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1. Figure 4 35 The Relay Configuration View Shown in Figure Figure 4 35 the Relay Configuration view allows specifica tion of both the closed and open intervals of the relay pulse generated when a Relay pulse function is executed from a cycle or procedure To set either the Closed or Open period for a particular relay select the relay drag through the existing value in an entry field and then type in the new value in seconds Times as long as 999 9 seconds are allowed 215 0 5 Closed Open Figure 4 36 Diagram of Pulse Duration 4 46 4 Editing the Synthesizer Window June 2002 Applied Biosystems As shown in Figure 4 36 a single pulse is specified using both a closed inter val in which a contact closure is provided and an open interval Specifying pulse duration using both closed and open intervals guarantees that several pulses executed in succession will produce distinct individual pulses rather than a longer pulse which is the sum of the pulses June 2002 4 Editing the Synthesizer Window 4 47 Applied Biosystems June 2002 5 Using OligoNet This section provides you with practical information useful in using Oligo Net with one or more synthesizers Contents Practical Use of OligoNet Special Program Symbols Entering Sequences Cycles and Procedures Creating Your Sequences Listing of Standard CE Sulfur and RNA Cycles Creating your own Cycles Procedures and Functions Pre synth
2. the 4th and 17th bases from the 3 end the subscript s is placed in the se quence to indicate phosphorothioate linkages and is not actually entered into the Edit Sequence view 5 Using OligoNet June 2002 Applied Biosystems June 2002 Since the phosphorothioate linkages to be placed between the 4th and fifth and between the 17th and 18th bases are added by the fifth cycle and the 18th cycle the multiple cycle synthesis option table is filled out as shown in Figure 5 2 SE aE DISS na ma er og ee e BASES CYCLE COL 1 COL 2 COL 3 COL 4 4 1 0 um CE 1 jum Sul fur 12 1 0 um CE 1 Jum Sul fur Insert Cycle Cycle 3 1 0 um CE vj Delete Cycle Number of bases h2 Figure 5 2 Filled Out Multiple Cycle Option for Sequence Example The procedure for entering multiple cycle information listed below as sumes that you have already entered the sequence using the Edit Sequence view and have then selected the desired sequence for just one column in the Set up view for a four column instrument The sequence you entered will not contain the subscripts shown above for the sequence 1 Select Multiple from the initial Cycle pop up menu to call up the multi ple cycle option You will need to scroll down to see the option You will initially see a default cycle name on line one of the table and in the multiple cycle pull down menu line 2 Use the cycle pop up menu in the multiple cycle area of the view to se lect
3. check boxes are grayed out unless one or more Synthesizer Windows are open and an instrument is selected on the Synthesis Order Entries made in the Column and Seq No fields must be re entered upon re opening a Synthesis Order after you have made entries and saved it e Advanced Feature s Two fields for Column and Sequence numbers When the check box for each field is selected These fields are used when multiple orders are sent using the Send All Orders command The fields are not needed when sending orders one at a time using the Send Order to Synthesizer command since the com mand provides a dialog box allowing the same choices choice of col umn and sequence Column field values 1 to 4 Specify on each Synthesis Order the column on which the order is to be synthesized Sequence field values 1 to 34 Specify on each Synthesis Order the loca tion containing the sequence to be sent The sequence resident in any of 34 locations may be specified For more information on sequence locations including default locations for a specific sequence see Edit Sequence View on page 4 5 This feature produces the following actions when no boxes or only a single box is checked and a location is specified a No boxes checked Use any available column and the default sequence slot for that column b Only Column box checked Use specified column with default sequence slot c Only Sequence box checked Copy to specified sequence
4. to provide both a control function and a display function for the option This view allows you to specify monitoring frequency choose the column to display and when the Interrupt option is selected enables you to specify the minimum average step wise yield acceptable for the synthesis The regular or default view is shown in Figure 4 13 An alternative view discussed in the next subsection provides a view of raw conductivity data as well as Average stepwise yield and Overall Yield A full discussion of AutoAnalysis is provided in the 392 394 Synthesizer User s Manual DE synthesizer Synthesizer 1 Choose function Trityl Monitor Y Monitor once every il couplings O Interrupt if yield falls below minimum Adinhivui guerade lep age uleie DESUUYE as Je Column 1 2 O3 O4 Number Aug Stepwise Overall from 3 Base Yield Yield Tr T ar r ma r Er 18 G 97 8 68 7 19 C 97 9 68 7 20 T 96 0 68 7 21 T 97 9 65 6 z2 A 98 0 65 6 23 G a8 1 65 6 24 G 98 2 65 6 igi Figure 4 13 Regular Trityl Monitor View Note The Trityl Monitor view is not available for 392 394 synthesizers which do not have AutoAnalysis Regular Monitor View As shown in Figure 4 13 the Trityl Monitor view displays the results of trityl monitoring during the run in the table at the bottom of the view The first column lists the number of each base addition monitored from the 3 end the second column lists
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6. 4 35 S y Applied Biosystems 4 36 5 Save the Synthesizer window to save the bottle change procedure you just created Your new procedure will be listed the next time you use the Procedure pop up menu in the Edit Bottle Change Procedure view Special Instructions for Creating Auto Dilute Procedures The five ABI auto dilute procedures 2g Double Auto 2 0 g Auto 1 0g Auto 0 5 g and Auto 0 25g can be used as is for diluting Applied Biosystems phos phoramidites in bottles containing these quantities In some cases however you may want to create procedures which explicitly specify the conditions of dilution rather than rely on software provided for this purpose in the synthe sizer This subsection includes the information you need to develop your own procedures If you examine an ABI dilute procedure as shown in Figure 4 28 you will notice that the functions that dilute A G C T 5 6 7 and 8 do not have step times When these procedures are used in the synthesizer the duration of acetonitrile delivery for any of the phosphoramidite positions is determined in the synthesizer by referring to a special Auto Dilute Calibration table developed during calibration of the instrument To develop your own pro cedures with explicit delivery steps you will need the information provided in the synthesizer Auto Dilute Calibration menu to use as a baseline 2S synthesizer synthesize 1 ES Choose function Edit Bottle Procedure
7. City CA 94404 USA Phone 1 650 638 5800 Toll Free 1 800 345 5224 Fax 1 650 638 5884 Worldwide Sales Offices Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches into 150 countries on six continents For international office locations please call our local office or refer to our web site at www appliedbiosystems com www appliedbiosystems com AS Applied D Biosystems Applera Corporation is committed to providing the world s leading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses Printed in the USA 06 2002 Part Number 902114D an Applera business
8. CycO4 user 15 CycO5 user 16 Cyc06 user 17 CycO user 18 CycO8 user 19 CycO9 user 20 Cyct0 user 21 Cycli Mac 22 Cycl2 Mac 23 Cycl3 Mac 24 Cycl4 Mac 25 Cyct5 Mac 26 Cycl6 Mac 27 Cycl Mac 28 Cyc18 Mac 29 Cycl9 Mac 30 Cyc20 Mac 31 Cyc21 Mac 32 Cyc22 Mac Synthesis setup information is entered into the Set up Synthesis view shown in the left side of Figure 4 5 as follows Single Cycle Synthesis Setup 1 Fora synthesis with a single cycle select a cycle from the Cycle pop up menu shown in the right side of Figure 4 5 Note When synthesizers are equipped with AutoAnalysis the cycles provided do not support fraction collection In order to collect sam ples with a fraction collector you will have to modify cycles as described under Modifying Cycle for Fraction Collection in Sec tion 4 of the 392 394 Synthesizer Manual Ver 2 00 Next select the number of Trityl samples to be collected using the Tri tyl Collect pop up menu shown in the left side of Figure 4 6 If you have the AutoAnalysis feature in your synthesizer this menu will not alter any setting related to AutoAnalysis Refer to the Trityl Monitor View for information on choosing AutoAnalysis feature options Select None if you plan on using AutoAnalysis 4 Editing the Synthesizer Window 4 9 Applied Biosystems 4 10 Note If you specify that the first two and the last two trityls are to be col lected and the
9. File Menu New Synthesis Order Open Open Synthesizer Send to Synthesizer Save a Copy In Send Copy to Synthesizer Import Export Edit Menu Undo Redo Can t Undo Cut Copy Paste Clear Select All Show Clipboard Read Selection Find Find Same Replace Replace Same Use Sounds Synthesizer Prepare to Start Interrupt Synchronize Clocks Enter Password Change Name Order Menu Send Order to Synthesizer Send All Orders Send Order to Synthesizer Send All Orders Copy from Synthesizer Window Menu 3 Overview of Commands and Capabilities 3 3 3 5 3 9 3 10 3 11 3 12 3 13 3 14 3 15 3 16 3 16 3 16 3 17 3 17 3 17 3 17 3 18 3 18 3 20 3 20 3 21 3 22 3 23 3 23 3 23 3 25 3 26 3 26 3 1 Applied Biosystems June 2002 File Menu File New Synthesis Order w Open 0 Open Synthesizer Sand io tunthesiver Save a Copy In Sand Cag ia tunthesiear Close W Rpg HE Sane fen impart Enpi faye Setup Prini Figure 3 1 The File Menu Note Besides choosing a command from a menu using the mouse it is possible to choose a command by using the Command key equiv alent listed on the menu by the command For example choose the Open command by holding down the key and then press ing O If you need information on using the standard Apple commands Close Save Save As Page Setup and Print see your Apple System Software User s Guide 3 Overview of Commands and Capabilitie
10. READ WRITE without password READ WRITE Figure 2 11 Communications Display with Correct Setting When you restart your synthesizer this way the display may indicate that a power failure occurred Press the Okay key and proceed 2 Installation and Start Up 2 11 Applied Biosystems Checklist of OligoNet Synthesizer Use The list below outlines the main activities needed for use of OligoNet with your synthesizer More practical use information is presented in Section 5 Using OligoNet 1 Start the OligoNet session as described in this section 2 If desired sequence is not already stored in the synthesizer prepare sequence information in one of two ways a Prepare Open a separate Synthesis Order for each sequence to be synthesized containing a listing of the sequence and the names of cycles and procedures needed for synthesis as described in New Synthesis Order in Section 3 b Write import sequences as needed as described in Edit Sequence View in Section 4 3 Optional write import custom cycles begin and end procedures needed for synthesis as described in Edit Cycle View in Section 4 4 On the Set Up Synthesis view set up the conditions required for a syn thesis as described in Set Up Synthesis View in Section 4 a Choose the sequence end procedure and DMT On Off state re quired for each column to be used If desired specify a Run ID op tional b Sele
11. activated it displays current format settings for these parameters EE synthesizer Synthesizer EEN Choose function Set Time Date Format v Date format Month Day Year v Time format Figure 4 34 The Set Time Date View Two choices are available for the date format Month Day Year or Day Month Year Changing between the two formats is done by selecting the non current setting from the pop up menu The time format may be set to either the 12 hour or the 24 hour format Changing between the two formats is also done by selecting the non current setting from the pop up menu Note To synchronize OligoNet with the synthesizer s clock use the Synchronize Clocks command in the Synthesizer menu To set the time and date in the synthesizer use Set Time and Date from the synthesizer s Main Menu une 2002 4 Editing the Synthesizer Window 4 45 S y Applied Biosystems Relay Configuration View The Relay Configuration view is used to set the pulse duration for the advance fraction collector and relay pulse functions placed in a cycle or pro cedure to drive the fraction collector and perform other external functions Note The pulse durations for both the Advance Fraction Collector and Relay 1 Pulse functions are both set by the Relay 1 settings Ti Synthesizer Syn th esiz er 1 EEN Choose function Relay Configuration v RELAY CLOSED Closed seconds Open seconds Ds
12. appear and flash at the top of any Synthe sizer Window It indicates that someone has operated the Synthesizer s front panel Click on the symbol to clear it This will present a dialog box which allows you to re read all information from the synthesizer if necessary A This symbol occurs near an entry field whenever you make an entry which is not allowed June 2002 5 Using OligoNet 5 3 Applied Biosystems 5 4 Entering Sequences Cycles and Procedures If you intend to use the standard 392 394 cyanoethyl sulfur or RNA chem istry you will have very little to do except enter or import sequences you want to synthesize The standard CE Sulfur and RNA cycles provided with the instrument are listed on the next page CE cycles are for normal phos phodiester DNA synthesis while Sulphur cycles are for phosphorothioate DNA synthesis Creating Your Sequences Probably the most important task in preparing for synthesis is to prepare the sequence or sequences you will be sending to the synthesizer The synthe sizer itself provides you with the capability of creating up to 34 sequences If you need to maintain a library of more than 34 sequences you can export multiple Synthesis Order files to increase your capability of maintaining a sequence archive Sequences can be input in one of four ways by selecting one of the existing sequences and then copying it or editing it by typing in a sequence by importing a sequence from a
13. cursor on the trityl collect value or on the begin procedure name and then using the Prev or Next key to make your selection You can initiate synthesis at this point by pressing either No or Yes No starts synthesis but circumvents the begin procedure Yes starts synthesis after executing the designated begin procedure Since the begin procedure fills the lines with fresh phosphoramidite you should use it if the instrument has been idle for more than 12 hours 6 hours in a humid environment As soon as synthesis starts you can monitor synthesis either at your synthe sizer or at your Macintosh using the Monitor view as described in a later sub section 3 Overview of Commands and Capabilities 3 19 Applied Biosystems 3 20 Interrupt The Interrupt command is the only command from the Macintosh which directly controls the synthesizer s valves since it performs an Immediate interrupt on a cycle in process Before you interrupt a synthesis be aware that the synthesis can only be resumed from the synthesizer Choosing the command while a Synthesizer window is active brings up the dialog box shown in Figure 3 18 A Do you want to interrupt the current synthesis Figure 3 18 The Interrupt Synthesis Dialog Box Click Yes to interrupt synthesis on the synthesizer represented by the active Synthesizer window or click No if you change your mind about interrupting synthesis Note An immediate
14. customer has not entered them Save the Synthesis Order to the To Do folder 2 Send Synthesis Order to the synthesizer a b Open the Synthesis Order Use the Send Order to Synthesizer command Order menu to send the order to the synthesizer A comment stating when the order is sent and the name of the in strument is automatically entered in the comment field Save the Synthesis Order still in the To Do folder 5 Using OligoNet June 2002 Applied Biosystems 3 Upon completion of synthesis a Give or send the oligo and a printout of the Synthesis Order to the customer b Drag order file to the Done folder 4 If customer requests a repeat order ask for the Run ID look up the file in the Done folder and repeat steps through 3 Processing variations The process of using Synthesis Order forms may differ from that outlined above Other possibilities are as listed below e Throw out Synthesis Order file when synthesis is completed rather than save it if it is known that the order is a one off or if repeat orders are never done e Distribute OligoNet Order program to customers and let them fill out Synthesis Order forms on line In this case you will still need to check out the information on the form after it is received especially additional information required like cy cle choice that the customer may not know When a customer has the order program it is also appropriate
