 14 3 Keyboard 14 4 M ethodbase 14 4 Peak_Frac Parameters 14 4 PumpGain 14 5 PumpT ype 14 5 Store 14 5 Time between samples 14 5 settings curve 14 5 display at start of run 5 40 specials group 14 4 shortcut keys ii Shortest baseline segment 9 11 Shortest baseline segment adjustment 9 18 sigma C 9 skim peak ratio calculation 9 7 Slope limit 9 11 Index Slope limit adjustment 9 14 slope value determination 9 28 slope values 9 28 Smooth 9 27 smoothing algorithms C 1 curve 8 13 parameter 8 14 software requirements A 1 sorting order of files 3 7 specials system settings instructions B 11 Spline through 9 32 stack curves 8 25 Stack O ffset 8 25 stacking and stretching curves 8 27 stand alone installation 2 10 A 2 Standard 8 51 start Unicorn 3 1 start protocol 5 38 6 3 starting a run 4 11 static electricity precautions 12 2 status bar text messages 6 17 Store in new chromatogram option 8 23 STORE ON F 3 Store setting 14 5 strategies what are strategies 2 1 strategy selection 13 2 Strateg
168. een the situations in Figures 9 12and 9 15 In Figure 9 12 no baseline segments were detected between the second and fourth peaks there are no blue crosses is this region when Edit baseline is selected The baseline follows the curve as a best fit and cannot be drawn above the curve unless Accept negative peaks is selected In Figure 9 15 baseline segments are detected on the up and down slopes of the peaks marked by blue crosses in these regions when Edit baseline is selected By considerably decreasing the Slope limit in Figure 9 16 a better baseline can be constructed leading to an improved peak integration Figure 9 16 Peak integration with decreased Slope limit Evaluating results 9 Y ou may also have to decrease the Noise window to get a perfect peak integration Figure 9 17 Correct baseline after decreasing both Slope limit and Noise window An alternative approach is to exclude the large peak from the peak integration as its presence affects the default baseline parameters and theretention and area of the large peak isin most cases not interesting Using the Operations Cut curve function the appropriate region of the chromatogram can be selected The peak integration can then in most cases be performed with default baseline parameters on the cut curve Y ou can not use the Peak window function to remove the large peak as the baseline is calculated for the entire curve Noise
169. efault report format Cancel Help Figure 8 42 Save Report Format dialogue 2 Enter a name for the format 3 Ifyou want the report format to be saved globally check the Save as global format option if you have Edit global lists authorisation 4 If you want the format to be used as the default format check the Save as default report format option 5 Click on OK Note If you selected the Save as default report format option the format name is changed to DEFAULT To save a copy of the format under another name select File Save As and enter a new namein the Save Report Format dialogue Exiting the Customise Report window To exit the Customise Report window select File Exit or click on the Exit button Y ou will be prompted to save unsaved formats When you have exited the window the newly created format is displayed in the Generate Format dialogue 8 7 2 Creating a new standard report format It you do not want to create a new report layout using a customised format you can instead use the fixed layout in the Standard report formats W ith Standard formats you can still select the objects that are included in the report and save the format for later use 8 47 8 Presenting results Selecting standard report options 1 Select the New button on the Generate Report dialogue This prompts the Create New Report Format dialogue 2 Choose Standard format and OK and then use the tabs to select the report compon
170. efautts z Browse Result fi 7 Browse All A Chromatogram B aseli a 3 Peak Skim E x Browse All aselin example eal im Example z m Found chromatograms Baseline example 2 Baseline example 1 Baseline example 3 Search Clear Select all Peak skim example id 18501 id 18502 ne ee r ee Figure 8 17 Open Chromatogram to Compare dialogue showing search delimiters 5 Select the chromatograms that you want to import If you click on the Select All button all of the displayed chromatograms are selected for importing If you want to clear the list of displayed chromatograms click on Clear 6 Click on OK and all selected chromatograms are shown on the Evaluation workspace Alternative B Importing using Open 1 Select File Open Chromatogram 2 Select the desired result file by double clicking on it and all of the chromatograms contained within will be displayed N ormally it is only one chromatogram and is named 1 3 Select the chromatogram s of interest and press the Select button Selected chromatograms are added to the Selected chromatograms list Chromatograms can be deselected by using the Remove command button 4 Repeat steps 2 3 for chromatograms in other result files 5 Click on OK 8 Presenting results 8 22 Open Chromatograms PJ c Default p Select chromatograms Name Available Selected i1932 Scouting Folder o test20hes 1
171. elected peak 2 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 Thenew left and right limits are now represented by a drop line above and below the curve respectively and the peak areas are automatically recalculated 3 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 peak table spreadsheet cell and edit the name directly in the cell Y ou will see the peak names in the chromatogram and the spreadsheet cell only if you have earlier accessed Curve Style and Colour tab in the Chromatogram Layout dialogue and selected Peak name for labelling the peaks N ote also that in order to have Peak name listed within the spreadsheet cell you must choose the Peak name option under the Peak Tabletab within the Chromatogram Layout dialogue 9 1 8 Measuring retention time and peak heights It is possible to determine the co ordinates of any point on a curve and thus obtain values for retention and peak height This is a useful tool for many other functions such as for measuring the parameters used in baseline calculations Co ordinates can be obtained in two ways
172. empts with the name of the user logging on or off including failed logon attempts Displays all system definition deletion and redefinition events Displays M ethodQ ueue start operations Displays sequence start operations System audit trails Select the system for which the audit trail is to be displayed from the drop down list and check the items you want to view AIl items are recorded in the audit trail the check boxes in the System field 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 beexamined from the local station without logging on to the network System audit trail files can be viewed from any computer in a network installation 13 15 1 3 Administration 13 16 98 02 28 06 39 32 Acknowledge Eror on System SYSTEM1 C Goba Bystent 98 02 28 06 46 34 Acknowledge Error on System SYSTEM1 E 98 02 28 08 46 01 Session start e 98 03 01 10 02 48 Acknowledge Error on System SYSTEM1 Explorer 00 98 03 01 10 38 14 Session start M Run End Manual VV Alarms Warnings M Enors Calibration r Renew log file Renew every 0 day of Set Now Bun Hours Open Backup Print Close Help Figure 13 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
173. en 0 00 M essages which areset 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 5 47 oe and editing methods 5 48 Set_mark Other text messages can be inserted into the chromatogram at set points using the Set_mark instruction The Set_mark instruction is also a convenient way of inserting a note into both the logbook and the chromatogram during a run This contrasts from M essage which is displayed on the screen and entered into the logbook only Set_mark can be used to insert manual notes for example when a problem occurs in the run The instruction can also be incorporated into a method as shown in the following example where Set_mark is used to highlight the start of detritylation in a method E Main 0 00 Message Fill your column with DNA T support Screen 0 00 Message Press CONTINUE when ready Screen 0 00 Pause 1 0 0 00 Base_Id _dT W 0 00 Block START_parameters E 0 00 Block Purge_G_ 2 W 0 00 Block Purge_C_ 2 W 0 00 Block Purge_T_U_V2 E 0 00 Block Purge_A_ 2 W 0 00 Block Purge_Tetrazole E 0 00 Block Purge_solvents_ox E 0 00 Block Column_wash W 0 00 Block Add_DNA_T_v2 Add__DNA_T_v2 0 00 Base Time 0 10 Block Detritylation Detritylation 0 00 Base Time 0 00 Set_mark Detritylation begins E m 0 00 Block Detrit_p
174. ending MethodQueues Use the list box at the top left corner of the dialogue to select a M ethodQ ueue The list shows pending and running M ethodQ ueues For the selected M ethodQ ueue Start at shows the time when the M ethodQ ueue is programmed to start The actual time of start is shown for a currently running M ethodQ ueue The buttons in the M ethodQ ueue display have the following functions Restart Restarts the currently running M ethodQ ueue if a start protocol has been terminated by Cancel End Exit MethodQueues Terminates a running M ethodQ ueue after the current step Any methods currently in operation will continueto run and must be terminated with End in the System control window if they are not to run to completion Clicking on End for a pending M ethodQ ueue deletes the M ethodQ ueue from the pending list Closes the M ethodQ ueue monitor panel 7 7 7 MethodQueues 7 8 Introductory material M ethods and runs System management Appendices Presenting results 8 8 Presenting results A result fileis automatically generated at the end of arun and contains a complete record of the run including method system settings curve data run log and Trityl data The Evaluation module offers extensive facilities for presenting synthesis run data This chapter describes how to e view the documentation from a run present the chromatograms and curves of your result file compare chromatograms and cu
175. ent 9 25 resolution and sampling frequency A 3 result 8 2 changing result name after start of run 5 40 result file copying from external 3 11 copying to external 3 10 Index specifying name 6 4 result files overview 2 4 result name 5 36 results after peak integration 9 3 copying results to the clipboard 9 43 export 9 41 opening a result file 8 1 presenting results overview 8 1 saving results 8 32 retention direct measurement of retention time and peak heights 9 23 measuring retention time and peak heights 9 23 retention multiply 8 30 run comparing different runs 8 18 control mode connection 4 11 data 6 5 documentation 8 51 editing method variable values 4 11 logbook 6 14 mehod file 3 6 messages during run 5 47 M ethodQ ueue 7 5 monitoring 6 5 performing 6 1 problems F 3 set mark during run 5 47 start protocol pages 4 12 starting 4 11 starting an instant run 6 3 viewing information about a run 8 9 run data layout 6 6 pressure units 6 8 window style 6 7 run documentation calibration 8 53 evaluation log 8 54 notes 8 52 variables 8 52 Run setup 5 8 run setup 5 26 S salt concentrations measuring in the fractions 9 32 Same header on all pages 8 44 Index sampling frequency A 3 save edited procedure 9 36 layout 8 10 method 4 10 5 40 method as a template 5 41 procedures 9 34 report format 8 46 8 49 results 8 32 scale changing scale of x axis 6 11 changing scale of y axis 6 10 scr
176. ents within the displayed Create Standard Report Format dialogue N ote that the tabs for the standard report format are similar to the individual dialogues listed for objects in Customised formats See Section 9 7 1 Create Standard Report Format x Header Method Documentation Chromatogram Evaluation Log Contents The selected items will be included in the report m Select Items Current user Run user Select all Clear H Cancel Figure 8 43 The Create Standard Report Format dialogue 3 You can preview the report contents by clicking on the Contents tab Clicking on the symbols next to the content headings reveals the contents of each section of the report Presenting results 8 Create Standard Report Format x Header Method Documentation Chromatogram Evaluation Log Contents This is the information included in this report format Current user Run date amp time Report title Result file name Page number Method Main Method Blocks Documentation Chromatogram o E e Print Preview Cancel Figure 8 44 Create Standard Report Format dialogue Contents tab Previewing and printing the report Within any of the tabs on the Create Standard Report Format dialogue you can preview the entire report printout by clicking Preview Similarly once you have made all of the desired adjustments to the report format you can click on the Print
177. equence in the sequence editor field of the Sequence page Create a method as described in Section 4 2 Save the 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 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 5 5 oe and editing methods 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 instruction editor The next level of advanced editing to create new methods uses Text instructions T his involves modifying the instructions within the blocks of an existing method and or adding new blocks to methods Note Users with the appropriate access authorization have global access to methods created by all users In such circumstances an existing method can be used
178. equire unattended M ethodQ ueue operation after the start of the first method step make sure that subsequent method steps do not include a start protocol The Condition setting for each step in a M ethodQ ueue determines the relative timing of the steps If successive methods are run on the same system the timing set in Condition applies from the completion of one method to the start of the next If successive methods are run on different systems the R eady instruction in one method can be used to trigger the start of the next method i e with theR eady instruction you will beableto start the next method already before the current method has ended The Condition setting then applies from the Ready instruction to the start of the triggered method This is useful for example in situations where a method on one system prepares the starting material for the next and then continues to wash the system 7 5 MethodQueues System 1 System 2 Apply sample Elute READY Apply sample Wash Elute 7 4 Displaying MethodQueues 7 6 Choose MethodQueue Display running to display pending and running M ethodQ ueues A pending M ethodQ ueue is one for which Run has been requested but which has not yet started either because the system is not available or because the Setup time has not been reached Running MethodQueue x fori 7 Friday 21 30 Oligo4 End of Sequence Hestar End Figure 7 6 Displaying running and p
179. er see steps 2 and 3 below By default the method will be saved in your home folder To change the folder double click on the appropriate folder icon in the M ethods panel vt Default For System Name System Size T Explorer Prev Folder E test20 OPII 312KB Method File Technique Anion_Exchange Cation_Exchange C J HIC Size_Exclusion Affinity Method name EEUE Chromatofocusing Figure 5 38 Save AS dialogue for saving a method If you have more than one system available choose the system for which the method is intended The method can only berun on the system for which it is saved Remember that different systems may have different configurations and control capabilities Note Each method is written for a specific strategy The function of the method cannot be guaranteed on systems having other strategies 4 Click on OK 5 7 2 Saving as a template 1 Y ou can save the method as a template if you have Edit global lists authorisation Choose File Save as Template T he Save As Template dialogue is displayed 5 41 B ieira and editing methods 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 field 4 Select the Technique from the lists as appropriate 5 Click on OK The templat
180. er for a user select the folder in the Home folder field in the User setup dialogue Any folder may be used as a home folder As a recommendation in network installations place the home folder on a drive which is addressed by the same drive letter from all computers in the network To create a new home folder at the same time as you create a user check the Create home folder option in the New user dialogue and enter the folder name and drive in the appropriate fields To create new folders under a home folder use the New folder command in the M ain menu see Section 3 2 This operation does not require special authorisation 13 3 6 Deleting home folders To delete home folders click on Delete in the Access field of the User setup dialogue Select a folder to delete in the Delete Folder dialogue that is displayed and click on OK Caution It doesnot matter which folders aremarked in the folders list in the User setup dialogue when you click on Delete This list shows the folders to which the currently marked user has access not the folders that will be deleted All methods result files and folders within a selected folder will be deleted when the selected folder is deleted Delete Folder x Folders Figure 13 9 Delete Folder dialogue 13 13 1 3 Administration 13 4 Audit trails 13 14 Y ou cannot delete ahomefolder to which a user is assigned To delete such a folder you must first either delete the user or
181. ers use the standard Windows function keys lt Ctrl gt or gt Shift gt 2 Click with the right mouse button on any file folder icon and choose the Copy or Move command or select File Copy or File Move T he Copy or Move dialogue is displayed respectively 3 9 Jaon and file handling 3 Select an available folder or the diskette drive to which you want to copy or move the file folder and click on OK Copied files and folders are user specific Note 1 Note 2 Note 3 N ote 4 N ote 5 Y ou cannot copy or move files between the Methods and Results windows of the M ain menu Explicit authorisation is required to copy or move files see Section 14 2 To copy a file within the same folder open the file in the relevant UNICORN module e g a method file in M ethod editor or a result file in the Evaluation module and use the File Save as command in the module to save the file with a different name from the original When copying to a diskette a use Copy to external so that the files are automatically compressed If you are moving a method to another system you must always use the Copy to external Copy from external functions this will give you the possibility of connecting the method to the appropriate system The extension for the method filename is used to identify the system for which the method has been created An incorrect extension may result in syntax errors in the method or the method not being visibl
182. ersion installed copyright and web address for obtaining more information Logon Logoff This allows you to log on or off UNICORN as appropriate Instant Run This opens the Instant run dialogue Fig 3 3 in which you can select the system to run technique and template Press on the Run button to view the Start protocol and to start the run see Chapter 6 3 3 Jaon and file handling 3 4 For system oru x m Template selection Technique Adi Column Flo Adj Column Res Column Packing Evaluation Fix Column Flowthrough Coupling Fix Column Recycling Coupling P50 Calibration Method notes Adj Column Flowthrough Coupling Template Method for Adjustable volume column to OligoPilot II This template is using Flowthrough for coupling of DNA and Recycling for coupling of RNA 2 DMe reagents Methods for Oxidized and Thiolated sequenses for each type of amidite is included SEQUENCE Start with saving the Test20 sequence in the Sequence editor This sequence can later be used as a template with it s built in Cross reference list The sequence is a test sequence 20 mer of Oxidized DNA that should give a purity of gt Figure 3 3 Instant Run dialogue Note Use of this function requires that templates are defined Standard systems are supplied with templates but custom systems require that the user makes templates New Method System Control Evaluation This immediately start
183. es can curve have any y axis unit They axis of the Target curve target curve position will bea position normalised scale without unit HISTOGRAM Creates a histogram from any non First source curve fraction curve source curve 1 anda_ Second source fraction curve source curve 2_ frac curve and stores the result in the target Target curve curve position If source curve2 isnot position a fraction curve a run time error will occur The y axis of the target curve position will be the same as that of the first source curve INTEGRATE Performs a mathematical integration Source curve of the source curve and stores the Target curve result in Result curve Thisinstruction position is not the same as Peak integrate which performs a real peak integration POOL_ Pools fractions from the source curve Source curve FRACTIONS and stores the result in the target Target curve curve position The fractions are position pooled from the first selected fraction First fraction to to the last selected fraction If source pool curve is not a fraction curve or First Last fraction to or Last is not an existing pool identification a run time error will occur RET_MUL M ultiplies the retention of the source Source curve curveby theM ultiplication factor and Target curve stores the result in the target curve position position M ultiplication factor RET_SHIFT Shifts the retention of the source Source curve curve by the Shift factor
184. es 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 Edit global lists authorisation 1 Choose File Delete template 2 Select the system and the template to delete and click on Delete 3 Confirm the action Note The templates for each system are common for all users Be restrictive in deleting templates 5 8 Printing the method 5 42 Y ou can print a copy of the method including items from the method documentation Run setup and the Text instructions window 1 Inthe M ethod editor select File Print or click on the Print toolbar button The Print dialogue is displayed The dialogue contains print UNICORN modules although only those available from M ethod editor can be selected Note Itis recommended that you select the print command from the text instructions view so that you have access to the text method print options Creating and editing methods 5 a Mv M Column data IV Method variables Jn Botferrrep Recipe Scoutra T Instruction set IV Text method T Method notes Prntcurenterpansion I Start notes in Evolude unused backs T Prerun Questions T Evaluation procedures T Reference curves I Start protocol I Result file setup I Sequence Cancel Help Figure 5 39 Print dialogue 2 Chec
185. es of instructions that use these variables as parameters To see and or change the instructions click on the Text Mode button on the M ethod editor toolbar or select View Text T instructions from the menu With the Text instruction editor you have complete facilities for designing and editing your own customised methods Y ou will also use the Text instruction editor for refining and modifying methods based on the standard templates e g Changing the method base column volume volume or time e Changing valve specifications for inlet and outlet if the templates do not suit your system configuration Creating methods from method templates 4 Adding or removing variables e Adding or removing instructions to change the method functionality e Adding 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 4 13 4 Creating methods from method templates Creating and editing methods 5 5 Creating and editing methods This chapter describes the complete facilities for creating and editing methods in UNICORN 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 see Chapter 4 Use the more advanced editing facilities d
186. escribed here for e changing selected instructions in the method templates e g changing the outlet valve position e adding blocks and instructions e g Watch instructions e changing method instructions to adapt to non standard system configurations creating new methods for applications which are not covered by the templates supplied Advanced editing facilities can be used at three different levels The chosen level is dependent on thetype and extent of changes to be made e modifications at the sequence editor level i e creating custom methods by assigning a specific base in the sequence editor to a freely selected block see Section 5 1 e adding new blocks to an existing method and or modifying instructions in existing blocks see Section 5 2 e writing anew method from scratch i e selection of No Template in the New Method dialogue 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 to 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 5 1
187. essential for local control of a system 1 To start UNICORN locate the program in theWindows NT Start button under Programs Unicorn Unicorn 3 10 Alternatively double click on the UN ICORN icon on the desktop if this option was selected during installation If UNICORN is already started and the previous user has logged off click on the Logon menu command or click on Logon Logoff button the in the M ain menu module Figure 3 1 The Logon dialogue Click on your username in the list and type your password Click on the OK button to log on If you cannot remember your password you cannot log on to UNICORN Ask your system administrator or other user with sufficient authorisation to give you a new password Note If UNICORN has been installed so that no password is required for logon you need only select you username and click on OK to proceed 3 1 Jaon and file handling Press the Cancel button to abandon the logon attempt Network installations In a network installation you must be logged on to the network before starting UNICORN Any computer station in the network with UNICORN software installed can be used to log on to UNICORN Y ou can log on with the same username and password on multiple computers simultaneously Each oligosynthesis system can have only one control mode connection at any onetime but it can have several view mode connections In a network installation the same or different users may establish
188. esult file is saved in the FAILED folder on the local station F 3 4 The Method System Connection dialogue keeps appearing If the Method System Connection dialogue keeps appearing you have some method s that you have not connected to a system most likely from imported methods using the Copy from external function in the M ain menu see Section 3 3 5 Connect the method s to the appropriate system and the dialogue disappears F 3 Troubleshooting F 4 F 3 5 The method editor window does not fit on the screen If the M ethod editor window does not fit on the screen and has scroll bars you may have the incorrect font size installed For pre installed Windows NT 4 0 the display screen resolution is set at 1024x768x65536 with Large fonts Y ou need to install the Small fonts which requires that you have available the Windows NT Workstation 4 0 CD ROM shipped with your Compaq computer Insert the CD ROM and follow the directions on the screen Note Be sure to always install the latest N T 4 service pack after installing something from the Windows NT 4 0 CD ROM F 3 6 There are red instructions in a method Red instructions i e instructions with a red dot in a method are syntax errors and may be due to the following e Themethod was connected to a wrong system i e the strategy of the system is incompatible with the method The method instructions do not correspond to the components you have chosen
189. etention and used by Pause_at_Cycle End to Pausea synthesis Also moves the marker in Synthesis Data in Run Control PeakCond1 Sets the minimum peak M inPeak N ot to be regarded as a disturbance when using the instruction Watch and the limits D_Baseline used by the instruction Watch Stable _baseline PumpError Enables disables the alarms from the pumps Scale Loading umol g weight g 10 250 umol g and 0 1 100 g respectively Integrate Start Indicates when the method can start looking for 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 CT_Capping Sets the contact time of capping B 1 4 Watch Instruction D escription Comment 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_O ff Cancels a watch on a specified monitor Watch_Efficiency 1 0 151 B 7 B Strategy for OligoPilot II B 1 5 Other Instruction Description Comment Base Defines the base for a block for Volume calculating breakpoints Each block Time must have a defined base Column volume Block Calls a block unconditionally Block name Continue Resumes execution of a paused or held method This instruct
190. ethod File basic10 307KB Method File basic10 303KB Method File explorer100 226KB Method File aiex798 iex1022 iex1023 ex2293 Figure 3 5 Icon and detail display modes illustrated for the Methods window Logon and file handling 3 Sorting order In the details list viewing mode files can be sorted in the window according to one of Name alphabetical order or reverse alphabetical order System alphabetical order or reverse alphabetical order Method window only Size smallest or largest files first Type alphabetical order of file extension type Modified last recently modified files first To change the sorting order choose Sort from the right mouse button menu or File Sort and choose the appropriate sorting order from the menu cascade Alternatively click on the column headers in the window for Name System Size Type and Modified to change the file sorting accordingly Click a second time on the same sorting option and thefiles are sorted in reverse alphabetical order increasing file size etc as appropriate to the selection Changing the sorting order affects only the currently active window Filter To restrict the files displayed according to file name or the system with which they are associated choose Filter from the right mouse button menu or select File Filter 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 fi
191. ethods Eoix GLENS Lol d default Copy to External Find v Large Icons Previous Level Smetlems Copy from External Figure 3 4 The Method window with right mouse button selected in the window left and the right mouse button menu for a selected icon within the window 3 5 Jaon and file handling w Epari Open Rename Copy Move Delete Copy to External 3 6 M ethod files can be run directly in the System control module Alternatively click on a file in the M ain menu Methods window and select File Run or click on the file with the right mouse button and select Run from the menu 3 3 3 Presenting files The way files are presented in the windows can be set from the File menu or from the mouse right button menu Presentation options are e view mode sorting order e filter for displaying only a chosen set of objects e g methods for one system View mode Y ou can select to display the contents of the windows in several Windows NT views from the View menu or View options in the right mouse button menus View the files either as a details list View Details a simple list View List large icons View Large Icons or small icons View Small Icons The details list includes a small icon identifying the type of object file name file type and the last modified date and time EIA E Methods ox Prev Folder basic10 303KB M
192. etrazole 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 Detrit_flow cm h The linear flow used in detritylation block Detrit_Pressure Flow bar M aximum flow pressure of detrit Efficiency_threshold M inimum acceptable threshold effi ciency Eq_oxidation DNA eq N umber of equivalents of oxidation Eq_oxidation RNA eq N umber of equivalents of oxidation Eq_DNA_amidite Eq N umber of equivalents of DNA amidite Eq_RNA _amidite Eq N umber of equivalents of RNA amidite Loading of support umol g To calculate the scale Recycle Time min Sets the time of recycling of amidite and tetrazole Weight_of_support g To calculate the Scale 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 Smoothing algorithms C 1 1 Moving Average For each data point in the source curve the processed curve is calculated as the average of the data points within a window centred on th
193. 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 T he calculated volumetric flow rateis shown during runs Breakpoint Instructions Parameters Flow ACN FlowRate 10 2000 Insert z E nsen co cv Rane Flow_Det Vat 0 00 cmh Flowpath Flow Reag iow Var Change AlaimstMon ae Beplace C Watch 5 CapCTE Flow C Other OxCTS Flow m 2e Figure 5 42 Setting linear flow rate in a method block The linear flow rate option is only available if the column diameter is defined in the method N ote If a column diameter has not been defined in the method linear flow will not be able to be used 5 10 How to use selected conditional method instructions Conditional instructions allow the progress of a run to be determined by the events during the run e g start collecting fractions when the first peak elutes or equilibrate the column until the eluent conductivity has reached a given value This is facilitated by the W atch instructions 5 10 1 Standard Watch conditions The system strategy includes Watch instruction for each monitor defined in the system A Watch is active from the po
194. for Periodicity enter the desired Pop up text and enter a Name for the warning type 4 Click on Save to save the warning The new warning is added to the specific component Maintenance manager Figure 6 22 Maintenance manager dialogue Warning tab with a defined warning set up for the UV lamp 5 Repeat theprocess for setting up warnings for the same component or other components in the list 6 Click on Close when you have made the appropriate selections 6 29 sorom a run 6 30 6 7 3 Viewing and zeroing the warning parameters A counter is set up linked to the Periodicity that you defined for a new warning message see above By entering the edit warning mode you are able to zero the warning parameters Y ou can do this by selecting the warning in the Warning tab followed by selecting Warning Edit from the menu Alternatively click with the right mouse button on the warning and select Edit from the displayed menu In the edit mode click on the Reset button which displays the Reset parameters dialogue Reset parameters x No of runs completed 1 Reset Next time out 3 29 1999 Reset Cancel Figure 6 23 Reset parameters dialogue Y ou can zero specific counters by clicking on the associated Reset button in the dialogue 6 7 4 Getting a warning When you end a method using an oligosynthesis system for which maintenance warnings have bee set up a warning message will be displayed once
195. formed 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 e Reports Comprehensive reports of the evaluation results can be generated for hard copy documentation of the synthesis process Generation and printing of reports may be included as an operation in an evaluation procedure to automate process evaluation and documentation 2 9 2 UNICORN concepts 2 7 Network considerations UNICORN can beinstalled on a stand alone PC workstation and or PC workstations in a network 2 7 1 Stand alone installation In a stand alone installation up to four oligosynthesis systems may be physically connected to and controlled from the workstation where UNICORN is installed a 0008 a EBDE Oligosynthesis systems PC with UNICORN wm o a Bila Bla a a Figure 2 5 Stand alone installation of UNICORN on a workstation which can control up to four separate oligosynthesis systems 2 7 2 Network control from a remote workstation In a network installation each oligosynthesis system is physically connected to a workstation but may be controlled from any workstation in the network on which the UNICORN software is installed A workstation to which a system is phy
196. from the right mouse button menu 2 Inthe displayed Properties dialogue select the Y Axis tab 3 Select the Pressure curve and then the appropriate pressure unit radio button Click on OK to implement the change Selecting the text alignment Y ou can select the way that text is aligned for the set mark curves and fraction curve 1 Select Properties from the right mouse button menu 2 Inthe displayed Properties dialogue select the Curve Style and Colour tab 3 Select the SetMark or Fraction curve as appropriate 6 13 sorom a run 6 14 4 Select the appropriate Text alignment option from Horizontal Vertical or Flyover Flyover displays the text only if you place the mouse pointer over the generated mark Click on OK to implement the change 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 Stretching a flow scheme The flow scheme can be stretched to fit the screen by selecting Stretch from the right mouse button menu Alternatively select the Properties option and the Flow scheme tab is shown in the Properties dialogue Check the Stretched box to display a stretched view of the window ACN Pump Detrit Pump mL min Press ACN Press Detrit Bar CM P Monitor HS em
197. fst JON JON JON OFF ON OFF 290 offset OFF JON JON OFF JON OFF 2A0 offset ON OFF ON JOFF JON OFF 300 offst ON JON JION JON J OFF OFF 310 offst OFF ON ON JON J OFF OFF 320 offset JON OFF JON JON OFF OFF Installation 1 2 330 offset OFF OFF JON JON JOFF OFF 340 offst ON ON OFF JON OFF OFF 350 offst OFF ON OFF JON OFF OFF 3E0 offst ON OFF OFF OFF OFF OFF UNICORN Control Board AT O ligosystems Alternative IRQs for this card are 3 5 10 and 11 The manufacturing default setting is IRQ 10 Thejumpers of the multinet card correspond to IRQs as follows Jumper IRQ setting W2 3 W3 5 W4 10 w5 11 Alternative addresses for this card are shown in the table The value that should be entered as an address in the UNICORN software installation is the number listed in the Address DualPort column The manufacturing default setting for the address is no 12 and for this setting D0000 should be entered as the address in the UNICORN software installation No S2 1 S2 2 S2 3 S2 4 Address DualPort 0 JON JON JON JON A0000 1 JOFF JON JON JON A4000 2 JON JOFF JON JON A8000 3 OFF OFF JON JON AC000 4 JON ON OFF JON B0000 5 OFF ON OFF JON B4000 ON OFF OFF ON B8000 12 5 12 Installation 7 OFF OFF OFF ON BC000 8 ON JON J
198. g 9 18 measuring noise level using curve co ordinates C 6 measuring shortest baseline segment using curve co ordinates C 5 measuring slope limit using differentiate and curve co ordinates C 6 morphological algorithm C 3 optimising the baseline parameters using a morphological algorithm 9 8 9 11 selecting baseline points C 5 selecting the baseline calculation algorithm 9 5 structure width 9 8 baseline problem baseline on top of peaks 9 17 baseline slope does not follow the source curve 9 12 batch runs 9 38 Index BioPilot methodbase instruction 5 44 block adding 5 11 calling 5 10 calling from an existing block 5 12 choosing base 5 12 controlling block and method length 5 45 cumulative time 5 46 deleting 5 13 entering length 5 12 log format 5 46 naming 5 12 renaming 5 14 strategy for creating 5 13 viewing 5 8 window 5 9 blocks copying 5 14 importing 5 14 in method templates 4 7 blocks and instructions relationship 2 5 blocks in methods 2 4 blue square beside text 5 17 bold text 5 17 breakpoint changing 5 21 moving instruction between 5 22 moving instruction within breakpoint 5 22 breakpoints 2 6 buffer capacity A 3 Cc calculating a baseline 8 16 9 1 calibrating monitors 6 24 calibration data display 6 4 calibration display of calibration settings at start of run 5 40 calling block 5 12 calling blocks 5 10 calls unconditional and conditional 5 11 capacity factor C 11 changing 13 12 chromatogram addin
199. g chromatogram object to report 8 37 comparing chromatograms from different runs 8 18 contents 8 2 copying curves into one chromatogram 8 26 displaying a grid 8 10 Index entering text 8 17 importing chromatograms to compare 8 19 8 21 optimising presentation 8 2 optimising the workspace 8 3 printing active chromatograms 8 32 renaming 8 18 temporary 8 2 viewing the curves 8 3 window adjustments 8 3 chromatogram layout changing curve names 8 5 choosing curves displayed 8 5 editing 8 4 opening the chromatogram layout dialogue 8 4 classic algorithm 9 5 Clear button 8 21 Close button 6 19 colour changing colour of curve 8 6 changing colour of text and background in run data window 6 7 changing curve colour 6 10 column choosing 4 2 columns display of definitions at start of run 5 39 communication failure consequences 6 20 comparing chromatograms from different runs 8 18 comparing curves 8 22 comparing runs 8 18 computer specifications A 1 Cond 9 32 Cond 9 32 conditional W atch instructions 5 10 conditional calls 5 11 conditional instructions 5 49 connecting oligosynthesis system to remote and local computers 6 21 connecting system controllers to oligosynthesis system 6 20 connection control and view mode connections 2 8 establishing 6 20 modes 6 21 security 10 1 system 2 8 connections are not available F 2 connections limit F 2 control capacity A 2 facilities 2 7 manual 6 16 software modules 2 2 Index control mode
200. ge the order of instructions within the same breakpoint in a block mark the instruction to move with the left mouse button and drag the instruction to its new location holding the left mouse button down Y ou can only move instructions in this way within a group of instructions at the same breakpoint W 0 00 Block Purge_Tetrazole E 0 00 Block Purge_Tetrazole Purge_Tetrazole Purge_Tetrazole Base Volume 0 00 Base Volume Flow_Reag 10 00 ml min 0 00 Solvent Tetrazole Waste Solvent Tetrazole Waste ne 0 00 Flow_Reag 10 00 ml min i Solvent ACN_3 1 Waste 4 00 Solvent ACN_3 1 Waste 8 00 Amidite ACN_A Waste 8 00 Amidite ACN_A Waste 12 00 Amidite ACN_A Waste 12 00 Amidite ACN_A Waste 16 00 Flow_Reag 0 00 ml min 16 00 Flow_Reag 0 00 ml min 16 00 End_block 16 00 End_block Figure 5 20 Instructions can be moved within the same breakpoint by dragging with the right mouse button Moving instructions between breakpoints To move an instruction to another breakpoint 1 Select the block to be moved using the right mouse button Select Cut from themenu The block is removed from thetext instruction window N ote A cut block does not mean that it has been deleted see Section 5 3 4 and can still be called from elsewhere in the method 2 Select the instruction line before which you want the cut block to be pasted Creating and editing methods 5 3 Click on the right mouse button and select Paste from the menu The pas
201. gure C 3 Width time or volume base Difference in retention between the peak end and peak start G A in Figure C 3 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 3 H eight M aximum amplitude above the baseline C F in Figure C 3 Peak endpoint reten time or volume base Retention value at peak start tion and peak end A G in Figure C 3 Width at half height time or volume base Calculated by taking the max imum height of the peak above the baseline then determining the peak width at half this value above baseline D B in Figure C 3 where BD bisects CF C 7 Cora functions and instructions Percent of total area time or volume base Peak area as a percent of the total area under the curve above the baseline N ote that this value may differ in time and volume base if the flow rate is not constant throughout the method Percent of total peak area time or volume base Peak area as a percent of the sum of all integrated peaks N ote that this value may differ in time and volume base if the flow rate is not constant throughout the method Type of peak limits Identifies the criteria for peak start and peak end as either the baseline intersection or drop line to the baseline Peak endpoint heights Amplitude above the baseline at left A in Figure D 3 and right peak limits E G
202. gy is appropriate for the synthesis system being defined Click on the Information button to display information about the selected strategy Administration 1 3 4 The Pipe server name field is the same as the Windows NT computer name L eave this value unchanged 5 Select the control unit number 1 4 in Control unit number This is the physical connection number for the synthesis system on the local computer see Chapter 13 6 Enter a valuein the Autosave interval field if you want UNICORN to save a copy of the result file at pre set intervals during a run This minimises 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 functions in UNICORN 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 14 3 System name System type Oligo fea Strategy Pipe server name MaR lA Control unit number 1 4 2 7 Auto save interval E minutes OK Cancel Help Figure 13 2 New system dialogue 13 3 1 3 Administration 13 1 2 Editing system definitions To edit an existing system select the system in the System setup dialogue and click on Edit Alter the parameters as appropria
203. h curve data samples is recorded 0 gt 1 000 s B 3 5 Method variables Variable Units Description Col_Diam mm Column diameter for linear flow rates Column_Volume ml Column volume for text method Conc_of_DNA_ amidite M Sets the DNA amidite concentration Conc_of_RNA_ amidite M Sets the RNA amidite concentration CT_Oxidation DNA min Sets the oxidation contact time CT_Oxidation RNA min Sets the oxidation contact time CT_Thio min Sets the Beaucage reagent thiolation contact time CT_Capping min Sets the capping reagent contact time CV ml Column volume for calculation of functions in the strategy CV_CT_Capping CV Sets the number of column volumes that the CT_Capping will last CV_CT_Ox_DNA CV Sets the number of column volumes that the oxidation will last B 13 B Strategy for OligoPilot II 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_Capping_wash CV Sets the number of column washes after Capping contact flow CV_Column_wash CV N umber of column volumes used in column wash block CV_Coupling_wash CV N umber of column volumes of wash after coupling CV_detrit_wash CV N umber of column volumes of detrit wash CV_tetrazole DNA Sets the of the column volume for tetrazole in DNA cycles CV_t
204. he default value in parentheses followed by the variable name e g 3 00 Recycle_Time_DNA Recyle 0 00 cm h OFF E 0 10 Block DNA_recycle DNA_recycle 0 00 Base Time 0 00 Recycle 195 cm h ON 3 00 Recycle_Time_DNA Recycle 0 00 cm h OFF 3 00 End_block Figure 5 22 Default values for variables appear in parentheses in Text instructions e when the instruction is shown in the Instruction box the VAR button beside the parameter field is active in capital letters i e VAR not Var Paramet wr Watch_PressACN rat most oe ump Watch_PressDet Less_than watch Cond Flowpath Value 1 0 151 0 c AlarmstMon 4 ctch_Int_Status var 75 Var PAUSE Instructions Watch C Other Ready Figure 5 23 Parameters with variable definition are identified by an active not greyed out VAR button In this example the Watch Efficiency Value is defined as variable and the Action 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 In the Text instructions window select the instruction where you want to define the variable The parameters for the instruction are shown in the instruction box 2 Locate the breakpoint or the required parameter in the instr
205. he block by selecting where the block will be inserted in the From field of the New block dialogue For blocks which are to be called conditionally with a Watch instruction first create the block and save it under unused by selecting an empty line in the From field Insert the W atch instruction into the text method and then make a call to the block The block will move from unused to beneath the Watch instruction Note If you call the block before inserting the Watch instruction a copy of the block will be created i e there will be two instances of the block in the text method 5 3 4 Deleting blocks To delete a block from the method using the right mouse button menu 1 Click on the desired block in the method with the right mouse button to display the menu 2 Select Delete A warning dialogue is displayed requesting if you want to totally delete the block instruction from the method Answer as appropriate YesThe block is totally removed from the method Blocks deleted in this fashion can not be called again in the method No The block is deleted from the method and transferred the Unused line Blocks deleted in this fashion can be called again in the method To delete a block from the method using the Block Delete command 1 Select the menu command Block Delete The Delete block dialogue is displayed with all blocks listed in alphabetical order 2 Select the block to be deleted and click on Delete 3 A warning dialog
206. he dialogue and then click on OK to view the inserted object The various object types and dialogues are as follows Free text T he Setup Free Text dialogue is used to define the desired text and the settings for the free text object Setup Free Text x Settings I Start on new page Font IV Size to content Cancel Help Figure 8 33 Setup Free Text dialogue In the dialogue you can 1 Enter text in the open field 2 If appropriate insert the text box on anew page by checking Start on new page 3 Automatically size the free text box by checking Size to content 4 Select the font type style colour and size by clicking on the Font button 5 Click on OK once you have made your selection The free text object is inserted Two of the report options warrant more detailed description namely Synthesis data and Chromatogram Presenting results 8 Synthesis D ata This option can be used to print out all of the 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 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 correlat
207. he 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 The Methodbase instruction Volumeor column volume base is calculated from the flow rate of the GradientPump AB or the sample pump selected with the instructions M ethodbase If no M ethodbase instruction is included in the method the default setting GradientPump will be used 5 9 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 0 00 Block 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 0 00 Call Normal Detritylation 0 00 Call Normal Detrit_wash 0 00 Call Normal Coupling_recycle_DNA_T Creating and editing methods 5 the instructions contained in 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 seq
208. he instruction If you want to suspend execution of an instruction temporarily e g during development work you can replace the breakpoint with a value after the End_block or End_method instruction Any instructions after the end of a block or method will not be executed Note You cannot delete the Base instruction at the beginning of a block Caution If you delete the End_block instruction the block will end at the last instruction in the block Creating and editing methods 5 5 4 4 Changing instructions There are three possibilities for changing an instruction e change the breakpoint e change parameters including variables see Section 5 5 select 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 Inthe Instruction Box make the required changes to the breakpoint or parameters or choose a new instruction 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 and Replace have different functions if the breakpoint is changed Change 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 You cannot change the breakpoint of an instruction to earlier than the nearest previou
209. he network 13 1 1 3 Administration 13 1 System definitions 13 2 System definitions set up the synthesis systems which are connected directly to the local computer in UNICORN installation This must be done for each new system installed In a network 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 14 2 and 14 3 To manage system definitions choose Administration System setup in the M ain menu To use this menu command you must have Audit trail System setup authorisation see above System Setup Systems E nm Close Figure 13 1 System setup dialogue x 13 1 1 Defining new systems To define a new system click on New in the System setup dialogue The New system dialogue is displayed 1 Enter the system namein the System name field The system name can be set only when defining a new connection and cannot later be edited since user access rights are linked directly to the system name N ames can be up to 30 characters long 2 Select a System Type either Chromatography or Oligo 3 Select a strategy for the system from the pull down list in the Strategy field Available strategies are determined when UNICORN is installed see Section 13 4 If you have several strategies installed make sure that the selected strate
210. he respective monitor 0 99900 uS cm Low alarm 0 99900 uS cm High warn 0 99900 uS cm Low warn 0 99900 uS cm H ysteresis 0 99900 uS cm Enabled D isabled Efficiency_ Sets the threshold for an acceptable Threshold Threshold coupling efficiency An efficiency 0 100 below this threshold will pause the Enabled D isabled synthesis pAlarm_Det Strategy for OligoPilot Il B pAlarm_ACN 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 warn ing message with the system in Run High alarm 0 20 bar Low alarm 0 20 bar High warn 0 20 bar Low warn 0 20 bar H ysteresis 0 5 bar Enabled D isabled B 3 2 Specials Instruction Description Parameters Tetrazole Percent tetrazole of the column volume Conc 1 100 CV AmiditeC onc Conc 0 01 0 5 M ColDiam Sets the column diameter in mm used Diameter in Lflow functions 10 100 mm CV Sets the column volume for calculation Volume of CT flows and volume of Capping 1 200 ml and Beaucage CV_Cap Column volume of capping Y2 CapA 0 1 10 CV and 2 CapB CV_Thiolat Sets the column volume for thiolation Volume 0 1 10 CV DelayVol The dead volume from port1 to the col Volume umn inlet 0 1 10 ml Eq_AM Concentration of amidite Eq 0 1 10 Eq_OX Equivalents of oxidation dependent on Eq the scale 1 10 B 11 B Strategy fo
211. he sequence Click on the Group button if you want the sequenceto bedisplayed in groups of three bases beginning from the 5 end 5 ATACCGAT TAAGCGAAGTTT a i B Figure 4 4 An ungrouped top and grouped bottom sequence in the sequence editor field of the Sequence page To save the sequence you have created click on the Save As button and type in a name for your sequence The name can be up to 256 characters in length Click on OK Thename 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 Place a check mark in those boxes beside the Optional method steps that you want to be included in your method Create the method for the sequence you have entered by clicking on the Create button The Create button serves four main purposes e 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 e to generate a method based on the sequence and cross reference list See Section 5 1 1 Creating methods from method templates 4 e update the method variables based on the generated method e display the Save As dialogue so that the method can be s
212. hecking the Synthesis D ata option provides information about the coupling efficiency of each base addition 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 8 53 8 Presenting results Documentation Figure 8 49 Documentation Logbook page Evaluation Log This lists all of the evaluation operations that you have performed for the current result file for all sessions including procedures executed at the end of the method Figure 8 50 Documentation Evaluation Log page Presenting results 8 8 9 Exiting Evaluation If you want to quit from the Evaluation module of UNICORN select File Exit Y ou 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 over written This may be undesirable if you have included the current result file within an evaluation procedure batch run see Section 10 3 5 8 Presenting results Evaluating results 9 9 Evaluating results This chapter will mainly describe how to integrate peaks automate evaluation operations export data and curves 9 1 Integrating peaks Using p
213. her C 22 D Feedback tuning D 1 Flow rate tuning D 2 D 2 Gradient tuning D 3 E File organisation E 1 Stand alone installations E 1 E 2 Network installations E 1 E 2 1 Local and remote computers E 1 E 2 2 N etwork server E 1 F Troubleshooting F 1 Logon problems F 1 1 Unableto log on to UNICORN F 1 2 Error message Strategy file error F 2 UNICORN access problems F 2 1 Unable to access certain UNICORN functions F 2 2 Connections are not available F 2 3 Run data Connection in System control displays a No x F 3 Method and run problems F 3 1 Cannot Q uit or Logoff from UNICORN F 3 2 Monitor signals do not appear in the system control Curves panel F 3 3 Error message Couldn t create result file F 3 4 The M ethod System Connection dialogue keeps appearing F 3 5 The method editor window does not fit on the screen F 3 6 There are red instructions in a method F 3 7 I ve logged out of Windows NT and then logged in again but can not get system connection in UNICORN only for local systems not remote F 5 F 3 8 Print screen does not send a copy of the screen to the printer F 5 maA nnn NA nmn N 1 I 1 1 1 1 1 Ww WO NNE Fr n a a 7TH a e MA a BBW WW F 4 Evaluation problems F 5 Contents F 4 1 Incorrect date and time F 4 2 Evaluation procedure aborts Index Contents M ethods and runs Evaluation System management Appendices J Introduction 1 Introduction UNICORN isa control syste
214. hich may be displayed in the System control workspace during the run can be selected here See Section 5 6 6 for a description of reference curve selection Evaluation Procedures Evaluation procedures which will be executed automatically after completion of arun can beselected here See Section 5 6 5 for a description of procedure definition and selection Method Information Information about the method being run System settings Displays the system settings for the run If the settings are not suitable cancel the method start change the settings with the System Settings command see Chapter 15 and restart the method Calibration Displays calibration data for system monitors If the calibration is not acceptable cancel the method start re calibrate the monitor s with the System Calibrate command see Section 6 6 and restart the method Result Name Thename of the result file is specified here This page is displayed if there areany other pages in the start protocol Thenames may be changed if this is permitted in the start protocol If any questions in the start protocol require authorised confirmation you will be asked for a username and password when you attempt to leave the screen containing the questions Only users with Confirm authorisation may authorise answers to such questions Each question that requires an authorisation must have a separate authorisation Note If thestart protocol for a method in the queue is c
215. ilename OPEN Result file defined in File name and Curve name stores it in target curve position If Target curve is entered as File name the position current result file will be used The File name parameter may includea path from the users root folder Evaluation functions and instructions C IM PORT_ Imports a curve to the current Chromatogram CURVE chromatogram from another name chromatogram in the current file source curve and stores it in the target curve Target curve position position IM PORT_ Imports a peak table to the current Chromatogram PEAKTABLE chromatogram from another name chromatogram in the current file Peak table source and stores it in the target curve posi Resulting peak tion table PEAKTABLE_ Opens the specified Peak tablein the Filename OPEN Result file defined in File name and Peak table name stores it in the R esulting peak table If is entered as File name the current Result file will be used The File name parameter may includea Resulting peak table path from the current users root folder C 4 4 Export Instruction Description Parameters EXPORT_ Exports the Source curve to the file Source curve CURVE_ defined in Export to Filein ASCII Left limit ASCII format In the part of source curve Right limit limited by Left limit and Right limit Every n gt sample Every lt n gt samples are exported Export to file EXPORT_
216. in Figure C 3 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 3 Sigma Standard deviation for a Gaussian shaped peak For definition see below Resolution Peak resolution For definition see Section 10 1 10 and below Capacity factor For a definition see below The Capacity factor will only be calculated when the chromatogram is in volume base The total liquid volume Vt must be entered in the Integrate dialogue for this parameter to be calculated Kav Gel phase distribution constant in gel filtration For definition see below Kav will only be calculated when a gel filtration column was used and when the chromatogram isin volume base The void volume V0 must be entered in the Integrate dialogue for this parameter to be calculated Plate height H ETP H eight equivalent to theoretical plate and plates metre The column height must be entered in the Integrate dialogue for this parameter to be calculated For definition see Section 10 1 8 and below Asymmetry Peak asymmetry indicator of column packing For definition see Section 10 1 9 and below Evaluation functions and instructions C Concentration Values calculated by the Analysis module For further details see the Analysis for UNICORN 3 10
217. ine i e no baseline subtraction at all Reusing an already existing baseline for the selected curve by selecting the Correlated baseline option This is the default alternative whenever possible 9 1 9 Evaluating results 9 1 2 Performing a basic integration To perform a basic integration 1 Select Integrate Peak integrate or click on the Peak Integrate toolbar button The Integrate dialogue is displayed Integrate x Chromatogram Baseline Target peak table LN Concistedbascine gt Baseline settings 02 test20_PressDet 03 test20_Press4CN I Accept negative peaks Peak table name Peak window Conan 01 PEAKI gt Reject peaks Column height jo cm I Peak skim fio ratio Column Vt fe fal Correlated baseline 17 3 year storage test dT frezer 990121 1_Cond1 01 BASEM Cancel Help Figure 9 1 Integrate dialogue 2 Choosethe source curve to be analysed usually the first UV curve and a peak table destination from the peak table list Any chromatogram can contain up to eight peak tables designated A H 3 M ake the appropriate Baseline selection from the above mentioned possibilities The Calculate baseline option with the default settings for the parameters is the most common choice Note There are two choices of algorithm for baseline calculation Morphological and Classic The default setting is Morphological See Section 10 1 3 for more details 4 Click on OK to perf
218. ing 5 24 in run setup 5 26 method 5 23 vertical cursor line 6 9 view mode 3 6 View Windows button 6 5 viewing blocks 5 8 view mode connections 2 8 Ww Watch conditions 5 49 watch instructions 5 10 B 7 wildcards used in file search 8 19 window Baseline box with Shortest baseline segment and N oise C 4 Create Curve chromatogram 9 31 curves 6 8 Customise Report 8 35 Cut 8 12 Edit Baseline chromatogram 9 19 Edit Peak Table chromatogram 9 21 flow scheme 5 23 general window techniques 6 5 hide 6 5 maximising or restoring 6 5 M ethod 3 5 M ethod editor flow scheme 5 23 N ormalise 8 29 Peak Window chromatogram 9 6 run data 6 6 text instruction 5 17 WindowsNT A 1 wizard report generator 13 18 WKS export format 9 42 X x axis adjustment 8 8 changing scale 6 11 xls 9 42 Index Y y axis adjustment 8 7 changing scale 6 10 Z Zero baseline 9 1 Ziegler N ichols method D 2 zoom function 6 12 undo 8 11 zoom function in chromatogram window 8 11 Amersham e Biosciences