15. desired value For example after changing a bot tle the cycles used should be changed back to zero Click the Alarm at 4 Editing the Synthesizer Window June 2002 Applied Biosystems cycle entry field to prepare for the next step Note Each time a new table line is selected the Alarm at cycle entry field is highlighted You need to click on the Cycles used entry field if you want a value other than the value shown Alternatively the Tab key can be used to highlight the Cycles used entry field During use if an alarm goes off and the bottle is changed using the Bot tle Change procedure in the synthesis the Cycles used field will auto matically be reset to 0 3 Type in the cycle for which you want the alarm to be set This will enter this value into the third column of the table 4 Click the Alarm on check box to set the alarm On appears in the last column for the table line 5 Repeat steps 1 through 4 for each bottle alarm to be set You can turn off any individual alarm which is set to On in the last column by selecting the appropriate table line and then clicking the Alarm on check box to turn the alarm off To turn all alarms Off click the All alarms off button June 2002 4 Editing the Synthesizer Window 4 17 Applied Biosystems 4 18 Trityl Monitor View The Trityl Monitor view is used on synthesizers equipped with AutoAnalysis
16. equipped and the level of access permission set at the synthesizer By using the various views of this window you can monitor your synthesizer as well as prepare all the operating information such as sequences cycles functions and procedures for use on your synthesizer Section 4 provides you with a detailed description of how to use this window Section 5 provides you with more general information on using OligoNet If for any reason you have failed to establish communication between Oli goNet and your synthesizer refer to the troubleshooting information pro vided in a later subsection to correct your problem Printing OligoNet provides you with the capability to print out the contents of two types of windows Synthesis Orders discussed in Section 3 and Synthesizer windows discussed in Section 4 on any Macintosh compatible printer con nected into the network in which the Macintosh participates You will not be able to access the RS232 printer connected directly to your synthesizer Think Jet printers used with the DNA synthesizers are not compatible with the Macintosh Synthesizer window views which may be printed include the Edit Sequence view The Set Up Synthesis view the Bottle Alarms view the Edit Cycle view and the Edit views for the Begin End Bottle Shutdown procedures and the 2 Installation and Start Up 2 9 Applied Biosystems 2 10 Edit Function view If you have AutoAnalysis installed on your synthes
17. file or by using cut and paste functions between two applications Once you have input a sequence you can use it as is or you can change it to its reverse complement or change the A G C and T letter representation to bottle position representation 5 6 7 8 Besides entry of the sequence in terms of the four bases sequences can be entered or imported using mixed bases as a designation for a base site or ambiguity characters can be used to designate a base site If either mixed bases or ambiguity characters are used in a sequence no reverse comple ment or change to bottle representation is possible Once you have created or edited a sequence read it back using the Read Selection command to ensure that it was entered correctly You can then print out a copy send the sequence to the synthesizer or export the sequence to a file for archival The term Mac when used in the one of four sequence names indicates that these are reserved for sequences transferred to the synthesizer from a Synthesis Order For more information on assigning sequence locations when sending Synthesis Orders to synthesizers see Send Order to Synthe sizer Send All Orders on page 3 23 For information on creating a sequence See Creating a New Sequence Importing a Sequence on page 4 6 5 Using OligoNet June 2002 Applied Biosystems Listing of Standard CE Sulfur and RNA Cycles Each cycle is provided in a form for both 2 and 4 column i
18. g Ifdesired change the Safe step designation of the step The default designation of the step in Figure 4 23 is Safe which designates the step as a safe place to stop the synthesis without harming the oligo nucleotide If the step you add is not a safe step click No after Safe on the left side of the view This will change the designation on the cycle line from Yes to No When you designate a step as a safe step you are stating that it is safe to resume synthesis after a power failure occurring during that step Note Safe Step Yes No If it is safe to resume the cycle sequence after power failure then specify Yes for Safe h Continue inserting function steps by repeating steps 8a through 8g 9 Delete unnecessary steps as follows a Scroll the cycle listing down to the area of interest b Select the cycle step you wish to delete or the first in a series you want to delete as shown in Figure 4 24 If you want to delete a number of consecutive steps hold down the shift key and drag downward to select a group of steps for deletion 4 Editing the Synthesizer Window June 2002 Applied Biosystems June 2002 Synthesizer Synthesizer 1 Choose function Edit Cycle v Cycle 11 1 0 um CE copy v Name 1 0 um CE copy PO ROMO FRET ION I ROM ID FRET ION 140 Column 1 On 141 Column 1 Off 142 Column 2 On 143 Column 2 Off 144 Column 3 On FUNCTION NAME NUM Column 1 Off 14
19. inter rupt in that it interrupts all active columns and is the same as Interrupt Immediately on the synthesizer Once initiated the synthesis can be resumed from the synthesizer but not from the Macintosh When you press Resume at the synthesizer all columns will start again simultaneously Using the command will produce the warning shown in Figure 4 10 allow ing you to back out if you desire The interrupted Monitor View will state Interrupted as shown in Figure 4 10 A Do you want to interrupt the current synthesis Figure 4 10 Interrupt Synthesis Warning Dialog Box 4 Editing the Synthesizer Window June 2002 Applied Biosystems Synthesizer Synthesizer 1 Choose function Monitor Synthesis 23 v th Interrupted Lotume Syunthesizing step C Funetien fLolumea 3 Sunthesizing tep arctan Column 1 Column 2 Inactive Inactive Step jfi Step Functien ijj Faaetien l aiii sess Base sees Base Figure 4 11 Interrupted Monitor Synthesis View Note Synthesizing changes to Interrupted after interrupting a synthe sis June 2002 4 Editing the Synthesizer Window 415 Applied Biosystems 4 16 Bottle Alarms View The Bottle Alarms view is used to set alarms that will be triggered when a bot tle has been used for a specified n
20. interrupt is a cycle interrupt and interrupts all active columns When you press Resume at the synthesizer all inter rupted columns start up again at the same time Synchronize Clocks The Synchronize Clocks command is used to synchronize the Synthesizer s clock to that of the Macintosh Clicking on the command brings up a dialog box like that shown in Figure 3 19 Synchronize Synthesizer s clock to Macintosh Synthesizer 1 1 91 11 57 05 PM Macintosh 1 17 92 11 36 50 AM Cancel OK Figure 3 19 Synchronize Clocks Dialog Box The first step in using the dialog box is to check the Macintosh day and time setting If it is not correct click Cancel to exit from the dialog and then set the current date and time correctly using the Control Panel General icon for System 6 General Controls icon for System 7 3 Overview of Commands and Capabilities June 2002 Applied Biosystems June 2002 After you have correctly set the Macintosh time and date or if your Macin tosh time and date were correct click OK in the dialog box This will reset the synthesizer s time and date settings over the network Enter Password The Enter Password command is used to prevent unauthorized editing or monitoring of a Synthesizer If you open a Synthesizer window for which a password has been set the dialog box shown in Figure 3 20 automatically appears In this case you must know the password to be able to gain full per mi
21. it This is indicated by a lock icon on the cur rent view Save a Copy In The Save a Copy In command is used to save all information contained in an open Synthesizer to a Macintosh file When this command is chosen from the File menu the message shown in Figure 3 7 is presented to inform you that this command will tie up your Macintosh for a time This makes a com plete copy of the sequences cycles procedures and settings of a DNA syn thesizer and is useful for backup purposes Do you want to save all the sequences cycles procedures and settings in this synthesizer to a file It may take a few minutes Figure 3 7 Save Synthesizer Information Message Note The Save a Copy In command makes a copy of the Synthesizer window rather than the synthesizer itself The copy represents the state of the synthesizer at the time of copying only if no changes have been made to the Synthesizer window since opening it If you have made changes in the Synthesizer window and want these changes to represent the state of the synthesizer use the Send to Synthesizer command and then use the Save a Copy In command When you click OK to initiate saving a dialog box like that shown in Figure 3 8 comes up allowing you to assign the file name and location 3 Overview of Commands and Capabilities June 2002 Applied Biosystems June 2002 Example O Exam yn0rd Save a copy in Synthesize
22. or api3 support sciex com Chemiluminescence Tropix tropix appliedbiosystems com Hours for Telephone Technical Support In the United States and Canada technical support is available at the following times Product Hours Chemiluminescence 8 30 a m to 5 30 p m Eastern Time Framingham support 8 00 a m to 6 00 p m Eastern Time All Other Products 5 30 a m to 5 00 p m Pacific Time June 2002 1 Introduction 1 7 Applied Biosystems 1 8 To Contact Technical Support by Telephone or Fax In North America To contact Applied Biosystems Technical Support use the telephone or fax numbers given below To open a service call for other support needs or in case of an emergency dial 1 800 831 6844 and press 1 Product or Product Area Telephone Dial Fax Dial ABI Prism 3700 DNA Analyzer 1 800 831 6844 then press 8 1 650 638 5981 DNA Synthesis 1 800 831 6844 then press 21 1 650 638 5981 Fluorescent DNA Sequencing 1 800 831 6844 then press 22 1 650 638 5981 Fluorescent Fragment Analysis includes GeneScan applications 1 800 831 6844 then press 23 1 650 638 5981 Integrated Thermal Cyclers ABI PRism 877 and Catalyst 800 instruments 1 800 831 6844 then press 24 1 650 638 5981 ABI PRism 3100 Genetic Analyzer 1 800 831 6844 then press 26 1 650 638 5981 BioInformatics includes BioLIMS BioM
23. pressure of the argon tank The low pressure gauge should read about 60 psi Check the waste level Perform the proper setup for collection of trityls and or collection of trityls using a fraction collector Note 8 392 394 synthesizers equipped with the AutoAnalysis option can not perform Step a below unless special cycles are developed for fraction collection See Modifying cycles for Fraction Collection in Section 4 of the 392 394 Synthesizer manual Ver 2 00 soft ware 392 394 synthesizers not equipped with the AutoAnalysis option cannot perform Step b below a Ifyou are going to collect trityls choose Trityl collect First 2 and last 2 or Trityl collect All available as part of synthesis start pro cess and fill the fraction collector with 10 to 15 mL tubes to collect the Trityl cation released at each detritylation step This can be done using OligoNet Set up Synthesis view or at the synthesizer Start Synthesis option b Ifyou are going to monitor trityls you may want to specify the threshold value for the interrupt and the coupling interval at which to monitor This can be done through OligoNet using the Trityl Monitor view or at the synthesizer using the Monitor Synthesis option from the Main Menu Ensure that columns and collection vials if used have been installed June 2002 5 Using OligoNet 5 7 Applied Biosystems 5 8 Saving and Retrieving Sequences Cycles Procedure
24. slot but do not load on column e Sequence name entry field 3 6 3 Overview of Commands and Capabilities June 2002 Applied Biosystems e Sequence listing entry field allows codon entry and form keeps track of total bases entered for the sequence as well as the number of A G C and T entries When all or part of the sequence in the Sequence entry field is selected using the Read Selection command Edit menu will read the sequence aloud in a digitized voice This feature is useful for proofreading sequences Note Entries other than A G C or T are flagged by the A symbol when entered as the last character to warn that such an entry is unacceptable The last position in a sequence which corresponds to the 3 end of the sequence cannot be ambiguous Besides the four bases valid entries include 5 6 7 and 8 bottle posi tions on 8 base instruments single character IUB ambiguity codes and ambiguity characters indicated by parenthesis As soon as a paren thesis is entered the codon grouping of the sequence is disabled e Comments for synthesis to be performed When Order is sent to the synthesizer a comment is placed in this field with the date time and synthesizer to which the sequence was sent as well as the Column and Seq numbers Tm and G C fields under Comments box as soon as a sequence is entered or imported into the Sequence listing field OligoNet calcu lates values for these parameters T
25. that on a Model 394 a four column instrument sequences can be assigned to col umns 3 and 4 In this case the 1 1 notation will also be presented in these columns Note The example shown in Figure 4 8 is that for a Model 392 instru ment but this portion of the view will look the same for a Model 394 instrument when columns 3 and 4 are not used SPERE E BASES CYCLE coL 1 COL 2 coL 3 COL 4 a Insert Cycle Cycle 2 2 um CE v Delete Eurie Number of bases mooo Figure 4 8 Multiple Cycle Portion of Set Up Synthesis View The cycle which is initially listed on line 1 in this table is the cycle which was present in the upper Cycle entry field when you chose Multiple as your cycle choice Entries are made in the portion of the Synthesis Setup shown in Figure 4 8 as follows 1 Select the first synthesis cycle from the Cycle pop up menu 2 Enter the number of bases which are to be added by the first cycle in the Number of bases entry field As soon as you type a value in this field you will see the same value entered under each selected column if all sequences contain at least that many bases For example if you entered 10 the column designa tions would each be 1 10 in this case If there were only five bases in the sequence assigned to column 2 the column designation would be 1 5 indicating that five bases will be synthesized using cycle 1 3 Click Insert
26. the base monitored the third column displays the average stepwise yield and the fourth column displays the overall yield The interrupt provided by the AutoAnalysis option is a cycle interrupt like that provided by the Interrupt command initiated from the Synthesizer menu of OligoNet or from the synthesizer and stops synthesis on all col umns simultaneously This is the only control that OligoNet has of a synthe sizer s valve operation When you resume synthesis after an AutoAnalysis interrupt however synthesis must be resumed individually for each column 4 Editing the Synthesizer Window June 2002 Applied Biosystems June 2002 An AutoAnalysis interrupt works in this way since only the user can deter mine whether the interrupt applies to all columns because of an empty bot tle or whether only a single column is affected for another reason Raw Data View Advanced Feature Besides the regular monitor view shown in Figure 4 13 an alternative view is available which shows the raw data for a particular base addition rather than the base designation A G C T The raw data num bers allow more diagnostic information for example to detect very low sig nal To bring up the alternative view do the following 1 Place the cursor directly on the Column designation button 2 Hold down the Option key and then click the mouse This will change the view so that it appears like Figure 4 14 The display shown in this display
27. the cycle to be used for the set of bases 3 Place the insertion point in the Number of bases entry field and enter the number of bases to be added by the first cycle If you enter 4 as needed for the example you ll see 1 4 bases 1 through 4 appear under the selected column in the table 4 Click the Insert Cycle button and repeat steps 2 and 3 for each line to be added to the table Note After you have entered a cycle line you can always edit it by se lecting it again The Delete Cycle button allows you to delete a cy cle line if you change your mind 5 Using OligoNet 5 11 Applied Biosystems 5 12 Sharing a Synthesizer with Many Macintoshes OligoNet supports the single user model This means that one user at a time should be logged onto a single synthesizer to avoid the potential of interrupting another person s work in progress Several users can share a synthesizer with multiple OligoNet nodes however if each individual agrees to aset schedule of sequential use and agrees to maintain a standard default Synthesizer file in addition to any custom Synthesizer file needed for the individual s work Caution We strongly recommend that two Macintoshes not be con nected to a synthesizer at the same time because when either tries to write to the synthesizer the results are unpre dictable and may be undesirable The Synthesizer window on the monitoring Macintosh may not be updated to ref
28. the pop up menu Multiple synthesis cycles When the Multiple box is checked multi ple cycles may be used for a single synthesis The single Cycle entry field is disabled and a new area becomes available for entry of multiple cycles and the number of bases to be added by each cycle as shown in Figure 3 3 See Performing Multiple Cycle Synthesis in Section 5 for more information on this type of synthesis Multiple cycles ingert pigie Oelele ugis BASES CYCLE Bases numbered fram 3 end Number of bases fi is Figure 3 3 Multiple Cycle Entry Area of Order Note Entries containing multiple cycles require that the number of bases to be added by each cycle be entered For multiple cycle listing base entries are counted from the 3 end even though they are entered from the 5 direction in the Edit View When the Syn thesis Order form is completed a copy can be printed for a hard copy record and the form should then be saved Name of End Procedure to be used for synthesis procedure name can be typed in If an instrument is selected the pop up menu for the End procedure field allows selection of an End procedure available on the chosen synthesizer Designation of DMT group on or off DMT group at 5 end When the Synthesis Order form is completed a hard copy can be printed and the Sequence Order can be transmitted to the synthesizer for synthesis 3 Overview of Commands and Cap
29. to enter numbers and the Prev and Next keys are used to toggle through the alphabet Change Name The Change Name command is used to assign a name to your synthesizer The name you assign will be used to open communication with the synthe sizer Open Synthesizer command and will be used to identify both Synthe sizer window s and Synthesizer Orders The name field in the dialog box will accept up to 32 characters Change synthesizer s name Synthesizer 1 Cancel OK Figure 3 22 The Change Name Dialog Box 3 22 3 Overview of Commands and Capabilities June 2002 Applied Biosystems Order Menu Order Send Order to Synthesizer Copy from Synthesizer Send All Orders Figure 3 23 The Order Menu The Order menu is used to send Synthesis Orders to a synthesizer Send Order to Synthesizer command Send All Orders command and receive Synthesis setups back from the synthesizer Copy from Synthesizer com mand See Organizing and Processing Synthesis Order Files in Section 5 for more information on handling Synthesis Orders Send Order to Synthesizer Send All Orders Send Order to Synthesizer The Send Order to Synthesizer command causes the sequence and setup choices to be transferred to the Synthesizer window and then causes all changes in the Synthesizer window to be sent to the synthesizer After using this command you still need to use the Prepare to Start command to initiate the starting proc
30. to send Synthesis Order files for completed synthesis to the customer along with the oligo and let the customer maintain the files In this case the customer would provide the Synthesis Order file rather than a hard copy of the Synthesis Order when requesting a repeat order e Synthesis Order files can also be read by database programs such as Ex cel FileMaker Pro etc which allows a customer to maintain a custom database of orders rather than the raw files Applied Biosystems can not offer technical support for such customer developed databases June 2002 5 Using OligoNet 5 17 Applied Biosystems June 2002 Appendix I Nucleotide Ambiguity Conventions Several less commonly used nucleotide ambiguity conventions and their translations are listed below The default convention for OligoNet is IUB GCG The other conventions Staden Sanger and Stanford are provided to enable you to translate these types of sequences Meaning IUB GCG Staden Sanger Stanford A A A A A C C C C C G G G G G T T U T T T U AC M 5 5 J AG R R R R AT W 7 7 L CG S 8 8 M CT Y Y Y Y GT K 6 6 K AGC V ACT H AGT D CGT B AGTC X N X The International Union of Biochemistry IUB convention will correctly translate DNA sequences received from NIH GENBANK University of Wis consin Genetic Computing Group UWGCG European Molecular Biology Laboratory EMBL and National Biomedical Research Foundatio
31. under the first heading below the Edit menu contains commands for the fol lowing tasks e Reading a sequence out loud for an existing sequence or portion of a sequence in the Edit Sequence view e Finding or finding again a particular sequence of bases in the Edit Sequence view e Replacing or replacing again a particular sequence of bases with a designated sequence of bases in the Edit Sequence view e Turning on or off the production of sounds to indicate communication between OligoNet and the synthesizer Note Besides choosing a command from a menu using the mouse it is possible to choose a command by using the Command key equiv alent listed on the menu by the command For example choose the Read Selection command by holding down the 38 key and then pressing K June 2002 3 Overview of Commands and Capabilities 3 15 Applied Biosystems 3 16 Undo Redo Can t Undo Cut Copy Paste Clear Select All Show Clipboard The Edit menu commands listed above are standard Macintosh commands for modifying files including cut and paste operations and the ability to undo editing operations These commands will not be discussed further If you need more information on their use see your Apple System Software User s Guide Read Selection K This command is used to check a sequence entered into the Edit Sequence view of a Synthesizer window or a Synthesizer file while examining a hard copy of the sequence allowin
32. 002 O OligoNet basic functions 1 3 ending a session 2 10 entry point and mass storage 1 3 installation and start up 2 1 kit number 2 3 menu bar 2 8 synthesizer software requirements 2 3 OligoNet Order reference 3 5 Open command 3 9 Open Synthesizer command 3 10 Order menu 3 23 Order menu commands Copy from Synthesizer 3 25 Send to Synthesizer 3 23 P password permission setting 3 21 Power Fail View 4 43 Prepare to Start command 3 18 pre synthesis list of tasks 5 7 printing 2 9 procedures creating 5 6 program symbols special 1 6 pulse duration relay 4 46 R read outloud 3 16 Read Selection command 3 16 Read write permission 3 22 Relay Configuration view 4 45 Replace command 3 17 Replace Same command 3 17 reverse complement button 4 8 S Save a Copy In command 3 12 Send All Orders command 3 24 Send Copy to Synthesizer command 3 13 Send Order to Synthesizer command 3 23 Send to Synthesizer command 3 11 sequence import export 3 14 sequence editing 4 8 Sequence field 3 6 sequence files vertical line in name 4 6 with Mac suffix 45 sequence naming 46 Sequence pop up view 4 5 sequence slots in synthesizer default 3 24 sequences creating a new 4 6 importing 46 naming 46 reverse complement 4 8 rules for entering 46 transmitting to synthesizer 1 3 use of Copy from button 4 7 service information 2 10 Set Time Date View 4 44 setting up a synthesis multiple cycles 4 12 single cyle 4 9 sing
33. 1 Column 2 On 142 18 to Waste 64 Block Flush 1 Block Vent 111 Tet to Waste 58 B Tet to Column 33 Tet to Column 34 B Tet to Column 33 Push to Column 43 Column 2 Off TIME ACTIVE AGCTS678 AGCTS678 AGCTS678 AGCTS678 AGCTS678 26 gt Column 3 On Safe Yes No fictive 4640708 OeoOe amp Ot OF OF 18 to Waste 3 Block Flush 4 AGCTS678 Block Vent AGCTS678 Figure 4 24 Edit Cycle View after Selection of Cycle Step c Click Delete to delete the selected step or steps 10 Repeat steps 8 and 9 as needed to add or delete steps to complete edit ing of the cycle 11 Save the Synthesizer window to save your new cycle You will see the new cycle you created the next time you pull down the Cycle menus in the Edit Cycle view of your Sequence window Note The new cycle you create will not be available in the synthesizer until you use either the Send to Synthesizer or Prepare to Start commands 4 Editing the Synthesizer Window 429 Applied Biosystems 4 30 Edit Begin Procedure View The begin procedure provided with the 392 394 synthesizer ABI Begin is a phosphoramidite purge procedure that fills all phosphoramidite and tet razole delivery lines from the reservoir to the reagent valve block with fresh reagent The begin procedure should be used prior to beginning a syn thesis when the instrument has been idle for more than 12 hours more than 6 hours in humid environments or if
34. 18 Mac 29 Cyc19 Mac 30 Cyc20 Mac 31 Cyc21 Mac 32 Cyc22 Mac As you can see by examining the Cycle pop up menu the application can store up to 32 cycles The first 10 cycles are permanent non programmable cycles for use in the synthesizer These cycles can be used as is or they can be copied into one of the 22 locations labeled user or Mac and then ed ited to create custom cycles Note Two different sets of cycles are obtained from 392 394 synthesiz ers The set available in synthesizers equipped with AutoAnalysis supports trityl monitoring but does not support fraction collection The other set is available from synthesizers which do not have AutoAnalysis The second set supports fraction collection but does not support trityl monitoring during the run See Monitoring Cycles for Fraction Collection in Section 4 of the 392 394 Synthe sizer Manual Ver 2 00 for information needed to modify cycles in AutoAnalysis option equipped instruments so fraction collection can be done 4 Editing the Synthesizer Window June 2002 Applied Biosystems June 2002 Both user and Mac cycles are for user created cycles Cycles labeled user may contain cycles entered at the synthesizer but can be edited in OligoNet Creating a New Cycle Editing Existing User Cycles Since the first 10 cycles are non programmable the first step in either creat ing or editing a cycle is to choose one of the user cy
35. 3 Overview of Commands and Capabilities June 2002 Applied Biosystems June 2002 If a message like this appears refer to your 392 394 synthesizer manual under Starting a Synthesis in Section 2 and follow the instructions pre sented below the figure which looks like Figure 3 16 If your reagents are present in sufficient quantity for synthesis or if the bottle alarms are turned off you will be presented with a display like Figure 3 16 Col 1 Install A Column Set Run ID gt 2 Intall T Column Base Source gt 3 Not Selected Print gt 4 Not Selected Start gt Figure 3 16 Start Synthesis Display The Figure 3 16 display indicates that your synthesizer is ready for synthesis using the downloaded synthesis setup if you have the proper columns installed as prompted on the display The column selections presented in this display should reflect your synthesis setup At this point press Start This will continue the start process by presenting a display like that shown in Figure 3 17 Check Waste Bottle Cancel Start gt Trityl collect All gt No gt Execute ABI Begin Yes gt Figure 3 17 Trityl and Begin Procedure Display Both the trityl collect setting and the begin procedure set with your synthesis setup should be represented on this display If you change your mind at this point you can use this display to change the trityl collect and begin proce dure selections This is done by placing the
36. 4 27 two types of bottle change procedures are available for each of the 16 bottles anon programmable procedure and a user defined procedure The non programmable procedure is named Botxx ABI where xx specifies the bottle to be changed The user defined procedures only exist if one has been created in either the synthesizer or in OligoNet and is named Botxx user In addition to these five Applied Biosystems auto dilution procedures are available as well as locations for 10 user specified auto dilute procedures The five Applied Biosystems auto dilution procedures are labeled Auto 1 0g Auto 0 5g Auto 0 25g Auto 2 0g and Auto 2g Double and are 4 Editing the Synthesizer Window June 2002 Applied Biosystems provided to automatically dilute bottles containing these quantities of B cya noethyl and RNA phosphoramidites Again the user supplied procedures only exist if one has been created in either the synthesizer or in OligoNet Two procedures are provided in this subsection The first provides general instructions for creating user supplied bottle change procedures from regu lar Applied Biosystems supplied procedures The second procedure pro vides special instructions required for creating usersupplied auto dilute procedures General Instructions for Creating User defined Bottle Change Procedures Since the Applied Biosystems bottle change procedures are non program mable the first s
37. Cycle to insert a new line and repeat steps 1 and 2 for each new line to be added For more information including an example on Multiple Cycle Synthesis see Performing Multiple Cycle Synthesis in Section 5 4 12 4 Editing the Synthesizer Window June 2002 Applied Biosystems June 2002 Monitor Synthesis View 3 The Monitor Synthesis view is used for monitoring the progress of the syn thesis on all columns two for the 392 and four for the 394 The view allows you to monitor each column individually by viewing the steps in the cycle and the sequence for that column Monitoring the synthesis allows you to determine that synthesis is proceeding properly S i TRITUI MANIN EEH lt lt Synthesizer Synthesizer 1 2 Choose function Monitor Synthesis 323 v Column 1 Column 2 Column 3 Column 4 2UMAA Inactive Inactive 2UMAA Step Sten Step Step Function Function Block Flush Block Flush TCAIICAG TCT GAT CTC GAT Figure 4 9 Monitor Synthesis View with One Active Column Base 16 16 ASWY 99 m GAT CTC GAT H alle Note The fact that synthesis is in progress is indicated in the view by the annotation Synthesizing Also notice that two columns are cur rently inactive Explanation of Monitor Synthesis View Fields As shown in Figure 4 9 example Monitor Synthesis View has two columns active This view shows the following information for the synthesis in progress e Cycl
38. FNCTION or User XX as the name of the function and print None for Valves Choose printing format Tent This function only All user functions O Window image E Allow for 3 hole punch Cancel OK Figure 4 32 Function Print Dialog Box June 2002 4 Editing the Synthesizer Window 4 43 Applied Biosystems 4 44 Power Fail View The Power Fail view shown in Figure 4 33 is used to set the maximum power failure time for functions designated as non safe steps and review the power failure history of the synthesizer The value which can be set ranges from 0 to 999 minutes in one minute increments If you enter 999 as the maximum power fail time the synthesis will always continue when the main power returns regardless of the duration of the outage Sa synthesizer Synthesizer 1 Mau safe power fail time minutes Power Failure History Monday February 24 1992 11 24 22 AM Figure 4 33 Power Fail View 4 Editing the Synthesizer Window Choose function Power Fail v June 2002 Applied Biosystems Set Time Date Format View The Set Time Date Format view is used to set the format for time and dates used in the synthesizer Time and date values are used to record when a power failure occurred and to provide dates on printouts on the printer attached to the synthesizer The actual Time and Date is set in the synthe sizer When the Time Date view is