219. inimum distance between data points This parameter reduces the total number of data points created from a curve Classic algorithm The Classic algorithm searches for all parts of the source curve which 1 4 are longer 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 have no point outside the Noise window The noise window is defined as a rectangular corridor parallel to the slope of thecurve and centred on the first and last points within the currently inspected segment slope less than the Slope limit This limits the maximum slope of the baseline to differentiate baseline segments from peaks are lower than the Max baseline level C 3 Cora functions and instructions 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 T he 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 D 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 Shortest baseline segment Noise Window
220. int at which it is issued until either 5 49 B ieira and editing methods 5 50 e the Watch condition is met anew watch is set for the same monitor e aWatch_Off instruction is issued for the monitor The conditions for which a Watch can be set are as 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 e g mAU min Slope less than Therate of change of the signal falls below a specified value expressed in monitor units minute e g MAU min Less than_or_valley Thesignal falls below a specified valueor a valley is detected A valley is detected only after a peak_maximum has been detected and the valley is defined by a local minimum followed by an increase to 102 of the local minimum value plus the Delta_Peak value see below Peak_max Thesignal fallsto a specified fraction of the most recent peak maximum minus the Delta_Peak value see below Factor 1 detects peak maximum Stable_baseline The signal is stable within the limits of the Delta_Base 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 sl
221. ion PEAK WINDOW om a Hedace File operation OFF gt lt Ampl gt Replace C Export SET_COLUMN_HEIGHT cq SET_COLUMN_VO Minimum peak width Delete Chom SET_COLUMN_VT C Other a OFF lt Ret gt Othe SET_SKIM_SIZE_RATIO Test Reject peak settings for peak integration Ea Close Figure 9 39 Editing procedures in the Procedure editor dialogue 9 35 9 Evaluating results There are several types of instructions as denoted by the options buttons e g Curve operation Integration File operation etc in the Instructions field The appropriate option button and instruction therein will be automatically selected when you select an instruction in the procedure The specific parameters contained within the selected procedure instruction are displayed in the Parameters field A simple definition of the selected instruction is displayed at the bottom left hand corner of the dialogue Y ou can also select an instruction and then press F1 or select Help Index Instruction A list of procedure instructions with fuller descriptions and parameters can be found in Appendix D 3 r Instruction COPY_PEAKTABLE NEGATIVE_PEAKS PEAK_INTEGRATE PEAK WINDOW C Curve operation Integration File operation r poa SET_COLUMN_HEIGHT SET COLUMN VO C Other SET_COLUMN_VT C Test xd Reject peak settings for peak integration Figure 9 40 Instructions field in the Procedure editor dialogue To edit the paramete
222. ion 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 Hold Places the system in Hold state This instruction has the same effect as clicking on theH old button in System control Loop Runs the instructions between a start No of loops and a number of loops 1 9999 Loop_end M arks the end of a loop M essage Generates a user defined message M essage which is recorded in the log book and M ode Screen may be displayed on the screen N oscreen N ew_ O pens a new chromatogram icon in Chromatogram chromatogram the result file All data collected after name the instruction will be stored under the new icon until another N ew_chromatogram instruction is issued Pause Places the system in the Pause state Time for the specified length of time 1 infinite 9999 9 in min utes Strategy for OligoPilot II B Ready Indicates that the next step in a process sequence may start Set_M ark Inserts a note into both the logbook and the chromatogram during a run Selected text End_Block Terminates a block and returns control to the point from which the block was called Inserts a comment in the method below the marked instruction Com
223. irst choice when trying to optimise the peak integration is to change the baseline parameters A brief description of the parameters and a suggested way of estimating settings for the Morphological baseline parameters from the source curve is given in Appendix D 2 The Morphological algorithm can be described in terms of a straight line that strolls along the chromatogram parallel to the x axis Data points for the baseline are created wherever the straight line touches the curve and the points are joined at the end to create a baseline Structure width W hen optimising the baseline parameters using a Morphological algorithm changes to Structure width will in most cases give the best improvement in results Structure width determines the length of the straight line see above which is set to a default value of the widest peak on the chromatogram multiplied by 1 5 In situations with drifting baseline for a curve the morphological baseline follows the curve faithfully Subsequent subtraction of the baseline thus creates a curve with the baseline at a more even level Evaluating results 9 Baseline example 1 1 SEB ATJMFO06 1_UV_215nm_Morphological mau E Figure 9 9 Fluctuating curve with a morphological baseline that follows the bases of the peaks at the different levels in the curve In some circumstances the default setting for the Structure width should be increased since a too low setting may result
224. ive routines such as user and system definition should be permitted only to the system administrator Installation of UNICORN 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 10 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 logon 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 1 0 Security features We 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 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 10 3 Data security 10 3 1 Network communication failure If the network communication fails while a method is running any control mode connection on a remote station will lose control of the system
225. ivity currently being performed in UNICORN 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 isin operation This feature allows for instance the System settings 1 4 pH warning threshold to be set to one value during process operation and another during system cleaning Error messages from the monitors and pumps are reported if the respective Error settings are Enabled Caution Alarms are not active unless the mode is set to Enabled This can be viewed for each instruction in the list The hysteresis setting for a warning determines the extent to which the signal can oscillate around the warning limits without re activating the warning Figure 15 2 Warning inn pe WARNING Figure 14 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 defined by the hysteresis setting above and below the warning limit This prevents repeated warnings from noisy or oscillating signals close to the warning boundary H ysteresis is only relevant for warning
226. k administrator must supply the Default Tree and Context to use Select the Default Tree and Context radio button enter the information from the network administrator and click on OK Connect the shared folder again this is necessary since each user has his own connected network drives Repeat steps 1 2 3 5 and 6 above and make sure the Reconnect at Logon option is checked Start UNICORN and set up system definitions and user profiles see Chapter 13 11 13 1 1 Network setup 11 14 Installation 1 2 12 Installation This chapter describes how to install UNICORN software 12 1 Installation summary The following installation procedures are required before UNICORN systems can be used 1 Back up files if migrating from UNICORN 0S1 10to UNICORN 3 10 2 Set up the network environment for network installations only 3 Install UNICORN hardware and software 4 Define access levels for the installation 5 Define users with home folders and access profiles 6 Check the system settings for the attached systems 12 2 Migrating from UNICORN OS 1 10 to UNICORN 3 10 12 2 1 Before migration Caution Before commencing with the migration from UNICORN OS 1 10 to UNICORN 3 10 ensure that all method files M ethodQ ueuefiles and result files are backed up on separate drive or medium and are separate to the UNICORN folders used on thenetwork When you access these files for the first time following installation of UNICORN 3
227. k the options that you want to print out 3 Click on OK to print 5 9 How to use selected unconditional method instructions This section provides recommendations for how to use some common programming featuresin UNICORN methods 5 9 1 Base instruction 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 base may be volume ml or depending on the scale defined in the system strategy time min or 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 Usethe base which most closely suits the purposeof theblock Column volume is recommended as the base for most steps in a run In some 5 43 B ieira and editing methods 5 44 situations it may be more suitable to use time or volume base for individual blocks 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 t
228. king 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 Any N ext select one of the available method templates in the Template 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 Select the Coupling list tag from the available 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 5 3 ons and editing methods Cross Reference List x Coupling Purge Solvent Purge Amidite Column wash Final detritylation Add _DNA A Add_sDNA_A Add _DNA_A v2 Add_sDNA_A_v2 Add_DNA_C Add_sDNA_C Add_DNA_C_v2 Add_sDNA_C_v2 Add_DNA_G Add_sDNA_G Add_DNA_G_v2 Add_sDNA_G_v2 Add_DNA T Figure 5 2 Cross reference list dialogue box 6 To change the assignment of a ba
229. l Sequence Method Notes ADJFLOT a Template Method for Adjustable volume column to OligoPilot II This template is using Flowthrough for coupling of DNA and Recycling for coupling of RN4 2 0Me reagents Methods H X Start Notes Run Notes Pig z Evaluation Notes Figure 5 32 The Notes page in Run setup 5 31 B ieira and editing methods 5 32 The notes are entered as free text and may be edited using standard Window editing functions to edit the notes W ords wrap automatically at the end of the field lt Ctrl X gt Cuts the marked text saving it to the clipboard lt Ctrl c gt Copies the marked text to the clipboard lt Ctrl gt Pastes the contents of the clipboard at the cursor lt Delete gt Clears the marked text without saving it in the clipboard 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 themethod notes describe thesystem setup required by the method e g eluent and sample inlets outlets column connections and so on Werecommend 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 th
230. l instructions are executed simultaneously some for example gradient and fraction instructions may take some time to complete in the liquid handling module The Delete button deletes selected instruction from the current list Only one instruction can be deleted at a time If you close the dialogue by clicking on the Close 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 6 3 3 Alarms and warnings The system settings see Section 15 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 a run a warning W or alarm A dialogue with a message is displayed on the screen e Therun continues if a warning is issued An alarm pauses the system Warnings and alarms are displayed regardless of the activity currently in progress in UNICORN you will be notified of an exceeded limit in a running system eve
231. lation 9 25 measurement 9 24 home folder deleting 13 13 new 13 13 selection 13 11 hysteresis setting 14 3 import chromatograms to compare 8 19 8 21 procedures 5 34 Importing 9 43 importing blank run curve 8 15 importing blocks 5 14 Insert button 6 19 installation address setting 12 3 hardware 12 2 operations required after installation 13 1 selecting the components 12 9 selecting the type of system 12 14 setting IRQ and address 12 3 stand alone A 2 UNICORN software 12 6 installation as stand alone or in network 2 10 installation of UNICORN on network 11 5 installing program files after the initial installation 12 18 software components after the initial installation 12 18 stategy and or template files 12 15 strategy files after the initial installation 12 19 system after the initial installation 12 19 template files after the initial installation 12 19 user information after the initial installation 12 19 installing IPX SPX on the computer 11 11 installing TCP IP on the computer 11 7 Index installing UNICORN 11 13 installing UNICORN NT domain with TCP IP 11 9 Instant Run button 3 3 6 3 instruction changing 5 21 deleting 5 20 insert 5 20 moving between breakpoints 5 22 moving within a breakpoint 5 22 instructions adding 5 19 alarms and monitors B 6 at the same breakpoint 5 44 conditional 5 49 flowpath B 4 manual 6 18 manual control B 9 miscellaneous B 8 pump B 1 system setting B 10 viewing 5 17 watch 5 10
232. le 2 Veight_of_support g 0 00 Message Press CONTINUE when ready 90 Loading_of_support um ml 0 00 Pause 1 0 0 00 DelayVol 2 00 ml 0 00 Block START_parameters 0 00 ColDiam 20 Colurmn_Diam mm 0 00 Block Purge_T_U 0 00 End_block 0 00 Block Purge_C 0 00 Block Purge_A Purge_T_U 0 00 Deck Purge G 0 00 Base Volume 0 00 Block Purge_Tetrazole 0 00 Flow Reag 5 00 ml min 0 00 Block Purge_solvents_ox 0 00 Amidite T U Waste 0 00 Block Colurnn_wash 100 End block 0 00 Block Add_DNA_T 0 00 Block Add_DNA_C 0 00 Block Add_DNA_C 0 00 Block Add_DNA_A 0 00 Block Add_DNA_G 0 00 Block Final_detritylation 0 00 End_method Figure 2 2 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 2 5 2 UNICORN concepts 2 6 Breakpoints Each instruction in a method block is issued at a specified breakpoint according to the method base Thefirst instruction in a block is always at break point 0 and all other breakpoints are counted from this point For example in the following instructions from a block 0 00 Base Time 0 00 Flow_Reag 5 00 ml min 9 00 Flow_Reag 0 00 ml min 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 Method
233. lems and click on Edit Check that the strategy pipe server name and the control number are correct according to installation at the local station physically connected to the system see Section 14 1 e If connecting to a system remotely check that the local station physically connected to the system is turned on and that the network is functioning at both the remote and the local station e Check that the limit of eight connections to the system has not been exceeded F 3 Method and run problems F 3 1 Cannot Quit or Logoff from UNICORN If you areunable to Quit or Logoff from UNICORN for aconnection you may be running a scouting method or a M ethodQ ueue These functions require a control mode connection in order to start subsequent cycles correctly F 3 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 isset can bechosen from the View Curve contents dialogue box for display in the curves panel F 3 3 Error message Couldn t create result file If you receive an error message Couldn t create result file Destination path could not befound at the end of a method the local computer was unable to access the folder specified in the result file path This may arise if the specified folder is on the network server and network communication has been lost see Section 11 3 1 The r
234. licking on the Baseline settings button in the resulting Integrate dialogue or ii selecting Integrate Calculate baseline These dialogues function identically H owever in the former case the baseline is immediately used in a peak integration r Chosen algorithm C Classic i Baseline Structure width 43 995 ml Noise window fo 47 mA Minimum distance between points 23 353 A Default Values Cancel Help Figure 9 4 Settings dialogue 2 Select the appropriate algorithm Classic or Morphological 3 Change the Baseline parameter values as appropriate Excluding peaks It is possibleto exclude peaks from integration based upon criteria you determine Click on the Reject peaks command button in the Integrate dialogue In the dialogue that is displayed 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 9 5 9 Evaluating results The default criterion is to include only the 20 largest peaks E i i 0 000000 mAU F Width less than 0 000000 min I Area less than 0 000000 min m U I Width more than 0 000000 min V Peak must be one of jo Largest Cancel Help Figure 9 5 Reject Peaks dialogue Selecting part of a curve for integration To select only a part of a curve for integration
235. line data points Drawing the new baseline When you are satisfied with your baseline point selection click on the Draw baseline button Thenew baseline curve will be drawn asa spline function based on the previous and thenew 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 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 9 1 7 Adjusting the peak limits Once a peak table has been generated using the appropriate baseline it is then possibleto split or join peaks and to manually adjust the peak start and end points The peaks will then be renumbered and the peak areas will be recalculated 1 It is recommended that you first access Curve Style and Colour tab in the Chromatogram Layout dialogue and select 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 volume or time for each peak 2 Select Integrate Edit peak table Select the desired peak table from the displayed dialogue N ote that name of the baseline on which the selected peak t
236. line select Marker from the right mouse button menu Drag the cursor line with the mouse Where the line bisects the curve the X axis and Y axis values are displayed at the top of the window 6 9 sorom a run 6 10 Changing the curve colours and styles The curves window displays graphs for the selected curves in different colours with any reference curves included with the method as dashed lines The curve colours and styles can be changed 1 Select View Properties or select Properties from the right mouse button menu The Properties dialogue is displayed 2 Select the Curve Style and Colour tab Run Data Groups Run Data Colour Curves Anis Y Axis Curve Style and Colour Flow Scheme Logbook Cond1 Text alignment Horizontal C Vertical C Fiy over Figure 6 8 Properties dialogue Curve Style and Colour tab 3 Select a curve from the list and then select an appropriate colour and style 4 Click on OK Changing the scale of y axis The y axis is automatically scaled for each of the curves To fix the scale of individual curves 1 Select View Properties or select Properties from the right mouse button menu The Properties dialogue is displayed 2 Select the Y axis tab Performing a run 6 Run Data Groups Fun Data Colour Curves Anis Axis Curve Style andColour Flow Scheme Logbook m Y axis options Auto oo ggggomii C Fined 449955 543345 uS cm
237. litate your selection as appropriate If appropriate click on the Font button to alter the font characteristics Add Free Text and a Report Title as appropriate Y ou can also change the font style for these options If you have a logo in bitmap format this can be added to the header Check the Logo option and then use the Browse function to locate your bmp logo file Select the alignment of the logo either Left Middle or Right Select if you want a Line under or Line over the header Click on OK to implement the selection Footer which allows you to select the components to be included in the footer 8 45 8 Presenting results Page Setup Run date amp time Report title Result file name Method file name Page number Layout name Figure 8 41 Page Setup dialogue Footer tab Footer options are similar to those for First Header so you can have all information in either the header or footer or split the information between them as required Printing the report To print the report select File Print or click on the Print button Select the page range in the displayed dialogue and then on OK Saving the report format To save the report format 1 Select File Save or click on the Save toolbar button If saving for i the first time the Save Report Format dialogue is displayed Presenting results 8 Save Report Format x Report format name EE I Save as global format I Save as d
238. llation 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 module to an oligonucleotide synthesis system open a System control that is not currently in use identified by the Disconnected icon in the System control tool bar and choose System Connect T he dialogue lists the systems to which you have access Select the system to which you want to connect and click on OK System Connect x Explorer OK Cancel Help Figure 6 16 System connect dialogue Performing a run 6 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 on to the network The UNICORN drivers must be running on the local station although the UNICORN program does not need to be running A local station can be used to control the oligonucleotide synthesis systems directly connected to the PC without logging on to the network Files stored on network drives will of course not be accessible This mode of working places UNICORN into an error state and you should not ideally work in this state since global files such as the user settings file musers30 mpm etc are stored on the network Any changes made to these files while
239. lt Tree and Context to use Select the Default Tree and Context radio button enter the information from the network administrator and click on OK 25 Network setup 1 1 Since the name and password of the user you logged on as are not a valid account on the N ovell server a message will be displayed after a 30 second timeout NetWare Authentication Failure C lick on Yes to keep the default tree and context Installing UNICORN 1 10 11 12 Start Windows NT Explorer with Start Programs W indows NT Explorer Connect the shared folder on the UNICORN file server with the designated drive letter You can do this by selecting T ools M ap Network Drive In the dialogue that is displayed select the drive letter in the upper drop down list Enter a valid domain user ID in the Connect As field In the Shared folders list at the bottom of the dialogue the network can be seen double click on the N ovell server with the shared folder Available shared folders on the selected server are displayed Double click on the shared folder UNICORN will use A dialogue appears with a request to give a valid user name and password The user name will be the ID previously entered in the Connect As field Give the password and click on OK Install UNICORN see Chapter 13 select Network version Re boot the PC and log on as one of the users that will run UNICORN The Select NetWare Logon dialogue will be displayed The networ
240. lt file will be stored press the Browse button double click on the required folder icon and press Close Note The result name may be specified as changeable in the Start protocol see Section 5 6 10 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 5 37 B ieira and editing methods 5 38 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 Open the Start Protocol page and check the items that are to be displayed Variables Questions Notes Reference Curves Evaluation Procedures Method Information Result Name Start Protocol Sequence Checked items are displayed before method is started IV Variables iS T Scouting nce curves I Text method I Evaluation procedures M Questions I Method information I Notes T Settings M Gradient T Calibration M Result name changeable I Sequence Scouting start protocol g uroni Aliru Figure 5 37 The Start Protocol page in Run setup Variables If this box is checked values for method variables will be displayed and can be changed at the start of the run These values will override the default values for the particular run and will be saved in the result file The default values stored in the method are however not affected If the Variables box is not checked the
241. lter affects the display in both windows Y ou can use standard Windows 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 test will display only 5 character names ending with test 3 7 Jaon and file handling 3 8 If a filter is active this is indicated in the title bar of the panel e g Results filtered igf To display all files choose Filter and click on View All Show method files for the following systems Explorer Show files and folders filtered on name Wildcard can be used Ex Phgrad Defaut Cancel Hep Figure 3 6 The Filter dialogue 3 3 4 Finding files To find a file 1 Choose Find from the right mouse button menu or select File Find In the displayed Find file dialogue enter a file name specification in the Search for files filtered on name field Y ou can use standard Windows wildcard characters in the file name specification see above under Filter 2 You can restrict the search further if required e Choose file type from the pull down menu for Type All Folders Method files or MethodQueue files for the Methods window All Folders or Result files for the Results window Click on Date range and use the slide bar to set the date limit
242. m developed and marketed by A mersham Biosciences AB for real time control of oligosynthesis systems from a personal computer The package operates together with OligoPilot II and OligoProcess from Amersham Biosciences UNICORN runs under the operating system M icrosoft Windows NT version 4 0 Functional features of UNICORN 3 10 include OnePC may control up to 4 oligosynthesis systems directly N etwork support allows up to 90 systems to be run from one PC M ethod templates providing method frameworks for most common applications eliminating the need to program methods from scratch M odular method definition in the method templates reflecting the separate steps in a process Dynamic graphical overview of active runs User definable alarm and warning limits for monitor signals Programmed sequential operation Batch operation and process documentation in accordance with the requirements of Good M anufacturing Practice GM P and Good Laboratory Practice GLP Comprehensive data evaluation software In addition UNICORN offers a comprehensive security system Password control for all users with access authorisation for other users method and result files Customised definition of access control levels Audit trail for system operation Note UNICORN must be correctly installed for stand alone or network operation before the software can be used N etwork considerations software installation and administration of sy
243. mbols E Main 0 00 Message Fill your column with DNA T support Screen 0 00 Message Press CONTINUE when ready Screen 0 00 Pause 1 0 0 00 Base_Id _dT E 0 00 Block START_parameters E 0 00 Block Purge_G 2 E 0 00 Block Purge _C_ 2 E 0 00 Block Purge_T_U_ 2 E 0 00 Block Purge_A 2 E 0 00 Block Purge_Tetrazole E 0 00 Block Purge_solvents_ox Purge_solyents_ox 0 00 Base SameAsMain E 0 00 Block Purge_Oxidation E 0 00 Block Purge_CappingAB 0 00 End_block 0 00 Block Column_wash 0 00 Block Add_sDNA_G_ 2 F H A E F E E Figure 5 8 Text instructions showing blocks in a method To collapse the view click instead on the symbol for that block Alternatively you can double click on the block name to view or hide the instructions see Section 5 4 1 In the block window The organisation of blocks in the method is shown graphically in the block window in text instruction mode Each block is represented by a grey bar with the block name and the length of the block The line is shifted down to indicate calls to other blocks In the example below 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 5 9 oe and editing methods E Main 0 00 Message Fill your column with DNA T support Screen 0 00 Message Press CONTINUE when ready Screen 0 00 Pause 1 0
244. ment text B 2 Manual control 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 Flow path group in method instructions Section B 1 2 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_ Threshold Can be used to set the watch on efficiency from off to on Pause_at_ Cycle End This sets the system to Pause at the next cycle start Active only one time B 9 B Strategy for OligoPilot I B 2 4 Other Instruction Description Parameters Block Calls a block Block name Next_ Jump to the next breakpoint in the breakpoint current method only relevant when a method or block is running Record_on Begins recording a run that has been started manually A result file will be generated End_Timer IT his is used to set the time out limit in Accumulated time time or volume for the End instruc r volume tion 0 9999 min or 0 9999 or Disabled B 3 System settings instructions B 3 1 Alarms Instruction Description Comments Cond1Error Enables disables all alarms Enabled D isabled Cond1Alarm Sets the alarm and warning limits for High alarm the signal from t
245. mentation pages R un notes may be entered in the N otes page O ther pages are displayed for information only The connection mode button has three states which indicate and change the connection mode see table below Button Connection mode Click to change mode m System control workspace Click to connect to a sys disconnected tem S Control mode connection Click to leave control of system View mode connection Click to establish a con pe trol mode connection if possible The status 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 O ther users may establish a view mode connection 6 17 sorom a run 6 18 Locked by lt user gt The indicated user has left the system in a locked state Users who can supply the required password may unlock the system and establish a connection N ote that the password is case sensitive Note Itis possible to unlock with the lock password or with the UNICORN logon password If using the UNICORN logon password the user must have the Unlock systems access rights The lock password is the password entered by the user who locked the system and is case sensitive System is available Any user may establish a connection System connections are described in more detail in Section 6 5 6 3 2 Manual instructions The oligonucleotide synthesis system can
246. method templates accessible by both the local setup and the remote UNICORN It is generally a good idea to use a directory on an NT server for easy backup The different network setups that UNICORN has been tested in are the following NT domain with TCP IP NT workstations connected to a Novell server Note Thesameversion of UNICORN must be installed on both the computers with the synthesis system and the remote control computers M ixing different versions will not work Establishing and maintaining a networked UNICORN installation requires some understanding of the working of the NT operating system and the concept of NT domains if used We recommend that a competent network administrator that will also be involved in the installation of UNICORN software should maintain the network The network administrator is not necessarily the same person as the system administrator for UNICORN Once the network is set up network functions are entirely transparent to UNICORN users and the network administrator does not need to understand the use of UNICORN for controlling synthesis systems Security is very important in a networked UNICORN 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 N etwork administrator UNICORN administrator Data storage Back up routines for Controlled user access to