39. Follow the voice instructions to order the documents you want Note There is a limit of five documents per request through the Internet for fax or e mail delivery a Access the Applied Biosystems Technical Support Web site at http Awww appliedbiosystems com techsupp b Under Resource Libraries click the type of document you want c Enter or select the requested information in the displayed form then click Search d In the displayed search results select a check box for the method of delivery for each document that matches your criteria then click Deliver Selected Documents Now or click the PDF icon for the document to download it immediately e Fill in the information form if you have not previously done so then click Deliver Selected Documents Now to submit your order Note There is a limit of five documents per request for fax delivery but no limit on the number of documents you can order for e mail delivery 1 Introduction 1 11 Applied Biosystems June 2002 2 Installation and Start Up This section provides you with installation and start up information includ ing instructions on testing your installation as well as service and trouble shooting information to aid you if you have problems with installation In addition the section provides you with a checklist for operation once you become familiar with the application Contents Checking Out the OligoNet Kit Synthesizer Requ
40. Gas CUB CUB CUB PAWWNN Figure 4 31 The Initial Edit Function View The Edit Function view provides three scrollable lists One list labeled FUNC TION LIST indicates which function is currently displayed A second list labeled VALVES OPEN FOR FUNCTION contains a valve listing for the current function The third list headed by the Add and Remove buttons is used to edit user lists and contains a list of valves available for creating a new func tion June 2002 4 Editing the Synthesizer Window 4 41 Applied Biosystems 4 42 Note The Advance FC Advance Fraction Collector and Relay1 func tions control contact closures between the same pair of terminals on the rear of the synthesizer labeled FC Advance FC and Relay 1 Pulse are identical and the pulse duration for both is set using the Relay 1 settings in the Relay Configuration view Procedure for Defining a User Function l Select one of the user function locations either by selecting it from the Function List or by entering the function number in the Function num ber entry field located in the upper right of the view Enter a number in the Function number entry field by clicking in the field dragging to select the existing number and typing in the desired function number Note When you list various functions you will see several abbreviations at the beginning of valve step names CVB stands for Column Valve Block FRVB stands fo
41. III 1 Applied Biosystems The following information can be entered on a Synthesis Order form in Oli goNet Order Customer a field to identify the requestor of the oligo Run ID entry field Two blank fields labeled 1 and 2 each of these fields provides room for entries of your choice For example these two fields could be used for Account No and for customer s phone number Sequence name entry field Sequence listing entry field allows nucleotide entry and form keeps track of total bases entered for the sequence as well as the number of A G C and T entries Note Entries other than A G C or T are flagged by the A symbol when entered as the last character to warn that such an entry is unacceptable The last position in a sequence which corresponds to the 3 end of the sequence cannot be ambiguous The Read Selection command is not available in OligoNet Order Besides the four bases valid entries include 5 6 7 and 8 bottle posi tions on 8 base instruments single character IUB ambiguity codes and ambiguity characters indicated by parenthesis As soon as a paren thesis is entered the codon grouping of the sequence is disabled Comments for synthesis to be performed Everything that an operator will need to complete the order in Oligo Net should be entered into this field Since the user may not know the exact name and spelling of Cycles and End Procedures to be used for sy
42. OligoNet 392 394 Synthesizer Application Software User s Manual BS 28 Be Plied ms Copyright 2002 Applied Biosystems All rights reserved For Research Use Only Not for use in diagnostic procedures Applied Biosystems June 2002 Contents Introduction General About This Manual Organization How to Get Started Quickly Special Text Usage Special Program Symbols How to Get Help Contacting Technical Support To Contact Technical Support by E Mail Hours for Telephone Technical Support To Contact Technical Support by Telephone or Fax North America Outside North America To Reach Technical Support Through the Internet To Obtain Documents on Demand Installation and Start Up Contents of OligoNet Kit Synthesizer Requirements Macintosh Requirements Network Installation Software Installation Program Cartridge Installation Testing Your Installation Beginning an OligoNet Session Setting Up Communication Initiating Communication Printing Ending an OligoNet Session Service Information Troubleshooting Service Information Checklist of OligoNet Synthesizer Use 1 3 1 4 1 4 1 5 1 5 1 6 1 6 1 6 1 6 1 7 1 7 1 7 1 8 1 9 1 10 2 3 2 3 2 3 2 4 2 4 2 5 2 6 2 6 2 7 2 9 2 10 2 10 2 10 2 12 Applied Biosystems 3 Overview of Commands and Capabilities File Menu New Synthesis Order Open Open Synthesizer Send to Synthesizer Save a Copy In Send Copy to Synthesizer Import Export Edi
43. Same G The Find Same command will find the second and other occurrences of the pattern specified by the previous Find command Replace 3 R The Replace command is used either after using the Find command or by itself to substitute a designated pattern Clicking the command brings up the dialog box shown in Figure 3 13 Find what string Replace with what string Replace All Find Cancel Figure 3 13 The Replace Dialog Box As you can see upon examining the dialog box this command allows you to find a designated pattern and then replace it with a second designated pat tern either the next single occurrence after the insertion point or all such patterns in the sequence Replace Same T The Replace Same command is used after the Replace command when the first command was used to replace a single occurrence of the designated pat tern Use Sounds The Use Sounds command is used to turn off or on the built in sounds the application produces to indicate communication over the network Each time the command is used it switches the sound function from on to off or off to on 3 Overview of Commands and Capabilities 3 17 Applied Biosystems The sounds are useful to verify that communication is occurring and you may find them entertaining but once OligoNet is running it is more effi cient to turn off the sounds since communication is noticeably faster with sound off Synthesizer
44. abilities June 2002 Applied Biosystems using the Send Order to Synthesizer command The Synthesis Order can also be saved to a file Note If more than one Synthesis Order is sent to a single synthesizer for example 4 orders one for each of the 4 columns in a 394 the cycle used for each column must be the same If not the last cycle sent will be the one used Open 3 O The Open command is used to open a saved Synthesis Order or a saved Syn thesizer file Choosing the command from the File menu brings up a file dia log box like that shown in Figure 3 4 Select the file you want to open and then click Open O Example O Examsynord Figure 3 4 Standard File Dialog Box June 2002 3 Overview of Commands and Capabilities 3 9 Applied Biosystems 3 10 Open Synthesizer The Open Synthesizer command is used to open communication with a syn thesizer on the network Choosing the command from the File menu brings up the Open Synthesizer Dialog box The dialog box may be like that shown in Figure 3 5 or may have a second list which allows you to select the zone on a multi zone network Open Synthesizer select a Synthesizer Synthesizer 1 Cancel Figure 3 5 The Open Synthesizer Dialog Box Note With the default application memory setting of 1024K 5 Synthe sizer Windows can be opened at one time If you need to open more windows see the instructions in Appendix II When you selec
45. ame is used Creating a New Sequence Importing a Sequence After placing the insertion point in the sequence entry field you can create a new sequence by typing in one of your own by cutting and pasting from another application or by importing from a file Once you have entered a sequence one or both of the other two buttons may become available The Reverse Complement button becomes available for both 5 base and 8 base position instruments allowing you to change the sequence into its Reverse Complement The AGCT lt gt 5678 button becomes available on 8 base position instruments allowing you to change the sequence from base calls to bottle positions To avoid error messages when entering sequences you must observe the fol lowing rules 3 End The 3 end of the sequence which corresponds to the column to be used in DNA synthesis must be an A G C or T Numbers mixed bases parentheses or ambiguity letters from translation table are not allowed Legal Bases The following single base entries are allowed A G C T 5 five or eight base 392 or 394 6 7 8 eight base 392 or 394 or letters encoded in the translation table see Appendix I Mixed Bases Parentheses may be used to designate a mixed base site A max imum of four bases may be included inside the parentheses Once you use a parentheses the codon grouping of characters is disabled and all entry is done sequentially with no spacing between groups Alternativ
46. ced by word processors The Export Sequence command is used to write a plain text file containing only the sequence for use as input to all programs that accept plain text in put Note The Export Sequence command is probably not the best way of maintaining a library of sequences since it provides just the se quence text It is better to use a Synthesis Order file to store each sequence since you are provided with fields for comments cycle or cycles and end procedure as well as other information For information on how to use Synthesis Orders to process sequences see Organizing and Archiving Your Files later in this section 5 Using OligoNet June 2002 Applied Biosystems June 2002 Exporting and Importing Cycles and Procedures Although the Export commands can be used to save a single cycle or proce dure and then read them back into a Synthesizer window most people will not have to use this feature since the synthesizer itself has room for up to 22 user cycles as well as for additional procedures These commands are useful of course if you find the need for storing and using more custom cycles or procedures than allowed by the synthesizer The Export and Import commands are useful in sending or receiving a sin gle cycle or procedure from a colleague using a disk or over a network in large laboratory settings If you have two synthesizers and want to copy a cycle or procedure from one to the other these co
47. cedure Edit Bottle Procedure View General Instructions for Creating User defined Bottle Change Procedures Special Instructions for Creating Auto Dilute Procedures Edit Shutdown Procedure View 4 Editing the Synthesizer Window 4 3 4 4 4 5 4 6 4 6 4 7 4 8 4 9 4 9 4 12 4 13 4 13 4 14 4 16 4 18 4 18 4 19 4 20 4 20 4 21 4 22 4 23 4 30 4 31 4 32 4 33 4 34 4 35 4 36 4 39 4 1 Applied Biosystems 4 2 Edit Function View 4 4 General Description of View 4 4 Procedure for Defining a User Function 4 42 Printing Functions 4 43 Power Fail View 4 44 Set Time Date Format View 4 45 Relay Configuration View 4 46 4 Editing the Synthesizer Window June 2002 Applied Biosystems June 2002 About the Synthesizer Window The Synthesizer window is the pivotal element in OligoNet and allows you to perform on your Macintosh most of the entry and editing functions nor mally done at the synthesizer This window allows you to do a number of tasks and includes the ability to create or edit off synthesizer the sequences cycles functions and procedures needed to perform synthesis This frees the synthesizer to perform synthesis The Synthesizer window also enables you to monitor one or more synthesiz ers in real time Because a Synthesizer window can be opened for each syn thesizer on a network this window is a key element enabling OligoNet to expedite the process of synthesis on a group of synthesizers opera
48. ch sequence and the instructions needed to synthesize it are con tained in a Synthesis Order using OligoNet can be thought of as processing Synthesis Orders The steps listed below or a similar set of steps should be followed for each sequence to be synthesized Note Before proceeding make a backup copy of the Synthesizer Win dow This is done upon first establishing communication with a synthesizer and backs up the contents of the synthesizer produc ing an icon for the file like that entitled Synth Copy in Figure 5 5 The contents of this file can be used later to restore your synthe sizer to its original configuration 1 Start OligoNet and prepare the Synthesis Order a C Create and fill out a Synthesizer Order for the sequence to be syn thesized when no order previous exists assigning the Run ID number to uniquely identify both the requestor and the particular order number Alternatively open a Synthesis Order created by a customer and check to see that the proper information is entered for the order Check by asking the following questions Is the identity of the customer clearly identified Is the Run ID clearly stated so that the customer can be identified without re opening the file Is the sequence entered correctly using OligoNet conventions Are the cycle and end procedure appropriate for the sequence re quested You will need to assign the correct cycle and end proce dure if the
49. cles from the Cycle pop up menu either an empty cycle or one you have already created The proce dure below is more detailed than that provided for the other Edit views to provide a tutorial for this type of edit view Note The most likely changes you will make to a cycle are 1 insert one or more new steps 2 delete one or more steps and 3 change the time or active base status set for an existing procedure step Creating a new cycle usually is done as follows 1 Choose a cycle from the Cycle pop up menu An example of an empty cycle is shown in Figure 417 Note In the present example we chose an empty user cycle Synthesizer Synthesizer EN Choose function Edit Cycle v Cycle 11 CycO1 Copy from y Cyc01 user wv hame Time a8 Functien 386 Phos Prep Cap Prep Hait Interrupt Start Detrityl J3 egin Safe es ONG fictive 640708 Oreos Ot 0 go Figure 4 17 An Empty User Cycle 2 Type inaname of your choice if you have opened an existing user cycle as a template and then proceed to step 5 If you are starting with an 4 Editing the Synthesizer Window 423 Applied Biosystems Go a o a empty cycle as done in this example proceed to step 3 without typing a name You can change the cycle name to one of your choice just by typing because the cycle comes up with the Name field highlighted for entry 3 Click the Copy from button to present th
50. ct the cycle s begin procedure and trityls collection option to be used by all columns 5 Optional Set Bottle Alarm choices as described in Bottle Alarms View in Section 4 6 Send synthesis information to the Synthesizer as described in Section 3 using the commands listed below a When using Synthesis Orders use the Send Order to Synthesizer command and then use the Prepare to Start command The Send Order to Synthesizer command sends the Synthesis Order to the synthesizer window and then sends all information in the synthesizer window on to the synthesizer The Prepare to Start command initiates the starting process on the instrument b When not using Synthesis Orders use the Prepare to Start com mand to send all synthesis information including sequences to the synthesizer and to initiate the starting process on the instrument 7 Set up instrument with reagents and columns etc Start synthesizer and specify whether to use begin procedure Monitor progress of synthesis using Monitor Synthesis view on the Mac intosh 2 12 2 Installation and Start Up June 2002 Applied Biosystems June 2002 3 Overview of Commands and Capabilities This section provides information on the command menus available from the Menu bar A general knowledge of these commands is required to oper ate OligoNet In addition under New Synthesis Order you will find infor mation about the Synthesis Order form Contents
51. d as a default 5 Save the Synthesizer window to save the bottle change procedure you just created Your new procedure will be listed the next time you use the Procedure pop up menu in the Edit Shutdown Procedure view 4 40 4 Editing the Synthesizer Window June 2002 Applied Biosystems Edit Function View General Description of View The Edit Function view is used to create and edit user functions and view the listings of function steps Applied Biosystems defines two types of functions for use in the synthesizer system functions and user functions System func tions are dedicated functions provided with the synthesizer They are perma nent and cannot be edited User functions functions 200 224 can be both viewed and edited Each function simultaneously opens a series of valves to perform a defined action during synthesis when executed from a cycle The valves opened as part of a function remain open for the time specified in the cycle step or oth erwise as controlled by software When first selected the Edit Function view looks like Figure 4 31 EU EE synthesizer Synthe siz er 1 SEN Function fi Trityl Flush Flush to Waste Flush to Colect VALVES OPEN FOR FUNCTION gt RUB to HP Gas RRYB to RUB FRUB to Waste CUB to Gas CUB to Waste CUB to Gas CUB to Waste CUB to Gas to Waste to Gas to Waste a CHOOSE VALVE TO ADD 00 gt CUB Vial 1 Trityl Col Waste Gas Vial 2 Trityl Col Waste
52. d for Creating a New Cycle Editing Existing User Cycles on page 4 23 Note The most likely changes you will make to a begin procedure are 1 insert one or more new steps 2 delete one or more steps and 3 change the time or active base status set for an existing procedure step The general procedure is as follows 1 Select an empty user Begin Procedure and then copy ABI Begin 2 Insert any additional steps needed to add functions to your procedure This is done by selecting the step above the desired new step inserting a blank step line selecting the desired function and changing the time for the new step if desired Note Time values can not be entered for some steps steps with blank values Except for the possible exception of the Time field and Active field all of the controls which are used to modify a begin procedure Insert Delete Time Function and Safe are open for entry as soon as a step is selected 3 Delete any existing step that you don t want 4 Type in a new name if you want a name other than ABI Begin copy 5 Save the Synthesizer window to save the begin procedure you just cre ated Your new procedure will be listed the next time you use the Procedure pop up menu in the Edit Begin Procedure view Note The new begin procedure will not reside in the synthesizer until it is sent there either by the Send to Synthesizer or Prepare to Start commands June 2002 4 Editing the Synthesiz
53. dit Cycle v Cycle 11 1 0 CE Copy from y 1 0 um CE copy Belete Name AEAU STEP FUNCTION NAME NUM TIME ACTIVE SAFE 1 gt Begin 2 18 to Waste 64 3 18 to Column 42 10 AGCTS673 4 Reverse Flush 2 AGCTS678 5 Block Flush 1 f AGCTS673 6 gt Phos Prep 101 7 Column 1 On 8 Block Vent 111 AGCTS678 Time g g Functian gg Phos Prep K Cap Prep Wait Interrupt Start Detrityl Soy in End Safe es ONG gge 160708 Oe Ot Of OF O8 9 Tet to Waste 58 10 B Tet to Column j AGCT5678 11 gt Tet to Column 34 AGCTS678 12 gt B Tet to Column g AGCTS678 13 Push to Column 43 14 gt Column 1 Off 15 gt Column 2 On Figure 4 20 Edit Cycle View Containing Copied Cycle 5 Type in a new name if you desire a name other than that assigned by the application The name present in the Name field was assigned by the application but is highlighted to allow you to enter another name by immediately typing 6 Determine the type of change you want to make to the cycle The most likely changes to a cycle include changing the time or active bases for an existing step proceed with step 7 inserting one or more new steps proceed with step 8 or deleting one or more steps proceed with step 9 7 To change the time for an existing step do the following a Select the desired step This will change the Edit Cycle view as shown in Figure 4 21 4 Editing the Synthesizer Window 4 25 App
54. duce custom cycles to perform synthe sis and then edit such cycles after you create them The Edit Cycle view ini tially appears as shown in the left side of Figure 4 16 The Cycle pop up menu shown in the right side of Figure 4 16 is used to select cycle or select a user cycle location for creating a new cycle Synthesizer Synthesizer 1 Cycle 1 40 nM CE vj Eppu from jasert Beiere Name 40 nM CE STEP FUNCTION NAME NUM TIME ACTIVE SAFE fime G gt Begin 18 to Waste gt 18 to Column d AGCT5678 Reverse Flush A AGCTS673 Block Flush A AGCT5678 Phos Prep Column 1 On Block Vent F AGCTS673 Tet to Column AGCTS673 B Tet to Column AGCTS673 Tet to Column AGCTS673 Flush to Waste 5678 18 to Haste g Block Flush AGCTS678 Column 1 Off Fane than 186 Phos Prep Cap Prep Wait Interrupt Start Detrityl Sayin Erd Safe es O Na fictive 160708 Oeot ot OF go Figure 4 16 The Initial Edit Cycle View an existing P D40 nM CE 2 2 um CE 3 1 0 um CE 4 10 um CE 5 2 um RNA 6 1 0 um RNA 7 10 um RNA 8 2um ulfur 9 Jum Sulfur 10 10umSulfur 11 Cyc01 user 12 CycO2 user 13 CycO3 user 14 CycO4 user 15 CycO5 user 16 Cyc06 user 17 CycO user 18 CycO8 user 19 CycO9 user 20 Cyc10 user 21 Cycli Mac 22 Cycl2 Mac 23 Cyc13 Mac 24 Cycl4 Mac 25 Cyc15 Mac 26 Cycl Mac 27 Cyci Mac 28 Cyc
55. e comple ment of your sequence where the complementary sequence is generated in the opposite configuration In other words the second strand of a duplex is created from the first Using AGCT lt gt 5678 changes the letter bases to their numbered base equivalents All A s are changed to 5 all G s to 6 all C s to 7 and all T s to 8 and vice versa After using this option you will notice that the 3 end base will still be called as A T C or G Note The AGCT lt gt 5678 function is only available on synthesizers with eight base positions Changing your sequence to numbered bases is one way of doing synthesis on base positions 5 through 8 using the default original bottle assignment for base sources See Base Source View on page 4 20 for more information 4 Editing the Synthesizer Window June 2002 Applied Biosystems June 2002 Set Up Synthesis View 2 2S synthesizer synthesizer 1 Choose function Edit Sequence 1 v Sequence 1 Seq01 v Name SeqO1 Clear Length 0 A 0 6 0 c 0 T 0 5 0 6 0 7 0 8 0 ne eee eats eee Rey Complement B a ob es REET S878 pees Copy from Figure 4 5 The Initial Set Up Synthesis View fo lt 3 Da Multiple 1 40 nM CE 2 2 um CE 3 1 0 um CE 4 10 um CE 5 2 um RNA 6 1 0 um RNA 7 10 um RNA 8 2umSulfur 9 Jum Sulfur 10 10umSulfur 11 CycOl user 12 CycO2 user 13 CycO3 user 14
56. e contact the Applied Biosystems Technical Support Group between 6 30 a m and 5 30 p m Pacific Standard Time except weekends and holidays or your local AB sales office whose phone numbers are listed on pages 1 8 through 1 10 1 Introduction 1 3 Applied Biosystems About This Manual For those who want to get started right away procedures can be found quickly by using the table of contents index and checklists Read How to Get Started Quickly in this section For those who want to learn by reading start by reading Section 5 and then read portions of Sections 3 and 4 as necessary in addition to the operational checklists The three checklists provided are referenced under How to Get Started Quickly Organization Section 1 Introduction contains an overview of the capabilities of OligoNet and information on how to use this manual and get help at Applied Biosystems Section 2 Installation and Start Up provides you with the information needed to install the application on your Macintosh and interconnect it and your synthesizer by network Also provided are two checklists one for troubleshooting installation problems and one which outlines the main activities involved in using OligoNet with your synthesizer e Section 3 Overview of Commands and Capabilities provides information on all the commands available from the Menu bar e Section 4 Editing the Synthesis Window describes how to perform all the editing requir
57. e dialog box shown in Figure 4 18 Copy steps from Synthesizer 1 40 nM CE Synthesizer 1 2 um CE Synthesizer 1 1 0 um CE Synthesizer 1 10 um CE Synthesizer 1 2 um RNA Synthesizer 1 1 0 um RNA Synthesizer 1 10 um RNA Synthesizer 1 2um ulfur Synthesizer 1 lum Sulfur Synthesizer 1 10um ulfur Cancel OK Figure 4 18 The Copy From Dialog Box The dialog box shown in Figure 4 18 contains only non programmable cycles but will show any non empty cycles Note If you have more than one Synthesizer window open cycles from all open Synthesizers will be available for copying In copying a cycle be sure to consider whether a cycle is compat ible with the synthesizer you are using If you attempt to use the Copy from button to copy a cycle from a synthesizer with a dif ferent number of columns or bases you will be presented with a warning like that in Figure 4 19 This cycle or procedure is from a model with a different number of columns or bases Are you sure you want to copy it It may not work as desired _Cancel_ Figure 4 19 Incompatible Cycle Warning Box 4 24 4 Editing the Synthesizer Window June 2002 Applied Biosystems 4 Select the cycle you desire and click OK Synthesizer 1 1 0 um CE was chosen for the present example This will change the Edit Cycle view so it appears as shown in Figure 4 20 SUES synthesizer Synthesizer S5 Choose function E
58. e in progress listed below the column number e Begin and End procedures will also be listed in the Monitor view e Step currently in progress listed both by number and name e Time that step has been active The secs field for the example shows that the current step has been completed since 0 seconds are left out of 3 seconds The total time for each step is shown by the right hand figure and the left value decre ments during the progress of the step 4 Editing the Synthesizer Window 413 Applied Biosystems 4 14 e Current base being added In the present example 16 18 indicates that currently base number 16 out of 18 bases specified in the sequence is being synthesized e Average stepwise yield this parameter is indicated by the value pre sented after the ASwy field Besides the numerical information provided in the top two thirds of the col umn area a list of the bases added is provided in the scrollable window at the bottom You can scroll up and down to see all bases currently synthe sized Interrupting a Cycle at a Safe Step To immediately interrupt a synthesis in progress use the Interrupt com mand from the Synthesizer pop up menu This will interrupt synthesis at the synthesizer stopping the incrementing of step information in the view The new status of the synthesis is indicated by the caption Synthesizing chang ing to Interrupted This type of interrupt can be considered a cycle
59. ect the step above This may require scrolling to bring the desired step into view b Click the Insert button Note Notice that an empty step appears after the step selected in step 78a to indicate where the additional step will be placed Figure 4 22 4 26 4 Editing the Synthesizer Window June 2002 Applied Biosystems June 2002 synthesizer Synthesizer EN Choose function Edit Cycle v Cycle 11 1 0 um CE copy w Time Tet to Column S Tet to Column 10 to Collect 11 to Column 12 to Column Cap to Column Safe Yes No Active M6K7h 8 BJA HJG HCH HS Name 1 0 um CE copy FUNCTION NAME NUM TIME Begin 18 to Haste 18 to Column Reverse Flush Block Flush Phos Prep Column 1 On Block Vent Tet to Waste B Tet to Column Tet to Column B Tet to Column Push to Column Column 1 Off AGCTS673 AGCT5678 AGCT5678 AGCT5678 AGCTS673 AGCTS673 AGCTS678 Figure 4 22 Edit Cycle View after Use of Insert Select the function you desire to enter If you know the number you can type it into the Function field If you don t know the number select the function from the Function list to the left of the view You may have to scroll the function list to see the one of interest As soon as you select the function it will appear as shown in Figure 423 If desired change the time of delivery Time column If desired change the active status of one or m
60. ed in a Synthesis Window Section 5 Using OligoNet provides all the practical information needed to use OligoNet e Appendix I Nucleotide Ambiguity Conventions provides a table enabling you to translate several commonly used ambiguity conventions into the form used by OligoNet e Appendix II Resetting OligoNet Memory tells you how to reset the mem ory allocated to OligoNet when you want to open more than 5 Synthesis Windows e Appendix III Using OligoNet Order provides information you need to operate OligoNet Order a companion application provided with Oli goNet The OligoNet application is intended for use on a Macintosh which is easy to learn and fun to use For the purposes of this manual it is assumed that you have used a Macintosh application and already know how to use the mouse and select items from menus If you are new to the world of the Mac intosh refer to the Macintosh System Software User s Guide or the Owner s Guide for more information 1 Introduction June 2002 Applied Biosystems June 2002 How to Get Started Quickly OligoNet must be properly installed before beginning use To get started quickly 1 Scan through the bulleted list at the beginning of Section 5 and read portions of the section about which you may need background infor mation e g multiple cycle synthesis sharing a synthesizer processing Synthesis Orders A checklist is provided in Section 5 for processing Synthesi
61. edure pop up menu shown in the right side of Figure 4 26 If you create a new cycle you may need to create a new end procedure to use with it Both user and Mac end procedures are for user created procedures End procedures labeled user may contain procedures entered at the synthe sizer but can be edited in OligoNet End procedures labeled Mac are intended to be downloaded into locations reserved in the synthesizer 4 Editing the Synthesizer Window June 2002 Applied Biosystems Creating a New End Procedure Since the first five end procedures are non programmable the first step in either creating or editing a procedure is to choose one of the empty user procedures from the Procedure pop up menu either a blank procedure or one you have already created If you need help because you are unfamiliar with editing this type of view refer to the more detailed procedure provided for Creating a New Cycle Editing Existing User Cycles on page 4 23 Note The most likely changes you will make to an end procedure are 1 insert one or more new steps 2 delete one or more steps and 3 change the time or active base status set for an existing proce dure step The general procedure is as follows 1 Select an empty user end procedure and then copy the desired non programmable end procedure 2 Insert any additional steps needed to add functions to your procedure This is done by selecting the step above the d
62. ely a letter that codes for a mixed base may be used translation table at the bottom of the view 4 Editing the Synthesizer Window June 2002 Applied Biosystems June 2002 Ambiguity characters may exist in a sequence imported from a file or you could use one of the ambiguity characters listed at the bottom of the view to define a mixed base site For such sequences you will be unable to produce the Reverse Complement or change base calls to bottle positions the but tons for these actions are grayed out in this case Before sending the sequence to a synthesizer confirm that the sequence is configured for the IUB nucleotide ambiguity convention For more information on converting other conventions to IUB Staden Sanger Stanford see the translation table in Appendix I Copying a Sequence Besides typing in a sequence or importing a sequence from a file you can create a sequence by using the Copy from button before you type in the sequence entry field or before you import a sequence This button allows you to choose any existing sequence either from your own synthesizer or from any open Synthesizer window As soon as you click Copy from a dia log box like that shown in Figure 4 4 appears Copy sequence from Synthesizer 1 207AULU A synthesizer 1 T3 Figure 4 4 Copy sequence from Dialog Box The dialog box lists sequences identifying the synthesizer on which the sequence is resident by presenti
63. ented with a dialog box so you can enter it The password can only be set from the synthesizer on the Communications Display accessed from Page 5 of the Main Menu but once set can be entered using the Enter Password command of the Synthesizer menu The current Read write permission information is also displayed on the Com munications display All of the other views of the Synthesizer window are accessed from the Com munication view using the pop up menu shown in Figure 4 2 HZ O Synthesizer Synthesizer 1 Choose function Communication 392 Edit Sequence Inst t iiae Set up Synthesis Model 392 Monitor Synthesis Base positions 5 Bottle Alarms Columns 2 Trityl Monitor Base Source ROM Version PA SONSES E ESES E Edit Cycle Trityl Monit A e oy Edit Begin Procedure Edit End Procedure Hemure controltrom Edit Bottle Procedure With password Edit Shutdown Procedure Without password Edit Function Power Fail Set Time Date Format Relay Configuration Figure 4 2 Choose Function Pop up Menu Note The Choose function pop up menu shown in Figure 4 2 is for instruments equipped with AutoAnalysis on non AutoAnalysis instruments Trityl Monitor is not available on Communication view and the command name is grayed out on the pop up menu 4 Editing the Synthesizer Window June 2002 Applied Biosystems June 2002 Besides choosing a command from a menu