247. mit and the number of points to be exported may be set by the Export every for example fifth point parameter EXPORT__ Exports the documentation in the ONOFF Variables DOC_WKS current result filein WKS format to ONOFF Scouting the file defined in Export to file If all parameters to this function are OFF then no documentation is exported If at least one of themis ON then the documentation will be exported and the corresponding parts will be included in the exported file ON OFF Start Proto col ONOFF Questions ONOFF RefCurves ONOFF EvalProc ONOFF Method Info ONOFF Method N otes ON OFF StartN otes ONOFF RunN otes ONOFF EvalN otes ONOFF Sys Settings ONOFF Calibration ONOFF LogBook ONOFF ResultN ame ONOFF Column Parameters N AM E Export to file C 19 Cora functions and instructions C 20 ile defined in Export to file If all Parameters to this function are O FF hen no documentation is exported If at least one of them is ON then the documentation will be exported and the corresponding parts will be included in the exported file EXPORT_ Exports the documentation in the ONOFF Variables DOC_XLS_ current result file in X LS format to ONOFF Scouting the file defined in Export to file If all O NOFF Start Proto parameters to this function are OFF col then no documentation is exported ONOFF Questions If at least one of them isON then the ONOFF RefCurves documentation will be exporte
248. more than fo I Peak must be one of fzo largest I Apply to all chromatograms Cancel Figure 9 3 Chromatogram Layout dialogue Peak Table tab Select the options that you want to be displayed from the Select peak table columns list M ost characteristics are automatically calculated 9 3 9 Evaluating results 9 4 for each integrated peak when the peak integrate function is used although only the selected items will be displayed in the peak table Changing peak labels As alternatives to using retention times as peak labels the peaks can be sequentially numbered or be marked with specific identification tags The choice of label type is made in the Curve Style and Colour tab in the Chromatogram Layout dialogue see Section 9 2 5 N ote that the labels may be displayed vertically for each peak by deselecting the Horizontal text option If you do not want to view the peak labels e g for presentation purposes select the No peak label option Filtering peaks from view It is possible to temporarily remove peaks from display in a peak table based upon the criteria you determine 1 Inthe Chromatogram Layout dialogue click on the Peak Table tab 2 Select check the filter criteria in the peak table and specify the values used to filter the peaks i e 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
249. multiple selected objects this function resizes the objects to the same size as the currently active selection in the group of selected objects For multiple selected objects this function resizes the objects to the same width as the currently active selection in the group of selected objects For multiple selected objects this function resizes the objects to the same height as the currently active selection in the group of selected objects Y ou have several viewing options availablein the View menu or on the toolbar Preview Edit One Page Two Pages Next Page Previous Page Zoom In This toggles between looking at the print preview mode and edit mode This toggles between viewing single pages or pairs of pages where there is more than one page This displays the next page or pair of pages where there is more than one page This displays the previous page or pair of pages where there is more than one page This increases the magnification of the view of the currently selected object or most recently selected object in a group of selected objects 8 Presenting results Zoom Out This decreases the view magnification of the currently selected object or most recently selected object in a group of selected objects Changing the page layout To change the page layout double click on the header region of the page to display the Page Setup dialogue There are three tabs for changing different aspects of the p
250. n 2 To view the information about a specific component click on it Theinformation can be viewed on the right hand side of the tab Maintenance manager x Information Warning info Waring System 3 pH Cond 40 gt General Specific Pump Sample Pump Uv Valve 1 amp Valve 2 Valve 3 Valve 4 Valve5 Valveb Valve f constant 40 00 1 cm D po 100 40 Zero offset 65 94 mv Cell 59 40 mv i resistance 257513 8 Ohm ae temp comp 2 00 E S Figure 6 20 Maintenance manager dialogue Info tab with information displayed for pH Cond cells Y ou can select to view General information which contains information such as serial number version number etc or Specific information which contains information such as how long the component has been used how many hours for example a pump has run etc 6 7 2 Setting up maintenance warnings To set up a maintenance warning 1 Click on the Warning tab All of the system components are displayed 2 Click on a specific component for which you want to set up a warning and select Warning New from the menu Alternatively click with the right mouse button on the specific component and select New from the displayed menu 6 28 Performing a run 6 Maintenance manager Figure 6 21 Maintenance manager dialogue Warning tab 3 Enter the appropriate values
251. n 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 curveis mAU min or AU min Co ordinates are based upon the position of the vertical 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 322401 1 BE 322401 1_UW1_280nm 322401 1_UV1_280nm 01 1dt 4 35 min 51 6000 Figure 9 34 Measurement of the Slope limit after differentiation 9 Evaluating results 4 Usethezoom function to magnify the curve over an appropriate area Place the vertical cursor bar at the beginning of a peak where you want the W atch conditions to be fulfilled i e where the slope becomes higher R ead the actual slope value in the active XY box In the M ethod editor enter the slope value as a parameter for the Watch instruction or enter the StartSlope and EndSlope values in the Peak_FracParameters instruction 9 2 3 Creating a curve It is possible to create a curve based on any external measurements 1 Select Operations Create Curve In the dialogue that is displayed select one or several help curve s 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
252. n 14 2 alarms and monitors instructions B 6 alarms and monitors manual instructions B 9 alarms and warnings 6 19 alarms system settings instructions B 10 algorithm autoregressive C 1 median C 2 moving average C 1 smoothing C 1 All button 8 20 All with this unit 8 8 amplitude multiply 8 30 answers input field 5 28 multiple choice 5 29 no answer 5 29 value 5 30 Apply to all chromatograms 8 4 ASCII export format 9 42 asymmetry C 11 measuring in peak 9 25 audit trail system setup 13 5 audit trails 13 14 backing up audit trail files 13 17 examining 13 14 global 13 14 Index printing 13 17 renewing audit trail files 13 17 system 13 15 viewing older audit trails 13 16 authorisation items 13 4 authorisation profile example development staff 13 7 process operators 13 9 process supervisors 13 8 system administrator 13 7 automated evaluation procedures 9 33 autoregressive 8 13 autoregressive algorithm C 1 autosave interval value 13 3 autoscroll in logbook window 6 15 axes fixed scale 8 9 8 12 B backup security 3 14 base choosing 5 12 instruction 5 43 baseline algorithm used for calculation 9 4 calculation 9 1 calculation theory C 2 classic algorithm C 3 default parameters values 9 11 defining baseline parameters C 3 deleting baseline data points 9 20 drawing the baseline C 5 drawing the new baseline 9 20 estimating the baseline parameters from the source curve C 5 insertion of baseline data points 9 19 manually editin
253. n if you are developing a method evaluating data or monitoring arun on a different system Warnings and alarms are also recorded in the logbook for the run In a network installation alarms and warnings are displayed on the controlling station and all stations viewing 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 19 arun 6 4 If communication fails This section summarises the consequences of system failure during a run See Section 11 3 for more details If the results of a run were to be stored on a server or another location and thereis network communication failure during a run that has been started from a remote station the run will continue and the results will be saved in the Failed folder on the local station A control mode connection can be established on the local station to control the running system 6 5 Managing system connections 6 20 UNICORN installed on a given computer may have up to four System control windows the actual number is determined when the software is installed see Chapter 13 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 insta
254. n is displayed and you must select the strategy for which you want to install the template files Select Strategy E100_110 OPII_110 OPI 310 Figure 12 14 Select Strategy dialogue 12 16 16 17 Installation 1 2 If you click on the Select button the template files will be installed to the folder where the selected strategy is located The Cancel button aborts the template installation In both cases you are returned to the Strategy amp Template Installation dialogue Choose to install additional strategies if you have more than one system configuration Strategies are installed independently of systems A strategy is assigned to a system when the system is defined The System Table Settings dialogue is only displayed when UNICORN is installed for the first time and only if a system table is not already present T he dialogue will be shown for each system defined in the System Setup dialogue see step 14 above Enter a name that you want to use for the system If you want to connect a strategy to the system select a strategy in the drop down list box where all installed strategies are visible When all selections have been made click on the OK button to save the system in the system table If you click the Cancel button the system will not be written to the system table and you have to enter it yourself from within the UNICORN software in the M ain menu Administration System setup System Table Settings x
255. n the Notes thumb tab in the R un setup to show the Notes page and read through the method notes Y ou can maximise each section in the notes pageto fit more of thetext 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 thetemplateand if relevant how the system should be connected for the method to work correctly 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 setup Variables Questions Notes Reference Curves Evaluation Procedures Method Information Result Name Start Protocol Sequence pleted Notes ml Column Recs ena Mathas dtor mae ace DligoPilot II oj a CVt 300 Contact Time valmax for Cap and Baaise has been nesaced in ardar ta sormpenaat for tha ie delay volume Start Notes Run Notes Evaluation Notes Figure 4 6 The Notes page in Run setup with the method notes maximised 4 9 4 Creating methods from method templates 4 6 Saving the method be saved under a method name before it can be run Click on the Save A new method created from a method template is untitled and must Method toolbar button or choose File Save to save the method c Default For System System
256. n the keyboard Enter a new sequence up to a maximum of 200 bases in the sequence editor in the 5 3 direction Remember that the synthesis reaction proceeds in the 3 5 direction so the 3 base position is always the first base on the solid support This base should be taken from the standard position and should be oxidated Use the radio button combinations to select the base type to be added for each position Y ou are able to select DNA or RNA whether the base is to be oxidated or thiolated and whether the base is taken from the standard or modified reagent position There are two extra physical reagent positions in O ligoPilot II labelled X and Y both in thestandard and modified positions The extra characters Z and Q are also provided The available combinations are as follows Radio button combination Bases as represented on the screen DNA O xidated Standard A C G T X Y Z Q DNA O xidated Modified A C G T DNA S thiolated Standard A C G T X Y Z Q DNA S thiolated Modified A C G T RNA O xidated Standard a cC g U X yY Z q RNA O xidated Modified a c g u RNA S thiolated Standard a C g U X yY Z q RNA S thiolated Modified a c g U 4 5 4 Creating methods from method templates 4 6 DNA Cs Modified C BNA O Standard Figure 4 3 Radio buttons in the Sequence page used for choosing the base type to be included in t
257. nd enter a breakpoint value 4 Click on Import to import the block The dialogue remains open until you click on Close so that you can import more than one block without closing the dialogue 5 15 ons and editing methods 5 16 Note If you use the 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 Theimported block may not have the same nameas 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 If the base of the imported block is defined as SameAsM ain the block will inherit the main base in the new method regardless of the base in the source method Also the pasted block is inserted with the same breakpoint value as the block selected for point of insertion Import block x c Default Select block DNA_parameters Oxidation RNA Block name Figure 5 13 The Import Block dialogue Creating and editing methods 5 5 4 Method instructions Use the Instruction box in text instruction mode to enter edit and delete instructions 5 4 1 Viewing instructions Instructions are displayed in the text Instruction box t Method E
258. nd target folders for moving or copying between folders Confirm Required for authorised confirmation of answers to start protocol questions Section 5 6 3 e Unlock locked system Permits a user to unlock locked systems by providing the user s own logon passwords locked systems can normally only be unlocked using the locking password see Section 6 5 3 We recommend that this authorisation is restricted to a few usersin an installation The user who locks a system does not require this authorisation to unlock the same system e Run methods Required for starting methods Section 6 1 13 5 1 3 Administration 13 6 Manual interaction Required for issuing manual commands in System control Section 6 3 Pause 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 authorisation Calibrate Tune Required for using the Calibrate and Tune commands in System control Section 6 6 and Appendix D System settings Required for changing system settings with the Settings command in System control Chapter 15 Any user may view the system settings but this authorisation is required to make changes to the settings Edit MethodQueue Required for using the M ethodQ ueue editor Sections 8 1 and 8 2 Run MethodQueue Required for running M ethodQ ueues Section 8 3 Edit global list s Required for saving a method as a meth
259. ne Style and Colour 8 6 Load M ethodQ ueue 7 2 Logbook page in Documentation 8 54 M anual instruction 6 18 M ethodQ ueue Editing 7 2 M ethodQ ueue Editor 7 1 M ethodQ ueue Setup 7 3 M ethod System connection 3 12 M ultiply 8 31 N ew block 5 11 New method 4 2 N ew system 13 3 N otes page in Documentation 8 52 Open chromatogram 8 22 Open Chromatogram to Compare 8 21 O pen chromatograms to compare 8 19 Open Curves 8 16 Open curves 8 26 Open Curves to Compare 8 23 Open Log File 13 17 Page Setup tab in Page Setup 8 44 Password and System Control Window Settings 12 12 Peak Table tab in Chromatogram Layout 9 3 Print 8 33 Print Options 8 33 Procedure editor 9 34 Procedure editor Editing procedures 9 35 Procedure editor instructions field 9 36 Reject Peaks 9 6 Rename Blocks 5 14 Run Data groups tab 6 6 Save Report Format 8 47 Saving a method 4 10 Select Components 12 9 Select Drive 12 10 Index Select Strategy 12 16 Settings 9 5 Setup Chromatogram 8 38 Setup Documentation 8 40 Setup Evaluation Log 8 41 Setup Free T ext 8 36 Setup M ethod 8 39 Setup Type 12 11 Shift 8 30 Smooth 8 14 Stack O ffset 8 25 Standard format 8 51 Start Copying Files 12 13 Strategy amp Template Installation 12 15 System connect 6 20 System Installation 12 13 System Setup 12 14 System setup 13 2 System Table Strategy 12 17 Toolbar Guide 2 2 3 3 Unicorn Setup 12 18 Unicorn Software License Agreement 12 8
260. nected to a different type of system using the copy from external facility If at any time you press on the Cancel button the Method System connection dialogue is closed H owever it will reappear each time you perform other copy to from external procedures for method files M ethod files that have been copied in and connected are displayed in the previously designated folder in the Methods window 3 3 6 Deleting files To delete a file or folder 1 3 Select the item s to be deleted in the Methods or Results window 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 Click with the right mouse button on any file icon and choose Delete from the menu or choose File Delete Confirm the deletion in the dialogue Note 1 Homefolders cannot be deleted by this method see Section 13 3 Note2 Explicit authorisation is required to delete files see Section 13 2 Note 3 A filethat has been deleted cannot be recovered except by restoring a back up copy 3 3 7 Renaming files To rename a file or folder 1 Select a file or folder to be renamed in the Methods or Results window of the M ain menu Click with the right mouse button on any file icon and choose Rename from the menu or choose File Rename T he Rename dialogue is displayed Jaon and file handling 3 Enter the new name for the file and click on OK 3 3 8 Backup security To pro
261. ng the run see Chapter 15 Calibration If this box is checked the monitor calibration settings will be displayed at the start of the run If the Calibration box is not checked you can still calibrate the monitors before the run is started by using the System Calibrate command in System control Result name changeable If this box is checked you can change the result name when the run is started Click on the Browse button to change the result folder If the Result name changeable box is not checked the result name will still be displayed but neither the name nor the folder 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 40 5 7 1 Saving a method Click on the Save button on the toolbar or choose File Save to savethe method The method remains open in the M ethod editor when it has been saved so that you can continue editing if you wish Once the method has been saved choosing File Save saves the current state of the method under the same name If you want to save a copy of the Creating and editing methods 5 method under a new name choose File Save As and enter the details as described below I Enter a name for the method M ethod names may contain letters A Z and digits 0 9 The case of letters is not significant The method name must be unique for the chosen system within the fold
262. ns 5 49 5 10 1 Standard Watch conditions 5 49 Contents 6 Performing a run 6 1 Starting a method 6 1 1 Starting from the M ain menu 6 1 2 Starting from System control 6 1 3 Starting an Instant Run 6 1 4 Start protocol 6 2 Monitoring a run 6 2 1 General window techniques 6 2 2 Run data 6 2 3 Curves 6 2 4 Flow scheme 6 2 5 Logbook 6 2 6 Synthesis D ata 6 3 Manual control 6 3 1 The toolbar 6 3 2 M anual instructions 6 3 3 Alarms and warnings 6 4 If communication fails 6 5 Managing system connections 6 5 1 Establishing a connection 6 5 2 Connection modes 6 5 3 Leaving and locking a system 6 5 4 Disconnecting a system 6 5 5 N etwork considerations 6 6 Calibrating monitors 6 7 Maintenance 6 7 1 Viewing system component information 6 7 2 Setting up maintenance warnings 6 7 3 Viewing and zeroing the warning parameters 6 7 4 Getting a warning 7 MethodQueues 7 1 Setting up a MethodQueue 7 1 1 Defining a M ethodQ ueue 7 1 2 M ethodQ ueue folders and icons 7 2 Editing MethodQueues PEE TPD PP ADMD P OBHOUN A WWNN P PPP o 6 30 7 1 7 1 7 4 7 4 Evaluation 7 3 Running a MethodQueue 7 3 1 Method execution in M ethodQ ueues 7 4 Displaying MethodQueues 8 Presenting results 8 1 Opening a result file 8 1 1 Chromatogram 8 1 2 Temporary chromatogram 8 2 Basic presentation of chromatograms 8 2 1 The chromatogram window 8 2 2 O pening the Chromatogram Layout dialogue 8
263. nteractive substance for example a small volume of acetone N ote that the injection must be at zero time In the Integrate dialogue typein the column height cm in the appropriate field area Perform the peak integration according to your other selected parameters To view the results of theintegration select Plate height HETP in the list within the Peak Table tab of the Chromatogram Layout dialogue 10 peak height Pan Evaluating results 9 Every peak will have a H ETP 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 x Vp Wp assuming a Gaussian peak where N no of theoretical plates L bed height in cm Vp peak retention elution volume or time wp peak width at half height expressed in the same units as VR 9 1 10 Measuring peak asymmetry This function can be used in combination with HETP to help assess column performance A perfect peak will have no asymmetry and after peak integration give a value of 1 0 Any valueless 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 select Asymmetry in the list within the Peak Table tab of the Chromatogram Layout dial
264. nu Do not double click on the method icon in the M ain menu as this will open the M ethod editor 3 Change the method variable values if required 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 4 Creating methods from method templates 4 Go through the rest of the Start protocol entering information where appropriate Use the Next and Back 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 Variables Adjust variable values as required for the run Questions Fill in answers to the questions Notes Read the method notes and enter start notes if required Evaluation Procedures Select the print_chromatogram procedure if you want the results to be printed automatically Oligosynthesis Sequence The sequence of the oligonucleotide to be synthesized Result Set the result file name and path folder as required The default result filename includes a 2 digit serial number 5 Thelast page of thestart protocol has a Start button Click on this button to start the run 4 8 Editing text instructions M ethods for most purposes can be created by adjusting the method variable values as described above The method is actually programmed as a seri
265. o optimise the size of exported files you may want to reduce the number of data points that are exported Y ou can do this by clicking on the Max no samples box and then adjusting the number of data samples up or down In addition the Reduce by factor function decreases the frequency with which data points are sampled for export For example reducing the number of data points by a factor of five will export every fifth point to the file Pressing the Export button will then prompt a dialogue where you can choosethenew filenameand destination The default name is the same as the name of the current file The extension of the exported file will be asc wks or xls depending on the file format chosen Evaluating results 9 Export Curves x Source chromatogram Curves to export p Cut curves From 9 ml 01 test20_Cond 02 test20_PressDet Io 4224 656 ml 03 test20_Press4CN Cut graphically m Reduce number of samples gt Reduce by factor E Max no samples 19 ps Select gt gt 1 v Erpart Cancel Help Figure 9 43 Export Curves dialogue 9 3 11 Copying results to the clipboard A related activity is Edit Copy to Clipboard where the contents of the active window are copied to the Windows clipboard and can be later easily pasted into other programs e g M icrosoft Word Curves and Documentation are copied as Windows enhanced metafiles emf while Peak tables are copied a
266. o 10 chromatograms can be made available at the same time on the evaluation workspace Evaluation oligo 1 BEE File Edit View Integrate Operations Procedures Quantitate Mol Size Adviser Window Help ossa Sia ols DA test20res 11 Mii E test20_PressDet test20_PressACN 416199901 1_Cond1 415199901 1_PressDet 415199901 1_PressACN 416199901 1_SetMank TROON 000 u maS I WY Bi M II i ia hia LAL W d 1000 1500 3000 360 o Ready E Ea Figure 8 15 Windows Tile function to display many chromatograms Alternative A Import chromatograms using Open to compare This method is useful for example when importing chromatograms from all files of a previous results folder 1 Click on File Open to compare Chromatograms The Open Chromatograms to Compare dialogue is displayed 8 Presenting results Open Chromatogram to Compare x Chromatogram selection Folder es defaults 7 Browse Result F 7 Browse All Chromatogram F x Browse All r Found chromatograms Clear Select all Baseline example 2 Baseline example 1 Baseline example 3 Peak skim example id 18501 id 18502 OK Cancel Help Figure 8 16 Open chromatograms to compare dialogue The search will take place in the displayed folder only To selec
267. o enter a short description of the problem the circumstances under which the problem occurs and the consequences caused by the problem Reproducibility See description above Attachments See description above O ne exception is that log book files can be attached instead of result files Generate Report See description above 13 23 1 3 Administration 13 24 System settings 1 4 14 System settings Each installed system has a set of default settings for example to define global system settings for alarms and warnings and to select which data will be stored in result files The default system settings are valid for all runs unless you change the settings in one of two ways you assign a new value to a parameter within a method The specific change that you make in the method is valid only until End After End the parameter returns to its default setting Some parameter settings however can be set to override the global settings for the duration of the method See Appendices B and C for settings defined in OligoPilot II system and other UNICORN System Controller strategies respectively e you assign a new value to the actual system setting This new value is valid for all runs and remains until you again change the value or return the setting to its default value Of course you can temporarily change the setting within a method as described in the previous point Changes to the default settings should be made when the
268. o parts the base address and the node address Valid base addresses for this card are any hex address between 0 and FFO the node address can be altered between 1 4 The manufacturing default setting should be updated to reflect the current default for the address 100 base address 100 and node address 1 N ote that this is computer dependent To change the settings for the address refer to the tables Node address O ffset S1 7 S1 8 1 0 ON JON 2 400h OFF JON 3 800h ON OFF 4 C00h OFF OFF 12 3 12 Installation 12 4 Base address S1 1 S1 2 S1 3 S1 4 S1 5 S1 6 100 offst ON ION JON JON OFF JON 110 offst OFF JON JON JON JOFF ON 120 offst ON JOFF JON JON JOFF ON 130 offst OFF OFF JON JON JOFF ON 140 offst ON JON JOFF JON JOFF ON 150 offst OFF JON JOFF JON JOFF ON 160 offst ON OFF JOFF JON JOFF ON 170 offst OFF OFF OFF JON JOFF ON 180 offst ON JON JON OFF OFF ON 190 offst OFF JON JON OFF OFF ON 1A0 offst ON OFF JON OFF OFF ON 1B0 offst OFF OFF JON OFF OFF ON 1C0 offst ON JON JOFF OFF OFF ON 1D0 offst OFF JON JOFF OFF OFF ON 1E0 offst ON OFF JOFF OFF OFF ON 220 offst ON OFF JON JON JON OFF 230 offst OFF OFF ON JON JON OFF 240 offst ON JON OFF JON JON OFF 250 offst OFF ON OFF JON JON OFF 260 offset JON OFF OFF JON JON OFF 280 of
269. od editor 4 1 4 Creating methods from method templates 4 2 These alternatives are equivalent When you choose the command from the M ain menu the M ethod editor is opened automatically For system fori i I No template r Template selection Technique Method notes Any hd Adi Column Recycling Coupling Template Method for Adjustable volume column to OligoPilot Il Template 1 ml Column Recycling Coupling Adj Column Flouthroush Coupling Adj Column Recycling Coupling Column Packing Evaon Fix Column Flowthrough Coupling Fix Column Recycling Coupling P50 Calibration This template is using Recycling for coupling of RN4 DNA 2 OMe reagents Methods for Oxidized and Thiolated sequenses for each type of amidite is includes SEQUENCE Start with saving the Test20 sequence in the sequence editor This sequence can later be used as a template with it s built in Cross reference list The sequence is a test sequence 20 mer of Oxidized DNA that should give a purity of at least 65 full length product and n 1 less than 5 A Figure 4 1 The New method dialogue L Choose the system for which the method is intended Theinstructions 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 for the technique The templates available for the selected technique will be displayed A list
270. od template Section 5 7 2 an evaluation procedure Section 10 3 a report format a column in the Column list or BufferPrep recipe also Quantitation tables and M ol Size tables if the Analysis module is installed as globally available It is also required for deleting method templates global procedures global report formats global columns or global BufferPrep recipes also global Quantitation tables and global M ol Size tables We recommend that this authorisation is restricted to only one user in an installation Maintenance Required to gain access to the System Maintenance command in System control Quit program Required for ending a UNICORN session with the File Quit program command in the M ain menu Administration 1 3 13 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 folder structure backup routines etc The administrator may not edit or run methods or M ethodQ ueues issue manual instructions calibrate monitors or change the system configuration Caution We 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 Access level name
271. 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 Typein a multiplication integer of 1 and click on OK 4 Shift the mirror image curve downwards for an improved 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 dialogue 8 Presenting results HWA test20res 1 BEB test20_Gond test20_Gond 01 MUL ey d 500 1050 1500 2090 25b0 3000 3500 4000 ml Figure 8 28 Two curves displayed in mirror image 8 5 Saving results Any changes to chromatograms including all new created curves and all imported or created chromatograms can be saved in either of two ways using File Save or the Save toolbar button which saves all changes in the original result file e File Save as which allows you to create a new result file in the specified target folder 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 The original curves can never be deleted 8 6 Printing active chromatograms To print
272. of the displayed available systems that have the same strategy as the system for which the method was created By default the system for which the currently selected method was created is selected checked Y ou can choose the default system or one of the other systems in this cascade list to run the method Note 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 module Click on the System control icon in the Windows NT taskbar If you have more than one system installed make sure you select the correct System control icon for the correct system From System control select File Run and double click on the method icon in the displayed dialogue Prev Folder 312KB Method File 4 28 1999 2 08 Figure 6 2 Starting a method from the System control menu Performing a run 6 For methods which are used frequently e g column cleaning methods 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 Themethod namewill appear as a command in the File menu and choosing the command will start the method 6 1 3 Starting an Instant Run Y ou can Start a method template directly by clicking on the Instant Run button in the M ain menu toolbar or by selecting File Instant Run in System control The method template will not howeve
273. ogram by selecting checking the Store in new chromatogram option This is recommended to keep the source chromatogram free of too many additional curves Select how theimported curves will be displayed by clicking on one of the buttons Overlay Stack or Mirror 8 Presenting results a b xy ie paw a 600 MM fi pH 4 0 400 200 A 0 J H 3 0 imAU 0 20 40 60 80 100 120 ml c Figure 8 20 Different presentation options for comparison of imported curves a overlaid curves b stacked curves c mirrored curves Presenting results 8 Overlay This presents the imported curves overlaid one on another Stack This presents the imported curves with a given offset y axis value so that the curves are stacked and distinct from one another Mirror This should be ideally used to view two imported curves O ne curve is inverted in the y axis and thus appears to mirror the other curve 6 When you have made you selection click on OK If you selected the Stack option see step 5 the Stack Offset dialogue is displayed Y ou can change the displayed value to increase or decrease the offset distance between the curves If the selected curves have different y axis units the dialogueis displayed for each curve Click on OK Stack Offset x Enter offset between curves m U we Figure 8 21 Stack Offset dialogue 7 Imported curves are displ
274. ogue Click on OK and return to the chromatogram window Asymmetry width B width A where A and B are the partial peak widths measured at 10 of the peak height with A representing the leading part of the peak and B the tailing part of the peak 9 1 11 Measuring resolution Resolution is a measure of the relative separation between two peaks and can be used to determine if further optimisation of the chromatographic procedure is necessary If the resolution value is 1 0 then 98 purity has been achieved at 98 of peak recovery provided the peaks are Gaussian and approximately equal in size Baseline resolution requires that the resolution value is greater than or equal to 1 5 At this resolution purity of the peak is 100 To view the resolution data select Resolution in the list within the Peak Table tab of the Chromatogram Layout dialogue Click on OK and return to the chromatogram window The resolution value for each peak shows the resolution with respect to the previous peak 9 Evaluating results _ Vag Vp X 1 177 Whit W R h2 where Va2 gt Vr_i Vp_z retention elution volume for peak 1 Vp_ gt retention elution volume for peak 2 Wh1 peak width at half height for peak 1 for Gaussian peaks Wh2 peak width at half height for peak 2 for Gaussian peaks See Appendix Section D 3 for alternative calculations of the resolution 9 2 Other evaluations 9 2 1 Peak purity and peak identification Ratios be
275. omatogram Zoom function To move around in the chromatogram at the current zoom scale use the cursor arrow keys on the keyboard To undo the last zoom step select Undo zoom from the right mouse button menu To reset all zoom steps at once i e no zoom applied select Reset zoom from the right mouse button menu 8 Presenting results Alternatively Use the lt Page Down gt and lt Page Up gt keys to zoom in and zoom out respectively on the whole chromatogram Fixed scale axes Another way to display only part of a curve is to choose or fix the minimum and maximum values of the y and or x axesin the Chromatogram Layout dialogue see Section 9 2 6 Cuiting curves Thecut 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 2 Select Operations Cut curve In the displayed dialogue select the curve s to be operated on Click on OK Theselected curve will now beshown 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 cut Mi E test20_PressAGN d 500 4000 1600 2000 2600 3000 3800 i mi Left limit fla76 7e985 Bightlimi 322253611 Cancel Hep Figure 8 10 Cut window To select the region to be cut either e drag the two cursor lines to define the left and right limits of the cut
276. on top of peaks Baseline slope does not follow the source curve Figure 9 12 Baseline calculation with default parameters Thecalculated baseline does not follow the source curve because short curve segments between peaks in the middle of the chromatogram are not identified as baseline segments If you decrease the Shortest baseline segment by 50 the following baseline is calculated Evaluating results 9 Figure 9 13 Baseline calculation with 50 lower Shortest baseline segment The baseline is however still unsatisfactory due to the high slope of the short segments in the region between the second and fourth peak which are still not identified as baseline segments If you increase the Slope limit by a factor 2 5 a correct baseline is calculated Figure 9 14 Correct baseline parameters Peak limits too high up on the peaks This peak integration problem is in most cases caused by a too high value for the Slope limit and or a too high value for the Noise window This can be encountered when the chromatogram includes a very large flow through or solvent peak The large peak affects the calculation of the default baseline parameters leading to too high values for Slope limit and Noise window 9 Evaluating results Figure 9 15 Peak start and end points on peaks caused by an excessively high Slope limit N ote the difference betw
277. ons Enter maximum and minimum limits Check the Integer box to if the question is to accept only whole numbers as answers 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 Creating and editing methods 5 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 Deleting a question To remove a question select the question and click on Delete To remove all questions click on Delete All 5 6 3 Notes N otes are descriptive comments that form part of the method documentation There are four separate notes fields for method editing start up run and evaluation respectively O nly 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 N otes page in the Run setup 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 Variables Questions Notes Reference Curves Evaluation Procedures Method Information Result Name Start Protoco
278. ope values use the Differentiate function in the evaluation module to measure the slope of the test chromatogram see section 10 2 2 N ote Creating and editing methods 5 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 O ligoPilot II Equal 0 Equal 1 N ote Air is not detected by the sensor Air is detected by the sensor To use the Watch_AirSensor instruction for air sensors the Alarm_AirSensor setting in Alarms amp Mon must be disabled use the M ethod editor to disable the alarm locally in a method or the settings to disable the alarm for all methods see Section 15 1 The Alarm_AirSensor setting overrides any Watch_AirSensor instruction and if the alarm is enabled the method will pause when air is detected 5 51 B icira and editing methods 5 52 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 fixrec 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 M ethods box of the M ain M en
279. orithm 9 1 5 O ptimising the baseline parameters using a c lassic algorithm 9 1 6 M anually editing a baseline 9 1 7 Adjusting the peak limits 9 1 8 M easuring retention time and peak heights 9 1 9 Measuring HETP 9 1 10 M easuring peak asymmetry 9 1 11 M easuring resolution 9 2 Other evaluations 9 2 1 Peak purity and peak identification 9 2 2 Finding the slope values for Peak Fractionation or W atch instructions 9 2 3 Creating a curve 9 2 4 M easuring salt concentrations in the fractions 9 3 Automated evaluation procedures 9 3 1 Recording a procedure 9 3 2 Editing an existing procedure 9 3 3 Renaming and removing procedures 9 3 4 Points to watch 9 3 5 Running evaluation procedures 9 3 6 Batch runs 9 3 7 Evaluation procedures and reports 9 3 8 Placing a procedure on the menu and running 9 3 9 Exporting data or curves 9 3 10 Exporting results 9 3 11 Copying results to the clipboard 9 3 12 Importing results and curves 8 34 8 47 8 50 8 51 8 55 9 28 9 30 9 32 9 33 9 33 9 35 9 37 9 37 9 38 9 38 9 40 9 41 9 41 9 41 9 43 9 43 Contents System management 10 Security features 10 1 Access security 10 1 10 2 Connection security 10 1 10 3 Data security 10 2 10 3 1 N etwork communication failure 10 2 10 3 2 Local station failure 10 3 10 4 Security recommendations for control stations 10 3 11 Network setup 11 1 Introduction 11 1 11 2 Requirements 11 4 11 3 Installation guide 11 5 11 3
280. orm the peak integration when you are satisfied with your selections Following integration the peaks in the chromatogram will be automatically labelled with their respective retention times T he start and end point of each peak will be marked by drop lines The peak table will be displayed underneath the active chromatogram 9 2 Evaluating results 9 test20res 1 test20_Gond test20_PressDet test20_PressACN stom JE 204 62425 54 627 88 833 40 255 4 kii 1681 30 111 20 545 23 2984 11 3420 6 863 49 iei ul EE ns ascension E asians waren 20172 7956 24286 9450 Figure 9 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 dialogue Peak Tables tab see Appendix D 3 Curve Style and Colour Edit Texts Layout Library Header CurveNames YAxs XAxis Curve Peak Table Select peak table to display Select peak table columns Peak name a 7 Fi Peaktable B Peaktable C Peaktable D Peaktable E Peak endpoint retentions Peaktable F Width at half height Peaktable G Percent of total area Peaktable H Percent of total peak area Type of peak limits Peak endpoint heights z Filter Peaks I Height less than pooo IT Width less than fo T Atea less than fo I Width
281. ot II select Oligo and click on the Change button to set up the system When all systems that are connected to the station have been defined click on the Next button to continue System Installation x Instrument settings for the installation Select a system and press the CHANGE button to edit the settings for the selected system When all systems physically connected to the computer have been defined press the NEXT button to continue m Select System C System C System2 C System3 C System 4 r Settings Control Board Tonio Unie IRQ Address Figure 12 10 System Installation dialogue 12 13 1 2 Installation W hen you click on the Change button the System Setup dialogue is displayed System Setup Instrument setup for the system installation Select the control board and set the resource settings for the system Press the OK button to save the changes and return to the System Installation Dialogue The CANCEL button ignores the changes The DEFAULT button gives the default resource settings for the selected system type and the CLEAR button clears all fields If you need additional help press the HELP button System System 1 Control Board Resources cu soo Control Unit fi x UNICORN Control ina ho Board AT Address 1100 Help Default Clear u E Cancel Figure 12 11 System Setup dialogue Select the type of control board that the system
282. ou choose CV enter a value for the column volume If you chose a specific column a column volume is entered automatically Length Y ou can enter a length for the block if required An End_Block instruction will automatically be inserted in the block at the corresponding breakpoint This field may not be left blank Call Y ou can call the new block from an existing block e g the M ain block The block is called by an instruction named Block Choose the block from which thenewly created block should be called 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 call the block e g when the block being created is to beactivated by a W atch instruction choose an empty line in the From field These blocks are placed last in the method in the Unused category Note 1 If the Block instruction is placed at the same breakpoint as the End_Block instruction the Block instruction will be placed immediately before End_Block Note 2 Do not call a block from within itself You will generate a potentially infinite loop which exceeds the maximum number of calls allowed in a method A loop symbol is displayed at the beginning of the line if this occurs Press OK to add the new block Creating and editing methods 5 Strategy for creating blocks For blocks which are to becalled unconditionally you have the option of creating the Block instruction at the same time as you create t
283. ource curve INTEGRATE sourcecurveand stores the resulting Resulting peak peak table in Resulting peak table It table is assumed that the baseline is sub tracted C 15 Cora functions and instructions PEAK_ Specifies which part of the source Source curve WINDOW curve that will be integrated Peaks Left limit between retention Left limit and Right limit Right limit will be detected if the OnOff OnOff parameter is set to On If OnOff is set to Off the whole curve will be used for integration REJECT_ Any combination of conditions is H eight less than PEAKS allowed If all parameters are O FF Width less than then every detected peak areincluded Width more than in the peak table Area less than Peak must beone of xx largest SET_ Sets the column height for the peak Column H eight COLUMN_ integration calculation of the HETP HEIGHT value The Column height parameter is the height of the column in centimetres If Column height is O FF then the H ETP value is not calculated for the following integrations SET_ Sets the void volume for peak Void volume COLUM N_VO0O Jintegration calculation of Kav SET _ Sets the total liquid volume for peak Total liquid vol COLUMN_V integration calculation of the capacity ume T factor SET _ Sets the Skim size ratio to be used in Ratio SKIM _SIZE_ thefollowing peak integration s RATIO C 4 3 File Operations CURVE_ Opens the curve specified in the F
284. out the open chromatogram s select File Print or click on the Print toolbar button If you want to print out several chromatograms ensure that these are open on the workspace before selecting File Print A dialogue appears allowing you to select the print format of the chromatograms Alternatively you can click on the Other option and a dialogue appears that allows you to select the number of chromatograms in each column and row to be used in the printed document Click on OK to print out the chromatograms Presenting results 8 Print format H OEH H Other T Use thick lines Cancel Help Figure 8 29 Print dialogue Chromatograms in each row il Chromatograms in each column no comet toe Figure 8 30 Print Options dialogue If the Documentation is open click on the Print button to print any components in the documentation Chromatograms can also be printed from the File R eport dialogue see 9 7 below 8 7 Printing reports 3 The Evaluation module provides you with extensive tools with which to create detailed reports Y ou can create and save report formats based on either Standard layouts or on Customised layouts To open the report generator interface select File Report or click on the Report toolbar button and the Generate Report dialogue is displayed 8 Presenting results Format Contents Global Chromatogram Global Chromatogram_3UV Run user Ne
285. plates that can serve as the starting point for creating customised methods These method templates are defined with variables for critical parameters in the synthesis so that customised methods can be created for most purposes simply by setting appropriate values for the method variables Different templates are provided for different system strategies This chapter describes how to create and edit methods at this level See Chapter 5 about advanced method editing facilities Briefly the steps in creating a method by editing method variables are as follows 1 Click on the New Method toolbar button in the M ain menu or select File New Method in the M ain menu or File New in the M ethod editor module Select a system technique template and column 2 Choose View Run setup or press the Run setup button 3 Adjust the values for the method variables 4 Read the method notes 5 Save the method Note The fastest and easiest way to run a method is to click on the Instant Run toolbar button in the M ain menu This function runs a method template and the method is not saved in the M ain menu Methods window The method may however be recovered from the result file 4 1 Creating a new method To create a new method do one of the following click on the New Method toolbar button in the M ain menu e select File New Method in the M ain menu click on the New Method toolbar button in M ethod editor e select File New in the M eth
286. played units I Click on the Pressure data with the right mouse button to display the menu Select Set Unit and then the appropriate unit either MPa bar or psi from the menu cascade The selected unit is now displayed 6 2 3 Curves The curves window displays monitor signal values graphically PresACN Curves d 500 1000 1500 2000 2500 3000 3500 obo Tml Figure 6 6 The Curves window Performing a run 6 To select the curves to be monitored on the screen 1 Select View Properties or select Properties from the right mouse button menu The Properties dialogue is displayed Select the Curves tab Y Axis Curve Style and Colour Flow Scheme Logbook Run Data Groups Run Data Colour Curves Axis m Display curves Select All Clear All Figure 6 7 Properties dialogue Curves tab Select the curves to be displayed from the list or click on Select All if you want to view all of the available curves N ote that curves will only be shown for components present in the oligosynthesis system To clear the selection click on Clear All The curves in this list are those for which Store is set to On in the system settings see Section 15 4 together with any reference curves defined in the method Note Fraction marks injection marks and set marks will always be shown and are not curves in the list 4 Click on OK Vertical cursor line To display a vertical cursor
287. ppropriate synthesis system manual for specific details B 1 Method instructions B 1 1 Pump Instruction Description 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 oxida tion reagents The flow rateis determined by CV and CV_Cap OXCT5_Flow Start flow with the reagent pump to create the contact time with the oxida tion 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 oxida tion reagents The flow rateis determined by CV and CV_Thio B 1 B Strategy for OligoPilot II CapCT6_Flow Start flow with the Pump P6000 to create the contact time with the oxida tion reagents The flow rate is determined by CV and CV_Cap OXCT6_Flow Start flow with the Pump P6000 to create the contact time with the oxida tion reagents The flow rate is determined by the scale and Eq_Ox ThioCT6_Flow Start flow with the Pump P6000 to create the contact time with the oxida tion reagents The flow rate is determined by CV and CV_Thio Flow_ACN Starts the flow with acetonitrile from Pump P6000 to the column or waste Flow_Det Starts the flow with detrit solution
288. process the run Additionally the methods are convenient starting points for developing customized methods Fully adequate customised methods for many applications can be created simply by adjusting the values of UNICORN concepts 2 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 Method base M ethod blocks are written in one of three method bases which defines the unit for the breakpoints in the block time min volume ml or according to the strategy column volume set by the user Different blocks 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 Note Theterm method base should not be confused with bases in a sequence or an oligonucleotide Instructions The method is a call to blocks with each block containing a series of instructions or sub routines see Figure 2 2 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 Main 0 00 Base CV 6 28 ml START_parameters 0 00 Message Fill your column with DNA T support 0 00 Base Time Screen 0 00 Sca
289. quence editor 5 1 5 1 1 M odifying cross references in the sequence editor 5 2 5 2 Text instruction editor 5 6 5 2 1 Run setup 5 8 5 3 Method blocks 5 8 5 3 1 Viewing blocks 5 8 5 3 2 Calling blocks 5 10 5 3 3 Adding blocks 5 11 5 3 4 Deleting blocks 5 3 5 Renaming blocks 5 3 6 Copying moving and importing blocks 5 4 Method 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 Method 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 N otes 5 6 4 Evaluation procedures 5 6 5 M ethod Information 5 6 6 Sequence 5 6 7 Result N ame 5 6 8 Start protocol 5 7 Saving the method 5 7 1 Saving a method 5 7 2 Saving as a template 5 7 3 Deleting a template 5 8 Printing the method Contents 5 13 5 14 5 14 5 17 5 17 5 19 5 20 5 21 5 23 5 23 5 24 5 24 5 26 5 26 5 26 5 26 5 27 5 31 5 32 5 36 5 36 5 36 5 38 5 40 5 40 5 41 5 42 5 42 5 9 How to use selected unconditional method instructions 5 43 5 9 1 Base instruction 5 9 2 Instructions at the same breakpoint 5 9 3 Block and method length 5 9 4 M essages and set mark 5 9 5 Pausing a method 5 9 6 Linear flow rates 5 43 5 44 5 45 5 47 5 48 5 49 5 10 How to use selected conditional method instructio
290. r be saved as a method file but it is possible to retrieve the method from the generated result file Note Use of this function requires that templates are defined Standard systems are supplied with templates but custom systems require that the user creates templates 6 1 4 Start protocol If the method is defined with a start protocol this 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 Back to return to the previous screen The last screen has a Start button to start the run At any stage click on Cancel to abort the method start The following start protocol items may be displayed see Section 5 4 4 for more details Variables All the variables defined in the method instructions organised by block Values for variables can be changed here for the current run Text Method Textinstructions for the method T heseare displayed for information only and cannot be changed at this stage Questions 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 authorised confirmation Notes M ethod notes are shown and start notes can be entered 6 3 Ox arun 6 4 Oligo Synthesis Sequence The sequence of oligonucleotide to be synthesized This cannot be altered Reference Curve Reference curves w
291. r OligoPilot II Int_Values Sets the level where an integration StartL evel starts and ends 2000 uS cm StopLevel 2000 uS cm P50Gain A scaling factor to calibrate the flow of Gain the P50 pump 0 75 1 25 PumpError Enables disables the alarms from the ACN Detrit pumps and Reagent each Enabled Disabled Scale Loading mol g weight g Weight 0 1 1000 g Loading 10 250 mol g CT_Thio Sets the contact time for Beaucage 0 1 10 min CT_Oxidate Sets the contact time for O xidate 0 1 10 min CT_Capping Sets the contact time for Capping 0 1 10 min B 3 3 Monitors Instruction Description Parameters Cond1Keys Keyboard Enabled D isplayed Peak_Det Sets the alarm and warning limits M inPeak Peak_ACN for the pressure on the Det and 0 20 bar ACN pumps An alarm will set the D_Baseline system in Pause A warning will 0 20 bar issue a warning message with the system in Run PeakCond1 Sets the minimum peak min peak M inPeak N ot to be regarded asa disturbance 0 99900 uS cm when using the instruction Watch D_Baseline and the limits D_ Baseline used by the instruction Watch Stable_baseline 0 99900 uS cm B 3 4 Curve Strategy for OligoPilot II B Instruction Description Comment Cond1 Set the specific signal curve on off ON OFF PressD et for storing in the Result file The Press ACN time between samples determines Time between the frequency with whic
292. r selected run parameters Values are updated at least every 5 seconds the actual interval is defined in the system strategy 6 5 sorom a run Instruments Connection Status Acc Volume Block Volume ii Time Block Time Figure 6 4 The run data window Run data layout The general display for the run data window can be selected using layouts by doing the following 1 Select View Properties or select Properties from the right mouse button menu T he Properties dialogue is displayed for all windows in System Control 2 Select the Run Data Groups tab Y Axis Curve Style and Colour Flow Scheme Logbook Run Data Groups Run Data Colour Curves XAxis Delete layout New layout men Figure 6 5 Properties dialogue Run Data Groups tab 3 Select individual run data parameters to view and click on OK Performing a run 6 Alternatively select a layout either an available layout Select this from the list on the left edit an available layout Select this from the list on the left and modify the included readings in the list to the right create a new layout Click on the New Layout button and enter a name for the layout in the displayed dialogue Finish by clicking on OK Select the readings that you want to view from thelist on the right Note For systems with optional components parameters are not shown for components that are not included in the system 4 To deletea layou
293. rams M any of the menu commands in UNICORN can be activated using the toolbar buttons keyboard shortcuts and the right mouse button menu The availability of these command options is dependent on the active field or window in which you are currently working The function of a toolbar button is displayed when you place the mouse pointer over a button Right mouse button menu commands are quickly found through use of the program Typographical conventions M enu commands the names of dialogue boxes and windows the contents of dialogue boxes windows and option buttons are written with a bold helvetica typeface M enu commands are written in the order of the menu name and then the command separated by a colon For example Select File Save As to display the Save As dialogue Locate the destination drive and folder and enter a file name Click on Save This directs you to click on the File menu and select the command Save As A dialogue called Save As is displayed in which you must locate the destination folder for the saved file and give the file a name Y ou then click on the button called Save in the dialogueto execute the save command A typewriter like typefaceis used for instructions as they appear in the text editor for methods and evaluation procedures These are normally entered automatically by UNICORN Some menu commands also have shortcut keys on the keyboard which are written within lt gt marks Contents Conten
294. rd Click on the Groups button In the dialogue that is displayed select the newly created group of UNICORN users and click on the lt Add button Finally click on the OK button in the Group membership dialogue Click on the OK button in the New User dialogue and the user is created W hen all users have been created click on Close in the New User dialogue Sharing a folder This is performed by the network administrator 1 Log on to the NT computer designated to be the UNICORN file server with administrative rights Start Windows NT Explorer with Start Programs W indows N T Explorer O pen the right mouse button menu for the folder that will serve as the UNICORN server disk Select Sharing In the dialogue that is displayed click on the Shared As radio button and enter a suitable share name eg UNICORN This name will be used when connecting to this folder Set up the permissions for the shared folder by clicking the Permissions button Select Everyone in the list box and click on the Remove button Click on the Add button and in the dialogue that is displayed make sure that the domain is selected in the top drop down list Select the newly created user group in the list and click on the Add button and finally select Full Control in the bottom drop down list and click on the OK button Select OK to accept Share in the Type of Access dialogue Network setup 1 1 Installing TCP IP on the computer This is performed
295. re already assigned the changes will apply to all users at this level At least one access level must have User setup Levels authorisation UNICORN will not allow you to remove this authorisation from all levels Click on the Print button in the dialogue to obtain a record of each authorisation level that you create The authorisation items are Method Editor Required for using the M ethod editor for creating and editing methods Chapters 4 and 5 Administration 1 3 e Evaluation Required for using the evaluation module for processing result data Chapters 9 and 10 e User setup Levels Required for defining and changing access levels and user Caution We recommend that only one user in an installation or network is assigned this access e Audit trail System setup Required for examining the audit trail and for defining connected systems Section 14 4 Caution Werecommend that only one user in an installation or network is assigned this access e Delete move Home only Required for deleting and moving files and folders within the user s home folder Sections 3 2 5 and 3 2 6 Does not authorise these operations on other folders Delete move Required for deleting and moving files and folders outside the user s home folder Sections 3 2 5 and 3 2 6 Also authorises these operations within the home folder Copy file s Required for copying files Section 3 2 5 The user must have access to both the source a
296. re also saved to disk at pre set intervals during arun minimising data loss in the event of power or communication failure The capacity of the data buffers is 16000 points for up to sixteen monitors as listed in the Curves group of System Settings in System control If a buffer is filled during a run the number of points is halved by deleting every second point and the sampling frequency for subsequent points is halved 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 Atan initial sampling frequency of 10 samples per second 10 Hz the following resolutions apply for the curves Duration ampling frequency No of points Resolution s point 0 27 min 10 Hz 0 16000 0 1 27 53 min 5 Hz 8000 16000 0 2 53 107 min 2 5 Hz 8000 16000 0 4 107 203 min 1 25 Hz 8000 16000 0 8 To ensure maximum resolution for part of a run issue a N ew_Chromatogram instruction at the beginning of the part This empties the data buffers and resets the sampling frequency to that specified in the system settings A 3 A Technical specifications A 4 B General strategy for Oligo Strategy for OligoPilot Il B Synthesis This appendix alphabetically lists the instructions for methods manual control system settings and variables supported by the standard strategy for OligoPilot Il The user is referred to the a