64. er Window 431 Applied Biosystems 4 32 Edit End Procedure View An end procedure is used to automatically cleave the oligonucleotide from the synthesis support at the end of a synthesis The synthesizer uses ammo nium hydroxide to cleave the oligo delivering the oligo into the collection vial along with the ammonia End CE an ABI end procedure is presented in the initial Edit End Proce dure view as shown in the left side of Figure 4 26 z0 Synthesizer Synthesizer 1 7 Procedure 1 End CE vj fopy from Name End CE 2 End CESS 3 End CE 10 STEP FUNCTION NAME NUM time gt Begin 106 4 End RNA 2 18 to Colum 42 5 End RNA 10 Function 3 Reverse Flush 2 6 EndOl user 7 EndO2 Mac 8 EndO3 Mac 4 Prep 10 115 5 10 to Collect 36 6 18 to Waste 64 we Begin wer End 108 Block to Col 109 Waste Port Block Flush 1 9 EndO4 Mac 110 Waste Bottle 8 Wait 103 10 End05 Mac Block Vent 9 10 to Collect 36 Tri ty Rdoance 10 18 to Waste 64 11 Block Flush 1 Safe es ONG 12 gt Wait 103 A 13 10 to Collect 36 gese 160708 14 18 to Waste ey Ok Ob Ot OF OF Block Flush Figure 4 26 The Initial End Procedure View End procedures are cycle specific and the appropriate one must be used with each cycle You can see the five non programmable end procedures provided by the synthesizer when you use the Proc
65. erge and SQL GT applications 1 800 831 6844 then press 25 1 505 982 7690 Peptide Synthesis 433 and 43X Systems 1 800 831 6844 then press 31 1 650 638 5981 Protein Sequencing Procise Protein Sequencing Systems 1 800 831 6844 then press 32 1 650 638 5981 PCR and Sequence Detection 1 800 762 4001 then press 1 for PCR 2 for the 7700 or 5700 6 for the 6700 or dial 1 800 831 6844 then press 5 1 240 453 4613 Voyager MALDI TOF Biospectrometry and Mariner ESI TOF Mass Spectrometry Workstations 1 800 899 5858 then press 13 1 508 383 7855 Biochromatography BioCAD Workstations and Poros Perfusion Chromatography Products 1 800 899 5858 then press 14 1 508 383 7855 Expedite Nucleic acid Synthesis Systems 1 800 899 5858 then press 15 1 508 383 7855 Peptide Synthesis Pioneer and 9050 Plus Peptide Synthesizers 1 800 899 5858 then press 15 1 508 383 7855 1 Introduction June 2002 Applied Biosystems June 2002 Product or Product Area Telephone Dial Fax Dial PNA Custom and Synthesis 1 800 899 5858 then press 15 1 508 383 7855 FMAT 8100 HTS System and Cytofluor 4000 Fluorescence Plate Reader 1 800 899 5858 then press 16 1 508 383 7855 Chemiluminescence Tropix only or 1 781 271 0045 1 800 542 2369 U S 1 781 275 8581 Applied Bio
66. esired new step inserting a blank step line selecting the desired function and changing the time for the new step if desired Note Time values can not be entered for some steps steps with blank values Except for the possible exception of the Time and Active fields all of the controls which are used to modify an end proce dure Insert Delete Time Function and Safe are open for entry as soon as a Step is selected 3 Delete any existing step that you don t want 4 Type in a new name if you want a name other than the copy name pro vided as a default 5 Save the Synthesizer window to save the begin procedure you just cre ated Your new procedure will be listed the next time you use the Pro cedure pop up menu in the Edit End Procedure view Note The new end procedure will not reside in the synthesizer until it is sent using either the Send to Synthesizer or Prepare to Start com mands June 2002 4 Editing the Synthesizer Window 4 33 Applied Biosystems 4 34 Edit Bottle Procedure View Bottle change procedures are used to remove empty bottles and replace them with bottles of fresh reagents These procedures are used either before beginning a synthesis or when an active synthesis has been interrupted either manually or by an alarm Change procedures are especially important for phosphoramidite and tetrazole bottles because they are the most sensi tive to atmospheric oxygen and water Bot01 ABI an App
67. esis Tasks Saving and Retrieving Sequences Cycles Procedures and Functions Backing Up the Synthesizer Setup Importing and Exporting Sequences Exporting and Importing Cycles and Procedures Performing Multiple Cycle Synthesis Example of Multiple Cycle Synthesis Sharing a Synthesizer with Many Macintoshes Setting Up a Multi synthesizer Network Organizing and Processing Synthesis Order Files Setting Up Your File System Processing Synthesis Orders Processing variations 5 Using OligoNet 5 3 5 4 5 4 5 5 5 7 5 8 5 8 5 8 5 9 5 10 5 10 5 12 5 14 5 15 5 15 5 16 5 17 5 1 Applied Biosystems Practical Use of OligoNet This section covers some of the practical information you will need to use OligoNet Using OligoNet can be as simple as dedicating a single Macintosh to one synthesizer or as complex as a number of Macintoshes on a network with several synthesizers It is even possible for several users to share a syn thesizer using multiple Macintoshes These as well as the following topics are covered in this section Entering Sequences Cycles and Procedures Pre synthesis Tasks Saving and Retrieving Sequences Cycles Procedures and Functions Performing Multiple Cycle Synthesis Sharing a Synthesizer with Many Macintoshes Setting Up a Multi synthesizer Network Organizing and Processing Synthesis Order Files Special Program Symbols Two special symbols may occur in a Synthesizer Window view This symbol may
68. esis on Column 2 Only the active columns are locked so that the Cycle Trityl and Begin procedure selections are locked and not accessible 4 Editing the Synthesizer Window June 2002 Applied Biosystems s 1 5eq01 2 Seq02 3 Seq03 4 Seq04 5 eq05 6 eq06 7 Seqo 8 SeqO8 9 S eqg09 10 Seqi0 11 Seqit Manual 12 Seql2 13 Seql3 1 End CE 14 Seqi4 15 SeqI5 16 Seqi 3 End CE 10 17 Seqi 4 End RNA ee 5 End RNA 10 e 20 Seq20 6 EndO1 user 21 Seq2t 7 EndO2 Mac be 8 End03 Mac 24 Seq24 9 End04 Mac 25 Seq25 10 EndO5 Mac 26 eq26 27 eq27 28 Seq28 29 Seq29 30 Seq30 31 eq31 Mac 32 eq32 Mac 33 Seqa3 Mac 34 eq34 Mac Figure 4 7 The Sequence Pop up Menu 5 Select the end procedure to be used from the End Procedure pop up menu shown in the right side of Figure 47 6 Change the DMT setting from DMT Off the default to DMT On ifyou want to manually detritylate your DNA 7 Type ina Run ID to help identify your synthesis setup une 2002 4 Editing the Synthesizer Window 411 S y Applied Biosystems Multiple Cycle Synthesis Setup For multiple cycle synthesis setup select Multiple from the Cycle pop up menu and complete all the steps for single cycle synthesis setup as described in the previous subsection When you are through scroll down to the bot tom of the setup window which will look like Figure 4 8 except
69. esizer Blue Label 6 synthesizer Jay s Machine Synthesizer Rocky synthesizer Zinnia Figure 3 26 The Window Menu The Window menu is used to show which Synthesizer Order documents or Synthesizer windows are currently open and to select the window you cur rently want to see When you select one of the windows listed on the menu that window will be displayed on top so that it becomes visible 3 Overview of Commands and Capabilities June 2002 Applied Biosystems June 2002 4 Editing the Synthesizer Window This section provides information on using the Synthesizer window This type of window is a very important part of OligoNet since it is used to view and modify synthesis information to be used in a Model 392 394 Synthe sizer Contents About the Synthesizer Window Communication View Edit Sequence View Naming a Sequence Creating a New Sequence Importing a Sequence Copying a Sequence Editing a Sequence Set Up Synthesis View Single Cycle Synthesis Setup Multiple Cycle Synthesis Setup Monitor Synthesis View Explanation of Monitor Synthesis View Fields Interrupting a Cycle at a Safe Step Bottle Alarms View Trityl Monitor View Regular Monitor View Raw Data View Base Source View Normal Bottle Replacement Customized Bottle Assignment Replacement Edit Cycle View Creating a New Cycle Editing Existing User Cycles Edit Begin Procedure View Creating a New Begin Procedure Edit End Procedure View Creating a New End Pro
70. ess in the synthesizer When you use the Send Order to Syn thesizer command a status window is presented briefly and then the dialog box shown in Figure 3 24 is presented Note If a number of orders are ready for transmission to synthesizers the Send All Orders command allows all open Synthesis Orders to be sent in a single operation if desired to different synthesizers on the network The proper entries must be made for Column number and sequence slot number Column and Sequence check boxes on each open Synthesis Order as described by Advanced Feature on page 3 6 Copy Order to Synthesizer EJ Place order on column Gi 2 O3 O4 Place sequence in 32 eq32 Mac vj Figure 3 24 Copy Order To Synthesizer Dialog Box June 2002 3 Overview of Commands and Capabilities 3 23 Applied Biosystems 3 24 The dialog box shown in Figure 3 24 allows you to choose which column on the synthesizer will be used for synthesis As a default your synthesis will be loaded into the location labeled Seq31 Mac for column 1 Seq32 Mac for column 2 Seq33 Mac for column 3 and Seq34 Mac for column 4 Note If you want the sequence sent to a labeled location but do not want the job placed on the column click the check box to deselect it If the sequence location you specify already contains another sequence your new sequence will replace the existing sequence When you click OK in the dialog box a stat
71. ew Begin Procedure on page 4 31 To create an End procedure See Creating a New End Procedure on page 4 33 To create your own Bottle change procedure See General Instruc tions for Creating User defined Bottle Change Procedures on page 4 35 To create a Shutdown procedure See Edit Shutdown Procedure View on page 4 39 To create your own Auto Dilute procedure See Special Instructions for Creating Auto Dilute Procedures on page 4 36 If you desire to create your own functions for use in custom cycles See Edit Function View on page 4 41 5 Using OligoNet June 2002 Applied Biosystems Pre synthesis Tasks Before beginning synthesis you should complete the steps listed below This list is a simplified version of a pre synthesis check list provided in Section 2 of the 392 394 Synthesizer manual and is reproduced here as a reminder since most of these tasks must be done at the synthesizer l Check reagent levels of all reservoirs Replace empty reservoirs with bottles of fresh reagents If not done prior to synthesis this step may be required in response to a synthesis interrupted by a bottle alarm Ensure that bottles are installed correctly with a tight seal on every position Empty bottles must be place on unused positions Check that all alarms are set correctly If new phosphoramidite bottles are required prepare phosphoramid ites using the auto dilution feature Check the
72. g you to find errors made in entry To use the command place the insertion point in the sequence and select the entire sequence using the Select All command or drag the mouse to select a limited portion of the sequence Then choose the command while viewing a hard copy of the source document for the sequence The applica tion will read out loud the order of bases as entered into your Macintosh If the shift key is held down while the Mac is reading aloud reading will stop and unread text will remain selected Find 3 F The Find command is used to locate a particular sequence of bases in the Edit Sequence view of a Synthesizer window or a Synthesizer file To use the command in the Edit Sequence view place the insertion point in the sequence prior to the area of interest and then click the command This will bring up the dialog box shown in Figure 3 12 Find what string Cancel Figure 3 12 Find Dialog Box Enter the pattern you want to find in the dialog box and then click Find The application will highlight the pattern in the Sequence view if it is found and will beep if the pattern is not found 3 Overview of Commands and Capabilities June 2002 Applied Biosystems June 2002 Note As a convenience both Find and Replace ignore differences between lower and upper case letters You may type everything in lower case OligoNet will automatically convert the text to upper case before using it Find
73. he sizer Window It indicates that someone has operated the Synthesizer s front panel Click on the symbol to clear it This will present a dialog box which allows you to re read all information from the synthesizer if necessary A This symbol occurs near an entry field whenever you make an entry which is not allowed 1 6 1 Introduction June 2002 Applied Biosystems How to Get Help Contacting Technical Support You can contact Applied Biosystems for technical support by telephone or fax by e mail or through the Internet You can order Applied Biosystems user documents MSDSs certificates of analysis and other related documents 24 hours a day In addition you can download documents in PDF format from the Applied Biosystems Web site please see the section To Obtain Documents on Demand following the telephone information below To Contact Technical Support by E Mail Contact technical support by e mail for help in the following product areas Product Area E mail address Genetic Analysis DNA Sequencing galab appliedbiosystems com Sequence Detection Systems and PCR pcrlab appliedbiosystems com Protein Sequencing corelab appliedbiosystems com Peptide and DNA Synthesis Biochromatography PerSeptive DNA tsupport appliedbiosystems com PNA and Peptide Synthesis systems CytoFluor FMAT Voyager and Mariner Mass Spectrometers LC MS apisupport sciex com Applied Biosystems MDS Sciex
74. http www appliedbiosystems com techsupp To submit technical questions from North America or Europe Step Action 1 Access the Applied Biosystems Technical Support Web site 2 Under the Troubleshooting heading click Support Request Forms then select the relevant support region for the product area of interest 3 Enter the requested information and your question in the displayed form then click Ask Us RIGHT NOW blue button with yellow text 4 Enter the required information in the next form if you have not already done so then click Ask Us RIGHT NOW You will receive an e mail reply to your question from one of our technical experts within 24 to 48 hours 1 Introduction June 2002 Applied Biosystems June 2002 To Obtain Documents on Demand Free 24 hour access to Applied Biosystems technical documents including MSDSs is available by fax or e mail or by download from our Web site To order documents Then by index number a Access the Applied Biosystems Technical Support Web site at http Awww appliedbiosystems com techsupp b Click the Index link for the document type you want then find the document you want and record the index number c Use the index number when requesting documents following the procedures below by phone for fax delivery a From the U S or Canada call 1 800 487 6809 or from outside the U S and Canada call 1 858 712 0317 b
75. iew can be used to assign different original bottles for all positions as well as assign which new bottle will be used to replace an original bottle Normally however synthesis is done using the default base bottle assignments shown in Figure 4 15 for original bottle positions and then using the other set of bottle positions for new bottle In this scenario bot tles 1 through 4 are used as original positions for bases A G C and T in this order and bottle positions 5 through 8 are used as original positions for bases numbered 5 through 8 Synthesis using positions 5 through 8 for orig inal positions would be used for bases in the sequence coded with these numbers rather than the usual letter designations 4 Editing the Synthesizer Window June 2002 Applied Biosystems June 2002 For normal bottle replacement as described above the ORIGINAL BOTTLE column of the table does not require changes and therefore the Original bottle entry field is not be used To program bottle changes for normal replacement for 8 base position instruments proceed as follows 1 Select the table line to be edited by clicking it 2 Click the Change to new bottle check box 3 Click the Number entry field and then type in the appropriate new bottle value as follows a For bottles 1 through 4 as original positions e For original bottle 1 enter 5 as the new bottle e For original bottle 2 enter 6 as the new bottle e For
76. ilute Calibration menu in synthesizer 4 37 auto dilute procedures creating 436 auto dilution procedures 4 34 AutoAnalysis difference in cycle requirements 4 22 presence indicated on Communica tion view 4 3 trityl monitor view 4 18 B Base Source View 4 20 bases Legal 4 6 mixed 4 6 begin procedure creating 431 bottle alarms procedure for setting 4 16 Bottle Alarms view 16 bottle assignment customized 4 21 bottle change procedures creating 4 35 editing 4 34 purpose of 4 34 bottle replacement procedure 421 C Caution 1 5 Change Name command 3 22 Checklists OligoNet Synthesizer use 1 5 2 12 pre synthesis tasks 1 5 processing Synthesis Orders 1 5 Choose Function pop up window 4 4 Column field 3 6 Command key equivalents hot keys Edit menu 3 15 commands and capabilities overview 3 1 Communication View 4 4 Communication view description of 4 4 Communications display incorrect setting 2 11 Communications view information displayed 2 9 core facility 1 3 correct Communications display 2 11 cycles creating 5 6 D default synthesizer name 2 8 DMT on or off designation in Synthesis Order 3 8 DNA Technical Support 1 7 Index 1 Applied Biosystems Index 2 E Edit Bottle Procedure View 4 34 Edit commands Find 3 17 Read Selection 3 16 standard Apple 3 16 Edit Cycle View 4 22 Edit Function View general 4 40 scrollable lists 4 40 Edit menu 3 15 Edit Sequence View 4 5 editing a bottle pr
77. in the three Instrument fields Instrument name model number and ROM Version SSS synthesis Order Untitled 2 LI Seq Na z Instrument model 394 08 ROM Version 2 00 Sequence name Sequence 5 Le 1 Ez Length 0 OA OG Oc oT Comments Tano BETE BD i Bef vj O Multiple End procedure vj DMT On O OMT Off Cycle Figure 3 2 The New Synthesis Order Window Note Use the Tab key to move from field to field or click to select the field of interest An additional application OligoNet Order is provided with Oligo Net and allows you to fill out orders off line Information on using this program is provided in Appendix III Besides information presented in the Instrument fields the default informa tion on the form includes the current date entered from the system when 3 Overview of Commands and Capabilities 3 5 Applied Biosystems you create the form Other fields allow you to enter the following informa tion e Customer a field to identify the requestor of the oligo e Run ID entry field Two blank fields labeled 1 and 2 each of these fields provides room for entries of your choice For example these two fields could be used for Account No and for requester s phone number Note The fields labeled Instrument Instrument model ROM Ver sion and the Column and Seq No
78. ion provides information on e How to use this manual e Howto get help Contents General About This Manual Organization How to Get Started Quickly Special Text Usage Special Program Symbols How to Get Help Contacting Technical Support To Contact Technical Support by E Mail Hours for Telephone Technical Support To Contact Technical Support by Telephone or Fax North America Outside North America To Reach Technical Support Through the Internet To Obtain Documents on Demand 1 Introduction 1 3 1 4 1 4 1 5 1 6 1 7 1 7 1 7 1 7 1 8 1 8 1 9 1 10 1 11 Applied Biosystems June 2002 General OligoNet is a software program that allows direct communication between certain Applied Biosystems DNA synthesizers Models 392 and 394 anda Macintosh computer using the AppleTalk network OligoNet is used to perform many of the operations normally performed at the synthesizer key board In addition OligoNet has special monitoring control and data man agement features This manual provides general instructions for using OligoNet in conjunc tion with one or more 392 394 synthesizers Although some users may ini tially not be familiar with the 392 394 synthesizer these instructions assume that the user understands the 392 394 and only needs some general sugges tions about how to use OligoNet If you are new to the synthesizer start with the 392 394 User s manual and not with this manual Also this manual assumes ba
79. irements Macintosh Requirements Network Installation Software Installation Program Cartridge Installation Testing Your Installation Beginning an OligoNet Session Setting Up Communication Initiating Communication Printing Ending an OligoNet Session Service Information Troubleshooting Service Information Checklist of OligoNet Synthesizer Use 2 Installation and Start Up 2 3 2 1 Applied Biosystems Checking Out the OligoNet Kit If any components are missing or damaged telephone Applied Biosystems at the Foster City number 1 415 570 6667 and ask for Order Administra tion to report your problem Synthesizer Requirements The synthesizers and software versions that OligoNet can communicate with are listed below OligoNet cannot communicate with Model 380A 380B 381A or 391 EP DNA synthesizers Table 2 1 Software Versions for Synthesizer Models Model Software Version 394 2 00 or higher 392 2 00 or higher If your synthesizer has an earlier version number than listed in this table you will need to purchase and install a new program cartridge Part Number 401469 before OligoNet can communicate with your synthesizer or synthe sizers Macintosh Requirements A Macintosh II family or Quadra family machine with a hard disk at least 4 MB of memory and System 7 0 or later is recommended The minimum configuration is a Macintosh SE with 2 MB of memory a hard disk and Sys tem 6 0 5 June 2002 2 I
80. is for the same portion of a synthesis as shown in Figure 4 13 except that the column labeled Base now contains raw conductivity data QS synthesize Trityl 1 SEEEN Choose function Trityl Monitor T A a Monitor once every couplings O Interrupt if yield falls below minimum Mihir anarade tep miee meii LESHEY E as Column 1 2 O3 04 Humber Avg Stepwise Overall from 3 Row Yield amp Yield amp Figure 4 14 Raw Data View 4 Editing the Synthesizer Window 419 Applied Biosystems 4 20 Base Source View The Base Source view is used on 392 or 394 synthesizers to automatically change the bottle position from which a particular base will be drawn after a designated number of cycles When 4 bases are used for synthesis on 8 base position instruments this allows all base positions to be changed to a new bottle after a number of cycles UE synthesizer Synthesizer SEEEN Choose function ORIGINAL NUMBER OF NEW BASE BOTTLE CYCLES BOTTLE Original bottle Number O Change to new bottle Number 1 after o cycle s Figure 4 15 The Initial Base Source View Note The Base Source view does not function for 5 base instruments Entries can be made for New bottle and number of cycles col umns only after the Change to new bottle check box is checked Normal Bottle Replacement The Base Source V
81. izer you will be able to print the Trityl Monitor view as well Ending an OligoNet Session End a session with OligoNet by choosing Quit from the File menu All open and unmodified OligoNet windows will close as the application quits If you have modified an open Synthesis Order but not saved it you ll be given a chance to save it before the application closes Ifyou have modified an open Synthesizer window without sending the changes to the synthesizer you will be given a chance to send your changes to the synthesizer before the appli cation closes Save changes to Synthesizer prompt Service Information Troubleshooting Service Information At the time you finish installation you may want to access the service infor mation provided with OligoNet and print out a copy of the information for possible use later This is done as follows 1 Hold down the 3 key Command key 2 Choose Show Service Info from the menu After a minute or so the program will show a window entitled Service Infor mation You can print out the window using the Print command File menu or save it to a disk file using the Save As command File menu Troubleshooting The troubleshooting checklist provided below is intended to cover the most common problems which might be encountered during installation of Oli goNet The symptom for a faulty installation will be no OligoNet to Synthe sizer communication e Fora network dedicated as show
82. le synthesis cycles Synthesis Order form 3 7 standard Apple commands reference for 3 3 start prepare to 3 18 starting OligoNet 2 7 starting quickly 1 5 Synchronize Clocks command 3 20 Syncom Plus installation and start up 2 1 starting the application 2 7 synthesis information preparing 5 4 synthesis interruption 4 14 Synthesis Order form 3 5 list of fields 3 6 listing of synthesizer name 3 8 use in core facilities 1 3 synthesis setup procedure multiple cycles 4 12 single cycle 4 9 Index 3 Applied Biosystems Index 4 Synthesis window editing 41 synthesizer starting a 2 6 synthesizer background importance of 1 3 Synthesizer commands Change Name 3 22 Enter Password 3 21 synthesizer commands grayed out 2 8 Synthesizer menu 3 18 synthesizer network connection 2 4 Synthesizers used with Syncom H 2 3 T Tab key use 3 5 Technical Support 1 7 technical support 1 3 telephone Technical Support 1 7 testing installation 2 6 text usage special 1 5 Think Jet printer 2 10 Tm and formulas 3 7 translation table reference 4 7 trityl data raw 419 Trityl Monitor view 4 18 troubleshooting installation 2 10 U Use Sounds command 3 17 user function defining 441 W Warning symbol duplicate sequence name 4 6 explanation of convention 1 6 improper character flagging 3 7 Warning symbol improper character flagging Appen III 2 Window menu 3 26 June 2002 Headquarters 850 Lincoln Centre Drive Foster
83. le using the Save a Copy in command One copy will become the custom copy and should be renamed to reflect its custom nature 2 Edit the copy to introduce custom cycles procedures and functions Also do any editing required for custom base source or bottle alarm settings 5 Using OligoNet June 2002 Applied Biosystems June 2002 3 At the start of a session using the custom Synthesizer file download it to the synthesizer using the Send Copy to Synthesizer command 4 At the end of the custom synthesizer session use the Send Copy to Syn thesizer command again with the standard Synthesizer file copy to re set the synthesizer Step 4 should be performed by the individual using the custom window but everyone should maintain a copy of the standard window for normal use An additional requirement for several individuals to share a synthesizer us ing multiple OligoNet nodes on a network is that they must share a common password if password protection is used Another requirement is that any custom Synthesizer file be clearly named to distinguish it from the standard file Also custom cycles and procedures should be named so that other users will not inadvertently use them The scenario of sequential shared use presented in this subsection should al low a number of users with different needs to share a synthesizer without in terfering with each others work The model enables use with the minimum effort by individ
84. lect the new synthesizer state In this scenario of sequential shared use at the start of every period of use the standard Synthesizer file should be resident on the synthesizer to enable users using default settings probably the majority to start work with the min imum overhead Anyone needing custom synthesis settings would then need to use the Send Copy to Synthesizer command to download a custom Syn thesizer file to the synthesizer at the start of a work session At the end of the work session the Send Copy to Synthesizer command is used again to download the standard Synthesizer file for use by the next user Everyone using the synthesizer should maintain a copy of the standard syn thesizer file in case someone forgets to reset the synthesizer to its default condition The main burden in this scenario is on individuals using custom settings since these individuals would have to maintain both types of synthe sizer files and do the downloading of synthesizer files at the beginning and end of his or her session A user who needs only the standard synthesizer file should use the Save a Copy in command once to save a copy of the standard file The Send Copy to Synthesizer command would be required for the standard user only when the prior user left a custom synthesizer file on the synthesizer Users needing a custom synthesizer file develop and use it as follows 1 Make two copies of the standard Synthesizer fi
85. les When the Multiple box is checked multi ple cycles may be used for a single synthesis The single Cycle entry field is disabled and a new area becomes available for entry of multiple cycles and the number of bases to be added by each cycle as shown in Figure Appen II 2 See Performing Multiple Cycle Synthesis in Section 5 for more information on this type of synthesis BASES CYCLE Bases numbered from 3 end Cycle name ST Number of bases fi z Figure Appen Ill 2 Multiple Cycle Entry Area of Order Note Entries containing multiple cycles require that the number of bases to be added by each cycle be entered For multiple cycle listing base entries are counted from the 3 end even though they are entered from the 5 direction in the Edit View When the Syn thesis Order form is completed a copy can be printed for a hard copy record and the form should then be saved For each cycle listed provide sufficient information for an operator to correctly assign an ABI or other cycle Name of End Procedure to be used for synthesis type in procedure name and provide sufficient information in the Comments field to enable the operator to correctly assign an ABI or other end procedure June 2002 Appendix II Using OligoNet Order Appendix III 3 Applied Biosystems Appendix III 4 e Designation of DMT group on or off DMT group at 5 end When the Synthesis Order form is completed the Synthesis Orde
86. lied Biosystems USS Synthesizer Syn th esiz er 1 SSMN Cycle 11 1 0 um CE copy v Name 1 0 um CE copy FUNCTION NAME NUM TIME ACTIVE SAFE Begin 18 to Waste 18 to Column A AGCT5678 Reverse Flush AGCTS678 Block Flush AGCT5678 Phos Prep Column 1 On Block Vent k AGCT5678 Tet to Column S Tet to Column 10 to Collect 11 to Column 12 to Column Cap to Column Safe Yes No Active K6 H 8 A Ke RICHI AS Tet to Haste gt B Tet to Column 33 2 5 AGCT5S678 Tet to Column A AGCT5678 B Tet to Column AGCT5678 Push to Column Column 1 Off Column 2 On Figure 4 21 Selecting a Cycle Step Note Time values can not be entered for some steps steps with blank time values Except for the possible exception of the Time field and the Active field all of the controls which are used to modify a cycle Insert Delete Time Function and Safe are open for entry as soon as a Step is selected b Click on the Time field to select it for entry placing the insertion point where you want to add a digit or dragging through the exiting value to select the entire entry c Type in the new time value d Change active base positions for the cycle by clicking one or more of the check boxes in the lower left corner of the view 8 Insert the new steps needed to modify the template cycle by perform ing the addition of each step as follows a Determine where you want to insert a step and then sel
87. lied Biosystems supplied bottle change procedure is pre sented in the initial Edit Bottle Procedure view as shown in the left side of Figure 4 27 1 BotO1 ABI 2 BotOl user 3 BotO2 ABI 4 Bot02 user 5 BotO3 ABI 6 BotO3 user 7 BotO4 ABI 8 BotO4 user 9 BotO5 ABI 10 BotO5 user EDES synthesizer Synthesizer SSSA 11 Boto6 ABI 12 Bot06 user 13 BotO ABI Lepy from Procedure _1 Boto abl r Poto ABST lasert Beiere a Name Botna sd tine STEP FUNCTION NAME NUM TIME ACTIVE SAFE Fuactian 3 pee Waste 54 ves 5 0 7 0 fH Begin 4 18 to A 70 3 0 Yes 5 0 5 0 0 0 33 Auto 1 0g 34 Auto 0 5g 35 Auto 0 259 36 Auto 2 0g 37 2g Double 38 AutOl user 39 AutO2 user WO End 5 Block Flush 1 Yes 108 Block to Col 6 Block Vent 111 k Yes 7 Flush to A 6 10 109 Waste Port Yes 40 AutOS user 110 Waste Bottle 8 Interrupt 104 Yes 111 Block Vent a Flush to A 6 5 0 Yes 41 Aut04 user 2 Ted ty Aches 10 gt Phos Prep 101 15 0 Yes 42 Aut05 user 11 A to Waste 50 2 0 Yes 43 AutO6 user Safe Yes ONa 12 18 to Waste 64 7 0 Yes 44 Auto user isaac i 13 gt Block Flush 1 5 0 Yes feuse O60 O18 14 eng 107 ves 45 Aut08 user Ok O amp O OF os ea 46 Aut09 user 47 Autio Mac Figure 4 27 The Initial Edit Bottle Procedure View List of Procedures As shown in the Procedure pop up menu shown in the right side of Figure