297. rectly by using the toolbar buttons A UNICORN Main Menu ae Launch the Evaluation module Immediately start a new run rm Control your system p i Create anew method Figure 2 1 UNICORN Toolbar Guide dialogue TheM ain menu toolbar buttons allow you to begin using UNICORN quickly for example to create a new method in the M ethod editor start an instant run open aresult filefor evaluation or execute manual instructions in System control see Section 3 2 2 2 2 Software modules UNICORN control software consists of four integrated modules The Main menu with functions for file handling and administrative routines such as definition of available oligosynthesis systems and maintenance of user profiles The Method editor where methods for pre programmed control of oligosynthesis systems are created and edited UNICORN concepts 2 e The System control module which permits manual or method based control of oligosynthesis 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 The Evaluation module with extensive facilities for presenting and evaluating stored results from synthesis processes These modules are present on the Windows NT taskbar To minimize a module to the taskbar click on the M inimize button at the right hand end of the window title bar To minimize the whole of
298. red 1 2 Select Operations Smooth Select the source curve to be smoothed and its target destination By default smoothed curves are given the suffix SMTH Select the Filter type to be applied in the smoothing operation This selection can be based on the following criteria e Choose Moving average if you have noise along most of the curve Smoothing with this filter affects peak height but not retention There is little effect on peak area e Choose Autoregressive if you have periodic noise along the whole curve Smoothing with this filter will affect peak height and retention although has little affect on peak area 8 Presenting results e Choose Median if there is only one or a few noise spikes e g caused by air bubbles or if the noise is confined to only a small part of the curve Smoothing with this filter may give flattened peaks and affect peak areas slightly but will not affect the retention Source chromatogram Target chromatogram Dt 01 test20_Cond 02 test20_PressDet 03 test20_Press CN Ie zi Curve name gt PressACN 03 SMTH Eee ype Light Hard Moving average fg 2 476 ml 1 151 C Autoregressive l 2 1 25 Median E t F 1 151 Cancel Help Figure 8 11 Smooth dialogue Select an appropriate smoothing parameter value from Light to Hard for the selected filter The smoothing effect increases with increasing parameter values Smoothing is always
299. 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 13 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 any Windows NT backup commands 13 17 1 3 Administration 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 Run hour values show thenumber of hours that the system has been used for manual or method controlled runs The Run hours record is useful in following up expected and actual lifetimes for liquid handling components Click on Reset to reset the accumulated run hours to zero The reset time is recorded in the audit trail 0 0 Hours Reset Figure 13 13 Run hours in the system audit trail 13 5 Report Generator Wizard 13 18 UNICORN 3 10 contains a report generator W izard for registration of errors or problems that you have detected or that occur during a run The Report Generator Wizard takes you through the steps to generate
300. result chromatogram and or curve name using the respective Browse command buttons Wildcard characters and can also be used to further specify the search parameters see Section 9 4 1 Alternative A Presenting results 8 User entered search filters to a maximum of 10 will be saved in the drop down menus for both Result and Chromatogram selections M ore than one string can be used by inserting a between strings as a search delimiter and search filters are automatically saved and stored within user profiles The UV curves are identified with number and sometimes wavelength For example UV1_280 UV2_280 and UV1_ 254 are all different curves To search for all UV curves enter UV in the Curve name text field Chromatogram selection aha cx deou SSS Blow Result ftest20res X Browse a Chromatogram fT Browse All Curve name Ff Browse All Curve options Overlay C Stack Mirror m Found curves Search Clear Select all iif Cancel Help Figure 8 19 Open Curves to Compare dialogue 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 curves located in the previous search Select the curves that you want to be imported Click on Select All if you want to import all of the curves You can import the curves into a new chromat
301. rogram is protected by copyright law and international treaties Unauthorized reproduction or distribution of this program or any portion of it may result in severe civil and criminal penalties and will be prosecuted to the maximum extent possible under law Figure 12 1 Welcome dialogue 12 7 12 Installation 6 Thelicense agreement must be accepted to install UNICORN Click on Yes to continue the setup program UNICORN Software License Agreement EG Please read the following License Agreement Press the PAGE DOWN key to see the rest of the agreement Licence Agreement and Warranty Statement Important Please read the Licence agreement below carefully The right to use this Amersham Pharmacia Biotech AB Software Product is sold only on the condition that the customer agrees to the following Licence If you do not agree to the terms of the Licence you may return the software for full refund terminating the licence as set forth below Licence Agreement In return for the payment of the one time fee for this Amersham Pharmacia Biotech AB software product the customer receives from Amersham Pharmacia Biotech AB a licence to use the product subject to the following terms and conditions zl Do you accept all the terms of the preceding License Agreement If you choose No Setup will close To install UNICORN you must accept this agreement lt Back Yes No Figure 12 2 Unicorn Software License Agreement dialogue
302. rs of a selected instruction enter the new value in the appropriate place in the Parameters field 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 Y ou can insert new instructions after the currently selected procedure instruction Select an instruction type and instruction in the Instructions field and enter the appropriate parameter values in the Parameters field 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 button To save the edited procedure select File Save or File Save as from the dialogue menu commands N ame the procedure Choose File Exit from the menu bar in the dialogue Evaluating results 9 9 3 3 Renaming and removing procedures Procedures that you have created can be renamed or deleted from the list of available procedures Note Itis recommended that you exercise caution in renaming and removing existing global procedures Renaming a procedure 1 Select Procedures Edit Rename 2 Select a procedure in the Rename Procedure dialogue 3 Click on OK Removing a procedure 1 Select Procedures Edit Delete 2 Select a procedure in the Delete Procedure dialogue 3 Click on OK
303. run will be executed with default values for all variables as defined in the method Text Method Displays the method instructions Double click on a Block instruction as denoted by the blue square and mark to display or hide the instructionsin thecalled block see Section 5 4 1 M ethod instructions cannot be changed from this display Questions If this box is checked questions defined in the method will be displayed at the start of the run Creating and editing methods 5 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 Notes 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 Thestart of therun isthe 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 Columns If this box is checked you can view the available column definitions Thecolumn definition used in the method run is selected with the Base instruction and may be changed at the beginning of the run on the Variables page if the columns parameter is defined a
304. rve to modify colour and linestyle for Select colour Select line style Peak label Fraction text alignment m Set mark text alignment Retention M Horizontal text C Vertical I Vertical C Number Horizontal Horizontal C Peak Name I No Peak Label C Fly Over C Fly Over Grid I Gi Help T Apply to all chromatograms Cancel Figure 8 5 Curve Style and Colour dialogue Presenting results 8 8 2 6 Defining and positioning curve text The Curve Style and Colour dialogue also allows for variable forms of peak labelling as well as alignment of text within Fraction and Set mark curves Peaks may be labelled according to Retention times the default label by sequential Number or by user defined Peak name see Section 10 1 6 The default Peak label text alignment is Horizontal but can be changed to vertical by un checking the Horizontal box In addition checking the No Peak Label box removes peak labels Both Fraction and Set mark text can beset to Vertical Horizontal or Fly Over alignment by checking the appropriate box Fly Over alignment sets text labels as hidden text which appear only when the cursor is carefully positioned over a curve line 8 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 scale is automatically displayed to show the whole run ml or minutes for OligoPilot and litres or minutes for O ligoProcess It is pos
305. rves e print reports 8 1 Opening a result file Jm rag 27466 8080 5178 200 26900 8651 5294 500 14561 6439 1980 900 26753 4695 4840 800 26402 7309 4276 400 1388 11 26391 1184 4099 500 1595 47 25997 9735 3772 700 1802 42 25862 6466 3714 000 2011 03 25542 2871 3525 800 2220 06 25202 9536 3398 400 2431 57 24987 3346 3347 900 2643 91 24810 5138 3369 600 Figure 8 1 Evaluation module view To open a result file either e doubleclick on a result file icon in the Results window of the M ain menu e select a result file icon in the Results window of the M ain menu and select File Open 8 1 8 Presenting results Temporary ajoj e click on the Evaluation icon in the Windows NT taskbar select File Open or click on the Open toolbar button and select a result file from the Open Result dialogue e click on the Evaluation toolbar button in the M ain menu and select a result file from the Open Result dialogue All contents of the opened result file are transferred to the Evaluation workspace By default the chromatograms in a run are shown as opened windows The chromatogram window on top is the active window There is also an minimized Temporary chromatogram window Note TheTemporary chromatogram window may be hidden from view to view it us the Window Cascade or Window Tile functions 8 1 1 Chromatogram A chromatogram includes a number of
306. s breakpoint in block Change E 0 00 Block Purge_Tetrazole W 0 00 Block Purge_Tetrazole Purge_Tetrazole Purge_Tetrazole 0 00 Base Volume 0 00 Base Volume 0 00 Flow_Reag 10 00 ml min 0 00 Flow_Reag 10 00 ml min 0 00 Solvent Tetrazole Waste 4 00 Solvent Tetrazole Waste 4 00 Solvent ACN_3 1 Waste 8 00 Solvent ACN_3 1 Waste 8 00 Amidite ACN_A Waste 12 00 Amidite ACN_A Waste 12 00 Amidite ACN_A Waste 16 00 Amidite ACN_A Waste 16 00 Flow_Reag 0 00 ml min 20 00 Flow_Reag 0 00 ml min 16 00 End_block 20 00 End_block Figure 5 18 Change moves the selected instruction and all subsequent instructions Replace 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 5 21 oe and editing methods 5 22 Replace E 0 00 Block Purge_Tetrazole E 0 00 Block Purge_Tetrazole Purge_Tetrazole Purge_Tetrazole Base Volume 0 00 Base Volume Flow_Reag 10 00 ml min 0 00 Solvent Tetrazole Waste Solvent Tetrazole Waste 4 00 Flow_Reag 10 00 ml min 4 00 Solvent ACN_3 1 Waste 4 00 Solvent ACN_3 1 Waste 8 00 Amidite ACN_A Waste 8 00 Amidite ACN_A Waste 12 00 Amidite ACN_A Waste 12 00 Amidite ACN_A Waste 16 00 Flow_Reag 0 00 ml min 16 00 Flow_Reag 0 00 ml min 16 00 End_block 16 00 End_block Figure 5 19 Replace moves only the selected instruction Moving instructions within a breakpoint To chan
307. s for the search Click on OK e Check Search all folders to search through all the folders to which you have access If Search all folders is not checked the search will be restricted to the current folder and sub folders below Logon and file handling 3 Find Files x Search for files filtered on name wildcard can be used Ex Phgrad iex1 Type All is Date range IV Search all folders From 1990 01 01 To 1998 02 02 Start folder fe default Found files d default iex1023 d default iex1022 Figure 3 7 The Find file dialogue 3 Click on Find when you have entered all parameters The result of the search is shown in the Found files box 4 Double click on a filein this list to return to the M ain menu with the selected file highlighted in the appropriate window If you click on Close with or without selecting a file you will return to the M ain menu with the window display unchanged 3 3 5 Copying and moving files and folders Y ou can copy and move files and folders to another folder that is specific to your user logon name Y ou can also copy or move files to and from an external driveand folders available on the network If you copy or move a folder all files within the folder will also be copied or moved Copying or moving files and folders 1 Select one or morefiles or folders in either the Methods or Results window of the M ain menu To select multiple files or fold
308. s pump B 1 methodbase instruction 5 44 M ethodbase setting 14 4 M ethodQ ueue adding more method steps 7 2 changing the start condition or method name 7 4 Condition command 7 4 condition setting 7 5 defining 7 1 displaying 7 6 editing 7 4 End 7 7 erasing a line 7 5 execution of methods 7 5 Exit 7 7 folders and icons 7 4 Immediate start command 7 3 inserting a line 7 5 overview 7 1 Ready instruction 7 5 Restart 7 6 running 7 5 selecting the method 7 2 setting a time interval for starting a selected step 7 3 setting up 7 1 Start at 7 6 Start time command 7 3 M ethodQ ueue facilities 2 8 methods concepts and principles 2 4 Migrating 12 1 migrating from UNICORN OS 1 10 to UNICORN 3 10 12 1 migration and post installation setup 12 2 minimum distance between points 9 10 mirror curves 8 25 M irror images of curves 8 31 modes connection 6 21 monitors calibration procedure 6 24 monitors system settings instructions B 12 morphological algorithm 9 4 moving average 8 13 moving average algorithm C 1 moving files and folders 3 9 multiply 8 30 multiply retention or amplitude 8 30 Index N naming method 4 10 network communication error 11 3 communication failure security 10 2 computer installations A 2 considerations 2 10 control access rights 6 24 controlling systems from local and remote stations 11 3 creating home folder 13 11 data transfer A 3 example 2 11 11 5 installation 3 2 A 2 installation of UNICORN 11 5 mainten
309. s 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 1 4 System settings 14 2 Specials OPII Specials Instructions x m Instructions Eq_OX Parameters C Alarms Eq 1 10 1 000 Equ r7 Specials Eq 1 000 Scale C Weight 0 100 g Monitors Loading 10 000 umol g XTetrazole Cone 1 000 CV AmiditCone Cone 0 020 M Curves CT Panninn Set Selected Parameter To Strategy Default Value _Sercel __Hele Figure 14 3 Special settings in System control Settings in the Specials group include Frac Parameters Peak_Frac Parameters FracNum bering Mode Keyboard Methodbase DelayVol determines the delay between peak detection on the UV monitor and the start of fraction collection Fraction marks in the System control display and result file are adjusted with the delay volume This value should be set to the volume of tubing between the UV monitor and the fraction collector outlet TubeChange determines the flow handling during tube changes Determines the parameters used for peak fractionation Determines whether fraction numbers will be reset after each run Reset or Continue
310. s Title and or Text 4 When you have made your selections click on OK in the Setup Documentation dialogue Details from the selected documentation menu are now inserted into the report in the documentation object m Presenting results 8 Evaluation log The Setup Evaluation Log dialogue is used to define the settings for the evaluation log object Setup Evaluation Log x r Eonts Title Text I Start on new page a Cancel Help Figure 8 38 Setup Evaluation Log dialogue 1 If appropriate change the characteristics of the Fonts by clicking on the buttons Title and or Text 2 If required select Start on new page 3 When you have made your selections click on OK The evaluation log is now inserted into the report Moving and resizing objects O bjects can be freely moved or moved and sized according to the various commands in the Layout menu or toolbar Note Resizing the width of the objects with Make same size or Make same width can only be performed on chromatograms and free text 1 Toselect an object for moving or sizing click on the Select toolbar button and click on the object of interest To select several objects hold down the lt Ctrl gt key while clicking on the objects 2 For free object placement of an object hold down the left mouse button and drag the object to its new position Similarly to free size the object click on one of the object border anchors either at
311. s a variable see Section 5 6 1 Note Not used for Oligo systems Reference curves If this box is checked the reference curves associated with the method will be displayed at the start of the run Y ou can add delete and rename curves at the start of the method All curves in the list can be displayed in System control during the run If the Reference curves box is not checked the curve settings saved in the method will apply 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 You 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 Evaluation Procedures box isnot checked the procedure settings saved in the method will apply at the end of the method 5 39 B ieira and editing methods Method information If this box is checked the method information including creator target system strategy information 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 Settings If this box is checked the settings including alarms monitors and curve configuration will be displayed for information at the start of the run To change settings use the System Settings command in System control before starti
312. s in the Temporary chromatogram 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 To copy curves 1 Select Edit Copy Curve 2 Select the source chromatogram and the curve of interest Select the target chromatogram Click on the Copy button to effect the copy Stay within the same dialogue to repeat this step for as many other curves you want from thesame or different chromatograms When you have copied all desired curves click on Exit 3 Open thedestination chromatogram and access the Chromatogram Layout dialogue Select in the Chromatogram Layout dialogue 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 9 2 6 Source Chromatogram Target Chromatogram gt z Temporary z 02 test20_PressDet 02 03 test20_PressACN Copy gt 03 gt 07 xl Curve name Conan 01 COPY Close Help Figure 8 23 Copy curve dialogue 8 4 3 Stacking and stretching curves Several tools are available to stack and stretch curves from different runs to better visualise the differences T hese tools are normalising curves shifting curves and stretching curves These allow you to manually reproduce the Stack and Mirror functions associated with the Open to compare Curves operation see
313. s must have the same y axis unit and not be fraction or injection curves or else a run time error will occur First source curve Second source curve Target curve position AMP_MUL M ultiplies the amplitude of the source curve by the multiplication factor and stores the result in the target curve position Source curve Target curve position M ultiplication factor AM P_SHIFT Shifts the amplitude of the source curve by the shift factor and stores the result in the target curve position Source curve Target curve position Shift factor CLEAR Clears specified curve from the work ing memory of the computer Source curve COPY Copies the source curve to target curve position Source curve Target curve position CUT Cuts out the part of the source curve between Left and Right Limits and stores the result in the target curve position Source curve Target curve position Left limit Right limit DERIVATE Differentiates the source curve first or second order and stores the result in target curve position The y axis of the target curve position will be a normalised scale without unit Source curve Target curve position First O rder or Second O rder C 12 Evaluation functions and instructions C DIV Divides two curves to gain a third First source curve curve which is the quotient of thetwo Second source curves The two source curv
314. s text In the latter case only the columns selected in the Chromatogram Layout dialogue will be copied 9 3 12 Importing results and curves You may import curves in ASCII text and Lotus 1 2 3 wks or xls spreadsheet formats and results in SMART M anager and FPLCdirector formats Select File Import and then the appropriate menu choice A new dialogue will then prompt you to choose the source folder and file Theimported curve or result fileis automatically opened to the screen 9 43 9 Evaluating results Introductory material M ethods and runs Evaluation Appendices Security features 1 0 10 Security features This chapter considers security features in UNICORN under the topics access security e system connection security e data security security recommendations for control stations 10 1 Access security Operation of UNICORN is restricted to authorised users defined in the system by the system administrator see Section 14 3 User access may be protected by a password The minimum password length for users is defined when UNICORN is installed see Section 13 4 In installations where access security is important passwords should be enforced and should be changed regularly see Section 14 3 2 Each user is assigned an access level and a folder access profile defining the operations that the user can perform and the folders the user is allowed to access see Section 14 3 For system security administrat
315. s the M ethod editor module and displays the New Method dialogue see Section 4 1 This activates the first connected System control and displays the Manual instruction dialogue see Section 6 3 2 This displays the Open result dialogue Select a result fileand click on OK to launch the Evaluation module see Chapter 9 Logon and file handling 3 3 3 UNICORN Main menu windows The two M ain menu windows display the folders to which you have access within UNICORN and the method and result files within the currently open folder respectively Y ou can only see method files written for systems to which you have access 3 3 1 Creating a new folder To create a new user specific folder 1 Select the appropriate window Methods or Results in which you want to create a new folder 2 Select File New Folder or New Folder from the right mouse button menu The Create New Folder dialogue is displayed 3 Enter the name of the new folder and click on OK The new folder is displayed in the appropriate window Any user that has access set to the main folder in which the new folder was created also has access to the folders and files contained therein 3 3 2 Opening and running method files To open and edit a method file in the M ethod editor click on a file in the Methods window and select File Open or click on the file with the right mouse button and select Open from the menu Alternatively double click on a file to open it amp M
316. sct gt gt Bemove i Baseline example 1 1173KB Result File Ul Baseline example 2 985KB Result File i Baseline example 3 929KB Result File id 18501 1160KB Result File ai id 18502 959KB Resul File i Peak skim example 998KB Result File i test20res 1164KB Result File SMART Manager example files User Folder Figure 8 18 Open chromatogram dialogue Viewing all chromatograms I Simultaneously display the chromatograms by selecting Window Tile or layer them by selecting Window Cascade Chromatogram windows can be individually sized and the presentation of the curves changed If you want to have the same scale on all of the chromatograms open the Chromatogram Layout dialogue for any chromatogram make the changes and select check the Apply to all chromatograms option Imported chromatograms cannot be shown with column volume as the x axis base 8 4 2 Comparing curves Curves from different runs can be imported or copied into one chromatogram for comparison Alternative A Importing curves using Open to compare Result files contained in the same folder can be automatically searched to locate all curves of a specified type for example all UV curves M oreover the imported curves can be automatically overlaid stacked or be presented as mirror images E Select File Open to compare Curves or click on the Open curves to compare toolbar button Select the search criteria for the folder
317. se to a different block in the method template first select the base in the Block field N ext click on the Block pull down list and select the method template block that you want assigned to the selected base The base in the Block field will now be associated with the block name that you chose Cross Reference List x Coupling Purge Solvent Purge Amidite Column wash Final detritylation Add_DNA_A_v2 Add_sDNA_A_v2 Add_DNA_C Add_sDNA_C Add_DNA_C_v2 Add_sDNA amp _C_v2 Add_DNA_G Add_sDNA_G Add_DNA_G_v2 Add_ sDNA_G_v2 Add DNA_T Figure 5 3 In the above example the base A has been cross referenced to the block Column_wash 5 4 Creating and editing methods 5 Y ou can select any block contained in the method template even those that you have modified or created see Section 5 3 Repeat this process for other bases as appropriate Y ou can also reassign the cross references for the optional method steps selected in the Sequence page The blocks for Column wash Final detritylation Purge Amidite and Purge Solvent can be assigned by clicking on the appropriate tab and then using the Block pull down list Cross Reference List x Coupling Purge Solvent Purge Amidite Column wash Final detritylation Column_wash Figure 5 4 Fields for cross referencing the column wash and final detritylation blocks in a method Click on Exit to implement the new cross references Enter the appropriate s
318. se window You can now use the following functions Size Allows the selected curve to be stretched along its y axis or x axis Click on Size and then drag the coloured box either along its y axis or x axis This is useful for comparison of curves with for example different gradient lengths Move Allows the selected curve to be moved to any position on the chromatogram Axes are automatically rescaled to accommodate the new positioning 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 Normalise The curve to be normalised will be adjusted to the help curve Thus the height of the highest peak on both curves will be the same and will occur at the same retention point The curveto benormalised 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 normalised curve O pen the Chromatogram Layout dialogue to select the normalised curve for viewing 5 Repeat the procedure for all curves you want to stack or stretch 8 Presenting results Alternative B Moving a curve using the Shift function If more precise positioning of curves is required then the shift function should be used This function is similar to Normalise Move except that each curve is repositioned by a precise value instead of by eye and the instruction logg
319. security back server and local disks home and shared folders up routines eg placing all home folders on a shared disk to prevent data from being scattered throughout the network Network M aintenance of user pass access security wordsand access rights to shared resources UNICORN M aintenance of user profiles access security Network setup 1 1 PC s with UNICORN software can be categorised as follows A local station isa PC to which synthesis systems are physically connected UNICORN software must be installed on all local stations in the network e A remote station is a PC to which often no systems are physically connected but which can control systems over a network link UNICORN software is installed with the Remote Only option on remote stations 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 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 folder on the local station A control mode connection can be established on the local station to control the run For a synthesis system to be accessiblein the network the local station must be switched on and logged on to the network The user interface for UNICORN
320. sentative of the baseline noise M easure the Y axis co ordinates using the appropriately 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 C 2 5 Measuring the Slope limit using Differentiate and curve co ordinates for Classic algorithm To measure the slope at any point on the curve 1 2 Select Operations Differentiate A dialogue will appear Select the desired source curve check that a First order calculation is selected and click on OK T he differentiated curve will appear in the active chromatogram M easurethe 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 see Section 9 3 2 Determine the highest slope value of the baseline non peak part of the curve Add 10 and insert this value as the Slope limit Evaluation functions and instructions C C 3 Peak table column components A Figure C 3 Diagram illustrating peak parameters See the parameter list below for explanations Peak name N ame of peak Retention time or volume base Retention at the peak maximum C in Fi
321. ser information Adviser Strategy amp template files and System installation according to which part s you wish to install The appropriate parts will be copied into the existing folder structure Note For network installations remember to log on to the network before installing any UNICORN software components Program files Before re installing the program files be sure that the UNICORN software is not running Check the Program files option in the Components list to re install UNICORN This will not affect any existing method or result files in the system The program is always installed locally even in network installations Installation 1 2 User information User information contains several sub components global procedures global report formats global BufferPrep recipes column files and the users file To reinstall these global files check each sub component Note Any changes to the files will be lost since they will be replaced with their defaults If for example the existing user definitions are damaged you can check the Users file to reinstall the default user Any other users defined in the system including users installed from other stations in a network installation will be deleted H owever method and result files will not be erased Y ou can regain access to these files by re defining users with appropriate folder access Users areinstalled on the network server in a network installation System install
322. setup dialogue 13 3 1 Defining new users 1 To define a new user click on New in the User field and enter the username in the displayed dialogue Create New User Figure 13 8 dialogue for defining a new user 13 10 Administration 1 3 2 Enter the Full name of the user and Position as appropriate 3 To createa new home folder for this user check the Create home folder box and enter the folder name and drive in the appropriate fields If you choose to create a home folder on a network drive make sure that the drive is always accessible Click on OK to create the user and return to the User setup dialogue Note A home folder can always be created on a network drive even if UNICORN is not installed for network control The computer only needs to be connected and logged on to the local network In general each user should have a separate home folder Caution In a network installation always create home folders on a network drive which is accessible from all computers If you create a home folder on the C local drive it will only be accessible from the computer on which it was created 4 Enter the user s password in the Password field The minimum number of characters in the password is defined when UNICORN is installed see Section 13 3 The program may also be installed without password protection The case of letters in passwords is significant Caution Leaving the password as default c
323. sibleto fix the minimum and maximum values for the axes of any curve and thereby select a specific part of the curve to be displayed Y Axis 1 Click on the Y Axis tab in the Chromatogram Layout dialogue Chromatogram layout 1 x Curve Style and Colour Edit Texts Layout Library Header Curve Names Y Axis Xasis Curve PeakTable Select curve to modify axis scale for 02 test20_PressDet Evi Max Min 03 test20_Press4CN eae vo 0 fi is em All with this unit Pressure unit C MPa bar C psi From strategy IF Apply to all chromatograms Cancel Figure 8 6 Chromatogram Layout dialogue Y axis tab 8 7 8 Presenting results 2 3 Select the appropriate curvefrom the list for which you want to fix the scale Click on the Fixed option Typein 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 scaled curve will be similarly scaled Click on OK Note On some systems Y Axis units for pressure curves may be changed by clicking on the appropriate Pressure unit M Pa psi bar The default Pressure unit is From strategy which is the unit defined in the original run strategy Note All with this unit will only beapplied 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
324. sically connected is referred to as a local station O ther workstations in a network installation are called remote stations During installation of UNICORN for the first time on a workstation in a network configuration certain files are copied to the network server These files include UNICORN user files and strategy files and are the global settings for all UNICORN users in the network see Chapter 13 However UNICORN program files and templates are NOT copied or located on the network server and as such the server cannot be used to control arun UNICORN program files and templates are instead locally installed on each workstation in the network and the network is used as the medium of communication to establish control with the oligosynthesis systems 2 8 Security UNICORN concepts 2 008 alee Oligosynthesis m mm system HEHHE aE BADE PC with UNICORN Network sever PC with UNICORN Oligosynthesis system EEEN Oligosynthesis system E E is NOT installed 90008 PC with UNICORN PC where UNICORN PC with UNICORN il Oligosynthesis system Figure 2 6 A network installation with 4 oligosynthesis systems and 5 workstations PCs The oligosynthesis systems physically connected to PCs 1 and 5 can be controlled locally Alternatively any of the PCs with UNICORN installed can be used to remotely control any of
325. so this skims the area under the second peak A B Figure 9 7 llustration of how a drop line A and a skimmed peak B affects the area under the main peak and the peak shoulder The Skim peak option can be checked in the Integrate dialogue Y ou can also set the Ratio value to determine when peak skimming should be applied to a peak instead of drop lines the default value for this ratio is 10 The ratio is based on the relationship hpi hy hyo hy gt skim ratio Figure 9 8 Skim peak ratio calculation 9 7 9 Evaluating results 9 8 Including negative peaks If you want to include negative peaks in the integration select check Accept negative peaks in the Integrate dialogue or in the Calculate baseline dialogue The negative peaks will be reported as negative areas in the peak table By default negative peaks are not included in the integration Manually editing the baseline In the event that the automatic baseline calculation does not produce a satisfactory baseline 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 10 1 6 Manually editing the peak table T he Integrate Edit peak table function see Section 10 1 7 allows you to manually adjust the peak start and end points split and join peaks 9 1 4 Optimising the baseline parameters using a mor phological algorithm The f
326. software which runs on an IBM compatible PC under Microsoft Windows NT 4 00 and hardware for interfacing the controlling PC to the synthesis module The manual is organised in 14 chapters and 6 appendices Introductory 1 Introduction material 2 UNICORN concepts 3 Logon and file handling Methodsand 4 Creating methods from method templates runs 5 Creating and editing methods 6 Performing a run 7 M ethodQ ueues Evaluation 8 Presenting results 9 Evaluating results System 10 Security features management 11 Network setup 12 Installation 13 Administration 14 System settings Appendices A Technical specifications B General strategy for oligosynthesis C Evaluation functions and instructions D Feedback tuning E File organisation F Troubleshooting Microsoft Windows NT and other M icrosoft products mentioned in this User M anual are trademarks or registered trademarks of the M icrosoft Corporation Preface Assumptions Two broad assumptions are made in this manual 1 You should be familiar with the oligosynthesis systems in your installation Refer to the appropriate System M anuals for details 2 You should be familiar with the general principles of using Microsoft Windows NT version 4 0 on your PC Although UNICORN isa self contained program package and does not require any direct interaction by the user with Windows NT the user interface principles follow the conventions set by Windows NT prog
327. specified named report layout and title If Titleis x then the title in the report layout is used If ReportLayout is thena default layout is used Report layout Report title RUN _ PROGRAM Starts a program as a separate process The Program name string contains the program name and parameters to start it with Program name Feedback tuning D D Feedback tuning Some systems allow you to perform PID feedback tuning of the pump flow rate using the System Tune command in System control O ther systems allow you to perform tuning with the insertion of a tune instruction in the method This appendix describes the principles of PID feedback tuning of pump flow rate to available systems UNICORN 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 O ligoProcess 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 e TheP 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 e Thel parameter eliminates the stationary error but results in
328. stallation you prefer then click Next The network installation will setup the software to work over a network If Remote only system is checked no systems will be installed on the target system The stand alone installation will setup the software to work locally on the target system If Demo system is checked no systems will be installed on the target system r Setup Type C Network installation M Bemote only system Server disk Stand alone installation I Demo system Figure 12 6 Setup Type dialogue 11 Enter the minimum number of characters required for passwords Valid numbers of password characters are 3 15 Alternatively if you do not require password protection check No password required With this setting users can be defined with or without passwords Note Ina network installation make sure that you enter the same password settings on each station in the network Choose the number of system control windows that should be available in the installation maximum 4 12 11 1 2 Installation 12 12 Password and System Control Window Settings Set the requirement for minimum number of password characters and the number of system control windows m Password Password required Minimum number of characters fs No password required System Control Windows 1 Ci C4 Figure 12 7 Password and System Control Window Settings dialogue
329. stem and user definitions are described in Chapters 11 12 and 13 1 1 1 Introduction UNICORN concepts 2 2 UNICORN concepts This chapter introduces the basic concepts that are specific to UNICORN For a description of how to work with the Windows NT operating system see your Windows NT system documentation M aterial in this chapter is divided into 8 sections dealing with e UNICORN control software e UNICORN user interface e Files and folders e Methods and method structure System control e Evaluation e Network considerations e Security and administration 2 1 UNICORN control software UNICORN runs under the Windows NT operating system and provides facilities for method controlled operation of oligosynthesis systems as well as real time monitoring and subsequent evaluation of the synthesis process 2 1 1 Strategies Part of UNICORN 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 independent of the strategy Strategy dependent instructions are listed in Appendix B 2 1 2 UNICORN concepts 2 2 UNICORN user interface 2 2 2 2 1 Toolbar Guide The UNICORN Toolbar Guide dialogue is shown after start up and logon reminding you about the M ain menu toolbar buttons Toolbar Guide x Begin your work di
330. stem3 lt home gt failed lt strategy gt E 1 Stand alone installations In a stand alone installation the entire folder structure resides on the local hard disk System and user definition files and system strategies are duplicated in the local and server folders E 2 Network installations E 2 1 Local and remote computers The bin dll and local folder structures are installed on each computer E 2 2 Network server The server folder structure is installed on the network server The local fil folder structure is also installed if home folders 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 stations can be used as stand alone systems in the event of a network communication failure E 1 E File organisation folder Description UNICORN The main UNICORN folder bin Executable modules dll Device drivers and dynamic linked library dll modules the drivers are installed in the NT system folder for device drivers winnt system32 drivers local H olds a copy of the global system definitions sys tem tab N ot available on remote computers fil H olds a copy of the files containing user definitions user30 mpm Column list columns cmn and BufferPrep recipes global rcp comp rcp N ot available on remote computers Global audit trail files are also
331. strategy H olds the folder structure for installed strategies lt strat egy gt H olds the original files for installed strategies There is one lt strategy gt folder for each installed strategy Each lt strategy gt folder also holds the corresponding template methods if installed Strategy files are copied to the local strategy folder according to the systems installed on the local station E 3 E File organisation E 4 Troubleshooting F Troubleshooting F 1 Logon problems F 1 1 Unable to log on to UNICORN 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 on using your correct username and password the USERS30 M PM file in the UNICORN SERVER FIL folder may be corrupt Restore the file from the latest back up copy or reinstall the default user see Section 13 4 2 If users are not available on a remote station in a network installation the user list in the Logon dialogue box is empty make sure that the computer is logged on to the network before starting UNICORN 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 on to the network before starting UNICORN Theuser list is stored locally on a local station and is updated automatically from
332. sult of the division is defined to be 1 0 The y axis of the curve will be the same as that of the first source curve Second source curve Target curve position Threshold1 T hreshold2 Evaluation functions and instructions C C 4 2 Integration Instruction Description Parameters CALCULATE_ Calculates a baseline from the source Source curve BASELINE curve The baseline is stored in the Target curve target curve position DEFAULT can __ position be selected in the Baseline parameters Noise Window which will then calculate default Shortest baseline baseline parameters for each new segment curve Slope limit M ax baseline level CALCULATE_ Calculates a baseline from the curve Source curve BASELINE_ crvSrc using a morphological method Destination curve MORPH The baseline is stored in curvecrvDst Noise Window Width Distance Between Points CLEAR_ Clears the peak table in Peak table Peak table source PEAKTABLE source from the working memory of the computer COPY _ Copies a peak table from Peak table Peak table source PEAKTABLE source to Resulting peak table Resulting peak table NEGATIVE_ Controls the baseline behaviour in OnOff PEAKS subsequent baseline calculations If OnOff is ON then the baseline may be drawn above the curve and negative peaks may be detected by PEAK_INTEGRATE If OnOff is OFF then the baseline is never drawn above the curve PEAK_ Performs a peak integration on the S
333. sults 9 amp Edit Baseline oix tes20_Cond test20_Gond 01 BASEM Point list lusiom J ml uS em 0 177 0 000 2111 9740 000 4224 4730 000 4224 4790 000 yd LL NL mn la A TIL x x 5 b 5d0 1000 1600 2000 2600 30b0 36bo z lt mi Draw baseline 2206 068 ml Delete Delete all Point mode 2274700 uS em Draw straight to next point Zoom mode Cancel Help Figure 9 23 Edit Baseline chromatogram window Insertion of baseline data points Select the Point mode button Click on the left mouse button to place a new baseline 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 To make your task easier you can click on Zoom mode and zoom in on specific regions of the chromatogram and then insert baseline data points The right mouse button menu allows you to undo the last zoom step with Undo zoom or to reset the default zoom scale with Reset zoom aoe ee Before Figure 9 24 Baseline before and after editing 9 19 9 Evaluating results Deleting baseline data points If you want to delete a data point from the Point list select the appropriate point in the 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 base
334. t another folder click on the Browse button and open the desired folder The search for chromatograms will take place in all result files within the selected folder as denoted by the asterisk You can instead select a specific result file using the Browse function M oreover you can use wildcard characters to search within result files with a specific name profile Y ou can use standard wildcard characters in the filename specification stands for any number of characters and for any single character For example iex will search files named iex iex will search all files with names beginning iex iex will search all files with names ending iex iex will search only 4 character names ending in iex User entered search filters to a maximum of 10 will be saved in the drop down menus for both Result and Chromatogram selections M ore than one string can be used as a search delimiter by inserting a between strings and search filters are automatically saved and stored within user profiles To return to the default setting to search in all result files click on All Presenting results 8 4 Click on the Search 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 Open Chromatogram to Compare x m Chromatogram selection Folder Joc Ad
335. t select it and click on Delete Layout 5 Click on OK to view the selection in the run data window The name of the layout selected replaces the default layout name Run Data 6 Toggle between the various layouts that you have created by selecting Next Layout in the right mouse button menu Y ou can choose run data items to display without using named layouts simply be selecting or deselecting items in the list N ote however that this will change the definition of the currently selected layout Run data style Y ou can change the colour of the text and background in therun data window To change the colour of the text or text background in the displayed readings boxes 1 Select Colour Settings Text or Colour Settings Background from the right mouse button menu cascade 2 Select a colour in the dialogue and click on OK 6 7 sorom a run Next Layout Colour Settings gt EN r bar Properties m 6 8 Alternatively 1 Select Properties from the right mouse button menu The Properties dialogue is displayed Select the Run Data Colour tab Click on the Text button or Background button and select a new colour Click on OK The result of the colour change is displayed in the tab M ake further adjustments to the colours as appropriate Click on OK to close the dialogue and apply the changes Run data pressure units If the Pressure data is displayed in the run data window you can set the dis
336. te see Section 14 1 1 for a description of the dialogue contents Remember that if you change the system strategy the arrangement of tubing pumps columns etc may need to be changed on the synthesis system An attempt to control a system using the wrong strategy may cause malfunction and damage to the synthesis system 13 1 3 Deleting system definitions To delete a system definition select the system s in the System setup dialogue and click on Delete A system definition can only be deleted if the system is idle and no users are connected to the system 13 2 Access levels 13 4 Access to UNICORN software is controlled by username and password authorisation see Sections 14 3 and 3 1 Within the program each authorised user is assigned an access level within the system which determines which functions the user can perform 13 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 select Administration Access levels from the M ain menu and select the level you wish to edit To use this menu command you must have User setup Levels authorisation see below T he levels are named Level 1 Level 10 by default to change the name of a level enter a new name click on the Rename button 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 a
337. tect important data against accidental deletion or loss in the event of hard disk failure backup copies should be taken at regular intervals This can be best achieved by having the UNICORN folders on the server if available and working directly from these folders Alternatively you can use the File Copy to external function to save files onto the network server It is standard practice for backups to be made of the server folders 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 3 4 Printer setup UNICORN 3 1 usesthedefault printer and printer settings installed on your computer To change the choice of printer either change the default settings in Windows NT or set up your choice of destination printer for the current working session by selecting File Printer setup in the M ain menu module and selecting the desired printer 3 4 1 Setting the margins The default margins for the printers can be changed 1 Locate the file UNICORN IN I found under CAUNICORN BIN for example by using Windows NT Explorer 2 Doubleclick on the file to open it and locate the following lines EVAL PrintMarginLeft 10 EVAL PrintMarginRight 5 Eval PrintMarginTop 5 Eva PrintMarginBottom 5 The values in the lines set the margins based as a percentage of the full width and height of the paper
338. ted block is inserted with the same breakpoint value as the block selected for point of insertion 5 4 5 The flow scheme window The flow scheme window displays the configuration of system components T his window isstatic and for information only useful for example in identifying valves for flow path instructions ACN Pump mL min Detrit Pump m min Press ACN Bar Press Detrit Bar CM P Monitor BS emn Figure 5 21 The Method editor flow scheme window 5 5 Method variables Variables can be assigned to many instruction parameters even including breakpoints Variable 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 Sample 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 arun Up to 64 variables can be defined in a single method 5 23 oe and editing methods 5 24 5 5 1 Identifying variables Parameters defined as variables are identified in two ways e in the text instruction window the parameter is given as t
339. ten for this system can be added using this button By default each method step will start as soon as possible ASAP after the completion of the previous method step To set the time interval for starting a selected step click on the Condition button In the Condition dialogue set the time when the step is to start Click on OK C As soon as possible Wait Hours f Minutes fo al Cancel Help Figure 7 4 Condition dialogue Thetiming of M ethodQ ueue steps performed on different systems can also be controlled by the R eady instruction see Section 8 3 1 in the method Click on Setup to define the starting time for the M ethodQ ueue Immediate start sets the M ethodQ ueue to start as soon as you request Run from the M ain menu Start time sets the M ethodQ ueue to start at a pre set time of day up to one w eek after Run is requested If no day is specified the M ethodQ ueue will start as soon as the preset time is reached i e within the next 24 hours MethodQueue Setup x Start Mode Immediate Start Start time 14 32 Figure 7 5 MethodQueue Setup dialogue 7 3 MethodQueues Note TheM ethodQ ueue setup time defines the starting time for the M ethodQ ueue as a whole Do not confuse this with Condition which defines the relative starting time for a step within a M ethodQ ueue 8 Click on Save to save the M ethodQ ueue Enter a M ethodQ ueue name in the dialogue Note Edit Method
340. the 3 base position is always the first base on the solid support before the start of the synthesis procedure 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 e Used specifically with the Fix Column Recycling Coupling method template you can save the method and directly perform a test run of UNICORN to synthesize the sequence see below Used with other templates containing the in built sequence you can replace the supplied sequence with a one 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 1 Create a new method according to Section 4 1 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 sequencein the sequence editor field 4 3 4 Creating methods from method templates 5 Variables Questions Notes Reference Curves Evaluation Procedures Method Information Result Name Start Protocol Sequence Sequence name TEST 20 from modified amidite positions DNA Cs C Modified Cc oE o BNA a dened ino O of 20 S ATACCG ATT AAG CGA AGT TT 7 4 r Optional method steps Open Purge amitide Create I Purge solvents M Column wash I Final detrityl
341. the corners or in the middle of a border and drag the box to re size it 3 For defined placement and or sizing of object s select from the following options 8 41 8 Presenting results 8 42 Align left Of Oe Align right 0 0 Align top a i El Align bottom les Oe Adjust to margins Adjust to left margin Adjust to right margin E Adjust to centre c For multiple selected objects on the same page this function aligns the objects by their left borders For multiple selected objects on the same page this function aligns the objects by their right borders For multiple selected objects on different pages this function aligns the objects by their top borders For multiple selected objects on different pages this function aligns the objects by their bottom borders For single or multiple selected object s this function resizes the width of the object s to span from the left to the right margin For single or multiple selected object s this function moves the object s so that the left border s are aligned with the left margin For single or multiple selected object s this function moves the object s so that the right border s are aligned with the right margin For single or multiple selected object s this function centres the object s M akesame size E M ake same width M ake same height a Viewing options Presenting results 8 For
342. the instruction box Press the VAR button 3 Enter a new variable name in the dialogue and click on OK The Run setup is a series of pages for defining the method properties To access the Run setup pages press the Run setup icon at the top of the M ethod editor toolbar To access a given page in the Run setup click on the respective tab 5 6 1 Variables The Variables page lists all variables used in the method with their default values organised by method block Y ou can change the default values to create a variant of the method see Chapter 4 Creating and editing methods 5 Variables Questions Notes Reference Curves Evaluation Procedures Method Information Result Name Start Protocol Sequence Main E Column_Volume mi 0 10 999999 00 START_parameters cv 6 30 mi 1 00 200 00 Weight_of_support fico fq 0 10 100 00 Loading_of_support fio dumala 10 00 250 00 Col_Diam feo dmm 1 00 100 00 C _Colurn_wash CV_column_wash fe0o cv 0 00 999999 00 Detrit_peak_start Detrit_flow 300 em h 0 00 2000 00 a 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 The changed values will be displayed for the
343. the oligosynthesis systems via the network In this example PC 4 is connected to the network but it cannot be used to control any oligosynthesis systems since it does not have UNICORN installed Note also that the server does not have UNICORN program files installed and is not involved in the control process per se Security features in UNICORN include e Access security Useof UNICORN is restricted to authorised users Each user is assigned an access level which defines the functions that the user is permitted to use e Connection 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 e Data security Result files can be saved automatically at pre set intervals during a run to minimise 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 10 N etwork and administrative aspects are discussed in Chapters 13 and 14 respectively 2 11 2 UNICORN concepts 3 1 Logging on ZN ay UNICORN 3 10 Logon and file handling 3 3 Logon and file handling When you start the computer you must log on to Windows NT before you can log on to UNICORN and begin working Logging on to WindowsNT will automatically connect you up to the network if NT has been so configured N etwork connection is not
344. the primary Domain Controller run Server M anager by selecting Start Programs A dministrative T ools Server M anager From the menu select Computer and then the Add to Domain item Select the Windows NT or Server radio button and enter the NT workstation name in the Computer Name field Click on Add The computer is added to the domain On the Windows NT workstation select the Domain radio button and enter the name for the domain The network administrator should supply this name Click on Next to continue Y ou will be welcomed to the domain Acknowledge and click on Finish Select the option to restart your computer now The computer is restarted Log on to the Domain with administrator rights Installing TCP IP on the computer This is relevant when network has been installed earlier 1 2 Log on to the computer with administrator rights O pen the control panel with Start Settings C ontrol Panel Double click on the Network icon In the Network dialogue enter the Adapters tab and click on the Add button In the dialogue that is displayed select the correct driver for your network adapter and click on the OK button Click on the Have disk button and insert the disk with the latest version of your driver and enter the path to it in the dialogue In the Network dialogue enter the Services tab and click on the Add button In the dialogue that is displayed select Server and click on the OK button Click on the Add b
345. the various options or else apply free placement and sizing To access the customised report interface click on the New button in the Generate Report dialogueto display the Create New Report Format dialogue Click on the Customised format radio button the default setting and then on OK The Customise Report window is displayed Presenting results 8 epori Preview L Pace Eev Peor _One Page Zoomin con tui AddPage Delete Page Ext ekme allele 5 Page 1 of 1 Edit Mode Figure 8 32 Customise Report window Adding and deleting pages To add new pages to the report click on the Add Page button A new page is added after the last page To delete a page while in single page mode click on the Delete Page button Confirm the deletion To delete a page in dual page mode click on an object on the page and then click on the Delete Page button Confirm the deletion Adding objects To add an object to the report 1 Select an object from the Insert menu or click on the appropriate toolbar button 2 Movethe mouse pointer into the page area of the window Y ou will notice that the pointer has an additional symbol according to the object type you selected to insert 3 Press down and hold the left mouse button and drag out a box to the desired size R elease the mouse button A dialogueis displayed 8 Presenting results specific to the type of object inserted M ake the appropriate selections in t
346. 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 baselineis 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 see Section 10 1 4 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 for Classic algorithm The baseline parameters can sometimes be difficult to set Rough estimates can be found by analysing the source curve Measuring 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 asa part of the baseline and measure the length of the segment using the XY box on the chromatogram see Section 10 1 7 Insert this value as the Shortest baseline segment value C 5 Cora functions and instructions C 6 Measuring 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 repre
347. tic baseline calculation algorithm When optimising the baseline calculation a change in the Slope limit will very often give the expected result An example of the effect of a too high Slope limit is that the up slopes of the peaks may be recognised as baseline segments Baselines that deviate substantially from the source curve may arise from a combination of a too long Shortest baseline segment value with a too high Slope limit A recommended strategy is to change the baseline parameters step by step and to check the resulting baseline after each change The size of the initial change depends on the cause of the peak integration problem but some general guidelines can be given When the desired effect is achieved it isadvisableto go back and check a parameter value in between the two last settings to avoid an unnecessarily low or high value The default baseline parameters can be restored by clicking on the Default command button Baseline parameter Recommended initial change Shortest baseline segment 20 50 Noise window 10 30 Slope limit 25 50 Max baseline level Usually not necessary to adjust 9 Evaluating results Below are some examples of common problems with peak integration and baseline calculation as well as suggestions for improvements The following problems will be discussed Baseline does not follow the source curve e Peak limits too high up on the peaks Noise detected as peaks e Peaks missing Baseline
348. ting Procedures Record Off Save the procedure N ow do a batch run see Section 10 3 5 on all the desired result files to get the printed reports The procedure can also be saved with a method to get automatic printouts at the end of arun Section 5 6 5 rocedures Record On Edit Run 0 Global Integrate_and_Print 1 Global Integrate_Full_ Report Evaluating results 9 Note If the selected report format is changed in File Report the new format will be applied when the procedure is run except in cases where the procedure has been imported to a method in these cases the procedure is saved in the method file and cannot by changed If the format is subsequently deleted the procedure cannot be run 9 3 8 Placing a procedure on the menu and running It is possible to choose a maximum of 15 created evaluation procedures to be placed 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 seethe procedurethat you added to the menu Select this procedure and it is automatically run for the active chromatogram The menu addition is remembered in UNICORN even if the results of the current evaluation session are not saved To take away a procedure from the menu deselect it from the Procedures Menu list 9 3 9 Exporting data or curves Data and curves can be exported to other file formats
349. tly open procedure i e you will not be creating a new procedure To create a new procedure select File New Procedure or Procedure Edit New to display the Procedure Editor dialogue To begin recording click on the Rec button or select the dialogue Control Record menu command 9 3 2 Editing an existing procedure Evaluation operations are represented by instructions see A ppendix D 4 in the Procedure Editor dialogue These may be modified to suit specific evaluation needs and be saved for later use Note You are recommended to be restrictive in editing existing global procedures It is recommended to open an existing procedure and save a copy under anew nameand use this copy to perform any editing procedures 1 Click on Procedures Edit Open and select the desired evaluation procedurefrom thelist T he Procedure Editor dialogue is displayed for the selected procedure 2 To view the parameters contained within a specific instruction in the procedure select it and view the information contained within the Instructions and Parameters fields of the dialogue Procedure Editor Integrate_and_Print Bm E File Contol Help ke e e REJECT PEAKS OFF OFF OFF OFF 20 NEGATIVE_PEAKS OFF CALCULATE_BASELINE 01 17 DEFAULT DEFAULT DEFAULT DEFAULT SUB 01 17 47 PEAK_INTEGRATE 47 4 Instruction Parameter Co tion NEGATIVE_PEAKS p Insert PR Eas on PEAK_INTEGRATE Minimum peak height 2
350. togram integrated using the morphological algorithm contains many data points when the Minimum distance between points parameter is set to a low value A The number of data points is reduced when the Minimum distance between points parameter is set to a higher value B Evaluating results 9 Noise window Sometimes you get too many peaks after the peak integration usually because noise on the baseline is erroneously detected as peaks The cause of this problem is that the Noise window parameter is too low Increase the Noise window parameter in the baseline calculation This may in some cases result in peak limits too high up on the peak slopes see Section 10 1 5 for a description of Noise window with the classic algorithm Another possibility is to use the Reject peaks function in the Integrate dialogue to reduce the number of peaks based on an appropriate criterion e g the number of peaks or the minimum peak height 9 1 5 Optimising the baseline parameters using a clas sic algorithm The first choice when trying to optimise the peak integration is to change the baseline parameters A brief description of the parameters and a suggested way of estimating settings for the baseline parameters from the source curve is given in Appendix D 2 The Integrate Edit baseline function may provide additional information about the cause of the unsatisfactory peak integration by revealing where the baseline points have been placed by the automa
351. tructions groups including Alarms Special Monitors and Curves T o view the parameters and parameter settings for a particular group click on the appropriate radio button 3 Theinstructions for the selected instruction group are listed Beneath each instruction is listed the parameters and the current setting 4 To changethe setting for a parameter select a parameter from the list and change the associated parameter setting The parameter is updated in the list 5 To restore a setting to its default setting as defined in the system strategy select the setting and click on Set Selected Parameter to Strategy Default Value 6 When all required changes have been made click on OK Choosing Cancel will cancel all changes made since the dialogue was last opened Caution Changes made to settings do not take effect until you click on OK to close the dialogue Alarms define upper and lower alarm and warning limits for process monitor signals e If 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 e If the signal exceeds the Warning limits a 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 act
352. ts When 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 removea noise spike the window width should in principle be slightly more than twice the width of the spike Thefilter algorithm only accepts odd integer parameter values between 1 and 151 If an even number has been given it is incremented by one C 2 Baseline calculation theory The baseline calculation can schematically be described in three steps 1 Defining baseline segments 2 Select baseline points 3 Draw the baseline C 2 Evaluation functions and instructions C C 2 1 Defining baseline segments In the first step baseline parameters are used to find the baseline segments T he parameters can be seen in the Integrate Calculate baseline function or by pressing the Baseline settings button in the Integrate Peak integrate function The default values for the parameters are determined from the source curve The baseline segments are found by different parameters based on the type of algorithm selected Morphological algorithm The Morphological algorithm searches for all parts of the source curve which 1 come into contact at both extremes of the Structure width This parameter is based on the widest detected peak in the curve fulfils the M
353. ts Introductory material 1 Introduction 2 UNICORN concepts 2 1 UNICORN control software 2 1 2 1 1 Strategies 2 1 2 2 UNICORN user interface 2 2 2 2 1 Toolbar Guide 2 2 2 2 2 Software modules 2 2 2 2 3 On line help 2 3 2 3 Files and folders 2 3 2 3 1 M ethod files 2 3 2 3 2 Result files 2 4 2 4 Methods 2 4 2 4 1 M ethod structure 2 4 2 4 2 M ethod templates 2 7 2 5 System control 2 7 2 5 1 Control facilities 2 7 2 5 2 System connections 2 8 2 6 Evaluation 2 9 2 7 Network considerations 2 10 2 7 1 Stand alone installation 2 10 2 7 2 Network control from a remote workstation 2 10 2 8 Security 2 11 3 Logon and file handling 3 1 Logging on 3 1 3 2 Toolbar Guide 3 2 3 3 UNICORN Main menu windows 3 5 Contents 3 3 1 Creating a new folder 3 5 3 3 2 O pening and running method files 3 5 3 3 3 Presenting files 3 6 3 3 4 Finding files 3 8 3 3 5 Copying and moving files and folders 3 9 3 3 6 Deleting files 3 13 3 3 7 Renaming files 3 13 3 3 8 Backup security 3 14 3 4 Printer setup 3 14 3 4 1 Setting the margins 3 14 3 5 Logging off 3 15 3 6 Quitting UNICORN 3 15 Methods and runs 4 Creating methods from method templates 4 1 Creating a new method 4 1 4 2 Saving and running a test sequence method 4 3 4 3 Creating a sequence and method 4 4 4 4 Editing method variables 4 7 4 5 Method notes 4 9 4 6 Saving the method 4 10 4 7 Starting a run 4 11 4 8 Editing text instructions 4 12 5 Creating and editing methods 5 1 The se
354. tween UV curves measured at different wavelengths give useful information about peak purity or peak identity The Operations Divide function can be used when you havea result file with run detected at more than one wavelength 1 Before dividing the curves you must make sure that both curves have a baseline close to zero AU This can be achieved with baseline subtraction 2 Create a baseline for each UV curve according to the procedures detailed in Sections 10 1 1 and 10 1 2 Subtract the baselines from their respective UV curves using Operations Subtract Alternatively you can subtract the corresponding blank runs from the UV curves if such exist 3 When you have achieved two curves with a zero baseline select Operations Divide and select the two curves for division Y ou have the option to set Threshold values by checking the option and setting the threshold value for each curve T his sets the quotient to 1 0 if either of the sample values is closer to zero than thethreshold value This is to prevent very high quotient values being created if division is performed with values close to zero Very low quotient values are also prevented Threshold values are suggested by UNICORN although these can be changed Evaluating results 9 Chromatogram Chromatogram Chromatogram E S i 01 test20_Cond 02 test20_PressDet ig test20_Press Ch 01 test20_Cond 02 test20_PressDet 03 test20_Press4CN oil 2I I
355. u window gt System Control 1 OPII Method d default HIO1 m01 Fie View Manual System Adviser Help Run Hold Pausen continual End alale 0 00 min Run 98 02 14 10 03 17 Method HIO1 Result qingrid HI01 res Manual For Help press F1 End Block Controlled By default Figure 6 1 The System control workspace with run data curves flow scheme and logbook displayed 6 1 Starting a method Y ou can only start a method if the system is connected and no method is currently running Y ou must have Run methods authorisation to start a method Before starting a method make sure that The 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 workspace 6 1 sorom a run 6 2 In the status bar information is displayed about the following traffic light with status text current running block M ethodQ ueue text connection status text If the correct system is not connected you can connect up to the system if it is free by selecting System Connect e The system monitors are correctly calibrated see Section 6 6 6 1 1 Starting from the Main menu Y ou start a method from the M ain menu by selecting the method in the Methods window and selecting File Run Alternatively you can click on a method file with the right mouse button and select Run followed by selecting one
356. uction box Click on the Var button Creating and editing methods 5 3 Enter a name for the variable in the dialogue and click on OK Variable name definition Variable name pa ooo OK Cancel Clear Help Figure 5 24 The Variable name definition dialogue Variable names may be up to 20 characters long and may contain letters A Z digits 0 9 and the underscore character Use underscore characters instead of spaces if you want to separate words in a name e g Flow_rate N ames must be unique within the method The case of letters is retained but is not significant The names Flow_Rate and FLOW_RATE are treated as identical W hen you definea variable the valuein the parameter field applies as the default value for the variable Note Only one variable which affects block length breakpoint or gradient length may be defined within each block Any number of other parameters may however be defined as variables within a block 0 10 Block C _Column_wash C _Column_wash 0 00 Base SameAsMain 0 00 _PFLow ACN 6 00 bar 8 00 CV_column_wash Flow ACN 0 00 ml min 8 00 Flow_ACN 0 00 ml min CV _Column_wash 8 00 End_block lt es 0 20 End block C _column_wash 200 Cv 0 00 999999 00 Detrit_peak_start 0 00 Block Detrit_peak_start h Detrit_flow 300 em h 0 00 2000 00 Detrit_peak_start 0 Base SameAsMain Detrit_wash DOO Cycle_Start 0 00 WasteDut Waste_Detrit Detrit_Pressur
357. uction line before which you want the copied block to be pasted Creating and editing methods 5 3 Click on the right mouse button and select Paste from the menu A dialogue requests if you wish to rename the pasted block Click on Yes to rename the block beforeinsertion or No to directly insert the copied block The pasted block is inserted with the same breakpoint value as the block selected for point of insertion Cutting and pasting blocks To cut and paste blocks within a method 1 Select the block to be copied using the right mouse button Select Cut from the menu The block is removed from thetext instruction window Note A cut block does not mean that it has been deleted see Section 5 3 4 and can still be called from elsewhere in the method 2 Select the instruction line before which you want the cut block to be pasted 3 Click on the right mouse button and select Paste from the menu The pasted block is inserted with the same breakpoint value as the block selected for point of insertion Importing blocks Blocks may be imported from other method files to which you have access including the current method file in which you are working 1 Select Block Import Block As 2 Choosethe method from which you wish to import and mark the block to import The name of the selected block is suggested in the Block name field 3 Select a block from the Call drop down list into which the imported block will be placed a
358. ue To change thestart condition or method name select the line to be edited and click on the Condition or assigned system button respectively MethodQueues 7 e To insert anew M ethodQueueline after the currently selected line click on Insert To erase the currently selected line from the M ethodQ ueue click on Delete In the dialogue box check the system s for which the method is to be deleted 7 3 Running a MethodQueue Before starting a M ethodQ ueuerun make sure that all systems used in the M ethodQ ueue are connected with control mode connections see Section 6 5 and arein End status The system associated with the first System button in the M ethodQ ueue definition must be connected to System control window 1 the second system to System control window 2 and so on The M ethodQ ueue will not start unless all required systems are connected in control mode To start a M ethodQ ueue from the M ain menu select the M ethodQ ueue icon and select File Run or select Run from the right mouse button menu The M ethodQ ueue will start in accordance with the conditions defined in theM ethodQ ueuesetup Y ou cannot start or end a M ethodQ ueue from the System control window Run MethodQueue authorisation is required to start a M ethodQ ueue 7 3 1 Method execution in MethodQueues The start protocol for the first and each subsequent method step in the M ethodQ ueue is displayed when the corresponding method is run If you r
359. ue is displayed requesting if you want to totally delete the block instruction from the method Answer Yes or No as described for this function using the right mouse button menu command above 5 13 oe and editing methods 5 14 5 3 5 Renaming blocks To rename blocks choose Block Rename Block or select a block in the method using the right mouse button and select Rename from the menu In both instances the Rename blocks dialogue is displayed Rename blocks x Select the block to rename Coupling_recycle_RNA_u_v2 Coupling_recycle_RNA_ Coupling_recycle_RNA_y Coupling_wash DNA_parameters Final_detritylation Ox_push Ox wash New name Figure 5 12 The Rename Blocks dialogue By default the block that is currently highlighted in the method text instructions is automatically selected in the dialogue Enter the new name in the New Name field and press Rename T he dialogue remains open until you press Close so that you can rename more than one block without closing the dialogue If the block you renamed is used in a Block or Watch instruction the block name in these instructions will be changed automatically 5 3 6 Copying moving and importing blocks Y ou can copy cut and paste blocks within a method or import blocks from another method Copying blocks To copy blocks within a method 1 Select the block to be copied using the right mouse button Select Copy from the menu 2 Select the instr
360. uence 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 9 3 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 SameAsMain 0 00 Scale 1 75 Weight_of_support g 93 Loading_of_support umol g 0 00 ColDiam 10 00 Col_Diamimm 0 00 Delay ol 1 3 Delay_volumet 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 e g START_parameters 0 00 Base SameAsMain 0 00 Scale 1 75 Weight_of_support g 93 Loading_of_support umol g 0 00 ColDiam 10 00 Col_Diarm mm 0 00 DelayVol 1 3 Delay_volume ml 4 00 End_block During a run the overall time or volume is determined by the sum of the block lengths Note Thelength 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 method length can be checked in the Gradient window of System control 5 45 ons and editing methods Depending on how conditional calls are used see Section 5 8
361. umentation The full documentation of a run is stored within the result file A few of these are described below To open the Documentation either select View Documentation or click on the View Documentation toolbar button To print documentation contents click on the Print button in the Documentation dialogue see Section 9 6 3 The contents can be saved as a new method by selecting Method page and clicking on Save as 8 Presenting results Variables These are the parameters that were used during the run Documentation x ae Std ston cain d upa l ent amme Main START_parameters_ Detrit_peak_start Figure 8 46 Documentation Variables page Notes This displays notes that you have made at various points during the run Y ou arealso able to enter new comments in the Evaluation Notes field Documentation x ResutName Stat Protocol Settings Calibration Logbook Evaluation Log ReferenceCurves Evaluation Procedwes Method Infomation E a ii E Figure 8 47 Documentation Notes page Presenting results 8 Calibration This displays what system calibrations were made when and by whom Documentation Figure 8 48 Documentation Calibration page 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 c
362. umol g 0 00 Block Purge_Tetrazole 0 00 ColDiam 60 Col_Diam mm W 0 00 Block Purge_solvents_ox 0 00 DelayVol 2 00 mi Ml 0 00 Block Column_wash 0 00 End_block 0 00 Block Add_DNA_T W 0 00 Block Purge_G W 0 00 Block Purge_C W 0 00 Block Purge_T_U 0 00 Block Purge_A W 0 00 Block Purge_Tetrazole W 0 00 Block Purge_solvents_ox W 0 00 Block Column_wash 0 00 Block Add_DNA_T Figure 5 15 Displaying and hiding block instructions General oligosynthesis instructions are listed in Appendix B 5 18 Creating and editing methods 5 5 4 2 Adding instructions To add a new instruction 1 In the text instruction window select an appropriate block and display the instructions within the block Select an instruction line in the block 0 00 Block Purge_A Purge_A New instruction 0 00 Base Volume at breakpoint 0 0 00 Flow _Reag 10 00 ml min will be added here gt 0 00 Amidite A Waste 1 00 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 selected instruction line is within the 3 block not the call to the block N ew instructions are added from the Instruction box O pen the Instruction box if not already displayed View Windows For the new instruction a Set the desired breakpoint in the Breakpoint field b Choose the instruction type from the five or six options depen
363. up to and including the last autosave time before the failure Results after this time will be lost 10 4 Security recommendations for control stations Oligosynthesis systems may be controlled without running the user interface modules In a network installation this situation can arise if a system is controlled from a remote station without starting UNICORN on the local station In a stand alone installation the situation can arise if a user quits UNICORN after starting a run In both cases it is not apparent from the desktop that UNICORN control softwareis actually running and thereis thereforea risk that Windows NT may be shut down and the computer turned off in the belief that it is not in use To prevent this e Do not quit UNICORN if you are controlling a system e Do not turn off local station computers in a network installation If possible start UNICORN 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 1 0 Security features 11 1 Introduction Network setup 1 1 11 Network setup UNICORN 3 10 can be installed in a network environment allowing synthesis systems to be controlled from any PC in the network where UNICORN isinstalled This chapter describes how to set up the network environment before installing UNICORN This chapter is only relevant if you will be running UNICORN to control synthesis s
364. utines 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 O ligoPilot II For other synthesis systems users are recommended to read the relevant system manual for calibration procedures Performing a run 6 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 Detrit solvent pump System Control 1 OPII Calibration Calibration procedure Monitor Set pressure to zero and press button bar 0 0000 Read value 1 1 Reference value 1 SR 2 Measured value l Read yalue 2 8 Reference VAA 2 0000 0 0 20 0 a Pad ale bar Measured 1 Figure 6 16 Dialogue box for pressure calibration of the detrit pump Select PressD et in the M onitor pull down list to calibrate the pressure reading of the D etrit 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
365. utton again 10 11 12 Network setup 1 1 In the dialogue that is displayed select Workstation and click on the OK button In the Network dialogue enter the Protocols tab and click on the Add button In the dialogue that is displayed select TCP IP Protocol and click on the OK button In the Network dialogue click on the OK button W hen the window is writing this new data to the hard disk the configuration dialogues for the TCP IP protocol will be displayed Information specific to the network must be entered This information should be supplied by the network administrator Installing UNICORN 1 Start Windows NT Explorer with Start Programs W indows NT Explorer Connect the shared folder on the UNICORN file server with the designated drive letter Y ou can do this by selecting T ools M ap Network Drive In the dialogue that is displayed select the drive letter in the upper drop down list If the current user ID does not have permissions to the shared UNICORN folder on the server enter a valid domain user ID in the Connect As field In the Shared folders list at the bottom of the dialogue the network can be seen double click on the server with the shared folder Available shared folders on the selected server are displayed Double click on the shared folder UNICORN will use A dialogue appears with a request to give a valid user name and password The user name will be the ID previously entered in the
366. utton to establish a control mode connec tion you must supply the locking password which is case sensitive or your logon pass word if you have Unlock locked systems authorisation A user has left the sys tem in an unlocked state Click on the con nection mode button to establish a control mode connection In all modes you can choose System Disconnect to disconnect the system from the control window 6 5 3 Leaving and locking a system A running or End state system with a control mode connection can be left and locked by clicking on the control mode button or selecting System Leave system W hen the system is left the connection becomes a view mode connection After leaving and locking scouting or M ethodQ ueue runs it is not possible to establish a control mode connection from another computer Performing a run 6 Leave Control of OPII x Leave system Unlocked C Locked Username default Password 3 Cancel Help Figure 6 17 Leave Control dialogue Y ou may leave the system unlocked or locked Unlocked 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 e Locked locks the system with the password specified in the dialogue A control mode connection can only be established by providing the correct password N ote that this password is independent of the user
367. variables Break point values and instruction parameters may be defined as variables This is a powerful facility for constructing a method which contains default parameter values These 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 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 Variable_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 3 The Run setup mode is displayed when the method is run allowing variable values to beset at the beginning of the run E Main Base CY 6 30 Column_Volume ml Any 0 00 Message Fill your column with DNA T support Scree 0 00 Message Press CONTINUE when ready Screen 0
368. w Global Chromatogram_Peaks Run date amp ti Global Chromatogram_pH LSAS Sma Delete Global Chromatogram_Quant Report title Global Full_ Report Result file name a Method file name Page number E Method E Documentation M Cheamskaneses x Preview Close Help Figure 8 31 Generate Report dialogue In the dialogue you can see the available formats in the Format field Y ou can select a format from thelist and directly apply it Alternatively you can create a new report format or edit the existing formats Note A report format saved with the Current chromatogram does not necessarily print the actual chromatogram as it appears on the screen in the Evaluation workspace 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 selected in the method templates 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 10 3 6 for details 8 7 1 Creating a new customised report format Y ou can create your own report format and save it for later use The customised report interface allows you to choose from a variety of objects including chromatograms methods documentation free text and more M oreover you can decide the placing alignment and sizing of the objects according to
369. y file error F 1 strategy for creating blocks 5 13 structure width 9 8 Subtract 9 26 subtracting a blank run curve 8 14 8 16 System 6 18 system audit trails 13 15 connection 6 20 connection modes 6 21 connections management 6 20 disconnect 6 22 disconnecting 6 23 leaving and locking 6 22 naming 13 2 requirements A 1 System button 7 1 System Control button 3 4 system control button 6 17 Index system control module overview 2 3 system control monitor signals not appearing F 3 system definitions defining new system 13 2 deleting 13 4 editing 13 4 management 13 2 System is available status bar message 6 18 system run hours 13 18 system settings accessing settings 14 1 display 6 4 instructions B 10 introduction 14 1 system type 13 2 T TCP IP installing on computer 11 7 template deleting 5 42 templates creating methods from method templates 4 1 method 2 7 temporary chromatogram 8 2 temporary chromatogram clearing contents 8 2 text adjusting free text object for report 8 36 defing and positioning text for curve 8 7 entry in chromatogram 8 17 text alignment selecting 6 13 text instruction window 5 9 text instructions editing 4 12 text instructions editor 5 6 threshold values 9 26 time and date incorrect F 5 Time between samples setting 14 5 tool bar in system control workspace 6 16 toolbar buttons 3 3 toolbar buttons 2 2 toolbar guide 3 2 trityl data 2 9 typographical conventions ii U
370. you have to deselect the options The difference between Filter peaks and Reject peaks is that the latter function permanently excludes peaks from the integration and affects the calculation of total peak area etc 9 1 3 Optimising peak integration If the results from the peak integration are unsatisfactory there are several possibilities to improve the results Morphological and Classic baseline calculation Y ou can use one of two baseline calculation algorithms depending on the type of peaks to beintegrated e The Morphological algorithm is set as the default and gives the best results in curves with drifting baseline and peak clusters O ptimising baseline calculation using the Morphological algorithm is also relatively easy since there are only three baseline parameters namely structure width noise window and minimum distance between points see Section 10 1 4 Evaluating results 9 e The Classic algorithm has long been used as the standard for calculating the baseline see Section 10 1 5 The Classic algorithm is particularly useful in integrating curves containing negative peaks Thus the Classic algorithm should be selected if the Morphological algorithm gives poor results from the presence of negative peaks or where quantitative data from negative peaks are important in the run To select the appropriate algorithm and change baseline settings 1 Display the Settings or Baseline Settings dialogue respectively by i c
371. your own report There are two ways of accessing the Report Generator Wizard you can generate a report in the M ain menu by selecting the menu command Administartion Create System Report when an error message appears in System control you can activate the report generator by clicking on the Report button in the message dialogue Administration 1 3 13 5 1 Generating a report from the main menu Generate Report Wizard This first dialogue gives an introduction to the report generator Generate Report Wizard This wizard will help you quickly generate a report on the UNICORN software To begin the report generation click Next Cancel ap Systems This dialogue is displayed only if you have accessed the wizard from the M ain menu This displays a list of the available systems for the logged on user In order to proceed further a system must be selected for which the report is to be generated Select system to make a report on Explorer 13 19 1 3 Administration 13 20 Description Y ou are required to enter a short description of the problem the circumstances under which the problem occurs and the consequences caused by the problem Reproducibility Y ou are required to giveinformation concerning the reproducibility of the problem If the problem is reproducible then you can describe how to bring about the problem Reproducibility Administration 1 3
372. ystems remotely The N etwork setup should be performed by someone with experience in Windows NT and network installations It isimportant to understand the basics of how UNICORN operates in a network setup to ease the installation A good start is to read the following explanations of some of the aspects of UNICORN ina network setup Explanation of some of the aspects of UNICORN in a network Storage of data Communication Log files in a net work setup N amed pipes com munication N etwork failure in the middle of a run Access to the net work drive while running M ethods and log files are stored in a folder shared between the local and the remote UNICORN The remote and the local UNICORN use named pipes to send commands and data between them W hile running the local UNICORN system writes logs on the local harddisk W hen the run is over it copies them to the network drive where log files are stored From the remote UNICORN commands are sent such as run method foo met From the local UNICORN messages and trend data is sent to the remote UNICORN Thelocal UNICORN will continue the run and the log file will be on the local harddrive when the run is over W hen a method is begun the method is copied from the network drive to a local directory and it is read from there during the run 11 1 1 1 Network setup 11 2 Server in a UNI UNICORN requires only a directory for log files CORN network and

UNICORN User Manual - GE Healthcare Life Sciences

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