88. m melting point of oligonucle otide and G C is the combined percentage of these bases in the sequence The user may want to add comments characterizing the DNA based on these parameters For Tm the following formulas apply a Sequences shorter than 14 bases Tm 2 A T 4 G C b Sequences 14 bases or longer Tessio Naa 202 A G C T A G C T In the second formula the assumption is made that Na is always 0 1 Molar In each of these formulas A number of A bases G number of G bases etc e Single synthesis cycle Cycle entry field Name of synthesis cycle may be typed in by hand If an instrument is selected the pop up menu for the Cycle field allows selection of the cycles available on the chosen syn thesizer and no typing is needed June 2002 3 Overview of Commands and Capabilities 3 7 Applied Biosystems 3 8 Note It is preferable to use the pop up menu to select the cycle If the cycle is typed in by hand it must be spelled EXACTLY as stored in the synthesizer otherwise OligoNet will be unable to recognize it later when it attempts to use this information When a single Synthesizer window is open at the time of opening the Synthesis Order the instrument name associated with that window is listed in the Instrument field When two or more Synthe sizer windows are open the instrument associated with the last Synthesizer window examined is listed and the other synthesizer name or names are listed on
89. mmands are not needed Instead open Synthesizer windows for both instruments from OligoNet and then use the Copy from button in the Edit Cycle view Caution If you use the Import command to bring a cycle into your Syn thesizer window be aware that you may import a non com patible cycle into your instrument a 2 column cycle into a 4 column instrument or vice versa since the Import command presents no warning before importing incompatible cycles It is much safer to use the Copy from button since a warning message is presented before an incompatible cycle can be brought into your Synthesizer window giving you the chance to back out When using Copy from to move cycles between open Synthesizer windows OligoNet checks the compatibility of the number of base positions and columns in each Synthesizer window 5 Using OligoNet 5 9 Applied Biosystems 5 10 Performing Multiple Cycle Synthesis Multiple cycle synthesis lets you use more than one cycle during the synthe sis of a single sequence This option allows you to make special oligos such as hybrid DNA RNA oligonucleotides or mixed normal phosphorothioate DNA hybrids Hybrid DNA RNA oligos require the use of two cycles because each type of base requires a different cycle Mixed normal phosphorothio ate oligos require the use of two cycles because each type of internucleotide linkage is produced by a different chemistry Another use for multiple cycles might be the pr
90. mn 42 60 0 Yes O8 O amp Of OF O18 15 Reverse Flush 2 60 0 Yes Figure 4 30 The Initial Edit Shutdown Procedure View If you need help because you are unfamiliar with editing this type of view refer to the more detailed procedure provided for Creating a New Cycle Editing Existing User Cycles on page 4 23 Note The most likely changes you will make to a bottle change proce dure are 1 insert one or more new steps 2 delete one or more steps and 3 change the time or active base status set for an existing procedure step The general procedure is as follows 1 Select an empty user shutdown procedure and then copy the desired non programmable bottle change procedure 4 Editing the Synthesizer Window 4 39 Applied Biosystems 2 Insert any additional steps needed to add functions to your procedure This is done by selecting the step above the desired new step inserting a blank step line selecting the desired function and changing the time for the new step if desired Note Time values can not be entered for some steps steps with blank values Except for the possible exception of the Time field and the Active field all of the controls which are used to modify a shut down procedure Insert Delete Time Function and Safe are open for entry as soon as a step is selected 3 Delete any existing step that you don t want 4 Type in a new name if you want a name other than the copy name pro vide
91. n NBRF The Staden convention is used to translate sequences received from the Cambridge University database The Stanford convention is used for the Stanford University database Appendix I Nucleotide Ambiguity Conventions Appendix I 1 Applied Biosystems June 2002 Appendix II Resetting OligoNet Memory With the default application size of 1024K OligoNet is limited to having 5 Synthesizer Windows open at the same time If you want more you need to add 140K to the memory assigned to the application for each additional win dow For example if you want to simultaneously open 7 Synthesizer Win dows you need to increase the assigned application memory to 1024K 280K 1304K This is done as follows 1 Single click on the OligoNet application icon to select it OligoNet 1 0 fe4 2 Go to the Apple menu bar and select the Get Info command from the File menu to bring up the Get Info box for the application ZOS ODligoNet 1 0 fc4 Info pacal Gligotet 1 0 fet Kind application program Size 638K on disk 702 539 bytes used Where SYNTH OligoNet Created Wed Jun 10 1992 2 07 PM Modified Thu Jun 11 1992 5 30 PM Yersion Oligotet 1 0 fect Comments po MEMO Y nennen Suggested size 1 024 K i C Locked Current size E Figure All 6 OligoNet Get Info Box 3 Drag to select the last three digits of the current size setting and then type in the new value of these digit
92. n in Figure 2 1 did you correctly insert the cable connector on each end On the Macintosh end the cable must be connected to the Printer port See your Macintosh manual for the correct location On the 392 394 Synthesizer end the cable must be connected to the port labeled APPLETALK The cable to the port labeled RS 232 will not provide communication e On your Mac did you forget to choose AppleTalk AppleTalk must be active even on a dedicated network for communication to occur 2 Installation and Start Up June 2002 Applied Biosystems June 2002 On connections to an existing networks contact your local network manager Do you have the correct Program Cartridge version and is the cartridge fully inserted into its slot Version 2 00 is required The Program Cartridge for Version 2 00 software is required to obtain the Communications display shown in Figure 2 5 Does the Communications display appear as shown in Figure 2 10 If so place the cursor on the R of RS 422 first line of display and press the Prev or Next key on the synthesizer This will restart your instru ment and change the Communications display so that it appears as shown in Figure 2 5 Communications RS 422 Receive 9600 Stop bits 1 Parity none Transmit 9600 Bits char 8 Figure 2 10 Communications Display with Incorrect Setting Communications AppleTalk Name Synthesizer 1 Access with password
93. n specify DMT On the first two trityls will be col lected but at the end of the sequence the synthesizer will collect the 2nd and 3rd trityls from the end rather than the last two Spec ifying DMT On instructs the synthesizer not to remove the dimethoxytrityl group from the 5 end v 1 ABI Begin 2 BegO1 user 3 Beg02 Mac First 2 and last 2 None Figure 4 6 Trityl Collect Pop up Menu Begin Procedure Pop up Menu 3 Select the begin procedure to be used from the Begin Procedure pop up menu shown in the right side of Figure 4 6 4 Select the number of columns to be used by selecting sequences as fol lows a Select the column line to be activated in the table by clicking on it b Select the sequence you want to assign to this column from the Sequence pop up menu shown in the left side of Figure 4 7 The caption on the column line will change from Not selected to Selected as you select a sequence Other messages you may see on a column line include Not Available for column lines 3 and 4 on 392 instruments and Active for a synthesis in progress Note When you select an active column line you will not be able to enter a synthesis setup and every menu on this view will be locked You may enter a synthesis setup for any column that is available and not active For example you can start synthesis on Column 1 and then return later while Column 1 is active and set up and ini tiate synth
94. ng a soft key beside the right side of the display a From the synthesizer Main Menu select Self Test to access the Self Test Menu b From the synthesizer Self Test Menu select Flow Rate Test to access the Flow Rate Test Menu c From the Flow Rate Test Menu select Set Dilute Flow to present the Auto Dilute Calibration menu This menu will look like Figure 4 29 Bot 1 4 92 gm 30 sec 2 4 91 gm 30 sec 3 4 93 gm 30 sec 4 4 92 m 30 sec 5 4 92 gm 30 sec 6 4 91 gm 30 sec 7 4 93 gm 30 sec 8 4 92 gm 30 sec Figure 4 29 392 394 Auto Dilute Calibration Menu 2 In Auto 1 0g used for the present example replace steps 7 through 38 as follows a For each bottle to be diluted include the following three functions as steps Bottlex On x bottle number 18 to x lt time gt x base letter e g A or bottle number Bottlex Off x bottle number b The time value for the second step for each bottle to be diluted is determined using the following equation Vx oe g ml 30 where V the desired volume in milliliters R the flow rate of 18 to the bottle in grams 30 seconds and T the delivery time in seconds T You should create a set of auto dilute steps in your new procedure for all existing bottle positions on your instrument even if you intend to une 2002 4 Editing the Synthesizer Window 4 37 8 y Applied Biosystems auto dilute bottles on only one or two positions because
95. ng the synthesizer name before the sequence name As soon as you select a sequence and click OK the sequence is entered into the sequence entry area You may now edit the sequence you copied as described in the next subsection Editing a Sequence The rules listed in the previous subsection Creating a New Sequence also apply 4 Editing the Synthesizer Window 4 7 Applied Biosystems 4 8 Note If you are going to copy a sequence it is important to use the Copy from button before making any other entries in the sequence entry field because the sequence copied in using the button will overwrite anything previously in the entry field wasting the effort of prior entries Editing a Sequence A sequence may be edited by simply selecting the portion you want to change and then performing the desired change such as deleting this por tion or typing in a new series of bases to replace the existing portion Note When all or part of the sequence in the Sequence entry field is selected using the Read Selection command Edit menu will read the sequence aloud in a digitized voice This feature is useful for proofreading sequences The Reverse Complement and AGCT lt gt 5678 buttons allow you to change the form of a sequence entered as letter bases These buttons are available after sequence entry except when you enter mixed bases or num bered bases Using Reverse Complement produces the revers
96. not placed on or assigned to a column By entering just the Column number you place the order on the designated column and specify that the default sequence slot for the column be used Default sequence slots are Seq31 Mac Seq No 31 for Column 1 Seq32 Mac Seq No 32 for Col umn 2 Seq33 Mac Seq No 33 for Column 3 and Seq34 Mac Seq No 34 for Column 4 3 Overview of Commands and Capabilities June 2002 Applied Biosystems June 2002 Copy from Synthesizer The Copy from Synthesizer command is used to copy the current synthesis setup sequence cycle and end procedure into the current Synthesis Order When you choose the command a dialog box like that shown in Fig ure 3 25 is presented Copy Order Information from Synthesizer Column 1 eq25 Oe Gs D 4 Cancel OK Figure 3 25 Copy Job Information From Dialog Box This box lists the column with the instrument s current synthesis setup including the name of the sequence When you click ok a status window is presented while reading over the network is taking place Both the dialog box and the status window allow you to change your mind about copying the job information Cancel button in the dialog box and Stop button in the sta tus window 3 Overview of Commands and Capabilities 3 25 Applied Biosystems 3 26 Window Menu Window Synthesizer Bufford synthesis Order Example synthesizer Barbara M synth
97. nstallation and Start Up 2 3 Applied Biosystems 2 4 Network Installation Your Macintosh and synthesizer must be interconnected by an AppleTalk network This can be as simple as a network consisting solely of a Macintosh and the synthesizer or your Macintosh and synthesizer may be intercon nected by an existing network For connection to an existing network plug in the appropriate cable between the connector labeled AppleTalk on the synthesizer located on the rear below the Program Cartridge and the network Then if your Mac intosh is not connected to the network connect the printer port of the Mac intosh to the network using the appropriate cabling Consult your local network manager for assistance with connections and needed cables To create a network consisting solely of a Macintosh and the synthesizer use the cable provided to interconnect the AppleTalk connector on the syn thesizer with the printer port of the Macintosh as shown in Figure 2 1 m Mac to 392 394 Cable Part No 201001 Figure 2 1 Macintosh to Synthesizer Dedicated Network Software Installation 1 Insert OligoNet disk Number 1 of 2 into the Macintosh disk drive 2 Double click the OligoNet disk icon to open the disk 3 Copy the application by dragging the OligoNet application icon to your hard disk 4 OligoNet operates via an AppleTalk network Select the Chooser from the Apple menu and make sure AppleTalk is set
98. nstruments 40 nm CE Cycle 2 um CE Cycle 1 0 um CE Cycle 10 um CE Cycle 2 um Sulfur Cycle 1 0 um Sulfur Cycle 10 um Sulfur Cycle 2 um RNA Cycle 1 0 um RNA Cycle 10 um RNA Cycle Ifyou need or want more information about the chemistry or cycles refer to Section 6 of the 392 394 User s Manual for more information about chem istry and to Appendix I of the manual for a complete listing of functions cycles and procedures provided with the synthesizer Note The synthesis cycles listed in Appendix of the 392 394 Synthe sizer manual are those available in 392 394 synthesizers equipped with the AutoAnalylsis option These cycles support monitoring of trityls by conductivity during the run but do not sup port fraction collection If you intend to perform fraction collection on AutoAnalysis option equipped instruments you need to modify cycles as described under Modifying Cycles for Fraction Collec tion in Section 4 of the 392 394 Synthesizer Manual Ver 2 00 June 2002 5 Using OligoNet 5 5 Applied Biosystems 5 6 Creating your own Cycles Procedures and Functions If you desire you can create your own synthesis cycles using the Edit Cycle View For instructions on editing cycles See Creating a New Cycle Editing Existing User Cycles on page 4 23 For information on creating your own procedures for use in synthesis see one of these references To create a Begin procedure See Creating a N
99. nt to the right of bottle position five or eight depending upon the configuration of the instrument This will present a display similar to the fol lowing on the synthesizer Applied Biosystems 394 DNA RNA Synthesizer version 2 0 0 4 Columns 8 Base Positions Press Main Menu to begin Figure 2 2 392 394 Synthesizer Start up Page 5 Press Main Menu as prompted by the Figure 2 2 display Main Menu Edit Sequence gt Start Sequence gt Monitor Sequence gt Page 1 of 5 Next Page gt Figure 2 3 Page 1 of the Main Menu 6 When Page 1 of the Main Menu appears as shown in Figure 2 3 press the Next Page soft key four times to go to Page 5 of the Main Menu Main Menu Relay Configuration gt Shut Down gt Communications gt Page 5 of 5 Next Page gt Figure 2 4 Page 5 of the Main Menu 7 Press the Communications soft key to bring up the Communications display as shown in Figure 2 5 2 Installation and Start Up June 2002 Applied Biosystems June 2002 Communications AppleTalk Name Synthesizer 1 Access with password READ WRITE without password READ WRITE Figure 2 5 Synthesizer Communications Display Note If this display indicates Communications RS 422 see the Trou bleshooting checklist near the end of this section Next follow the procedure below to determine if your OligoNet installation is successful Initiating Communica
100. nthesis sufficient information should be entered in this field to allow the operator to correctly assign ABI or other Cycles and End Proce dures in response to the entries in the Cycle and End Procedure fields If multiple cycles are to be used information should be pro vided in the Comment field for all cycles Tm and G C fields under Comments box as soon as a sequence is entered or imported into the Sequence listing field OligoNet calcu lates values for these parameters Tm melting point of oligonucle otide and G C is the combined percentage of these bases in the sequence The user may want to add comments characterizing the DNA based on these parameters For Tm the following formulas apply Appendix III 2 Appendix IN Using OligoNet Order June 2002 Applied Biosystems Multiple cycles inser pupie feipie Eurie a Sequences shorter than 14 bases Tm 2 A T 4 G C b Sequences 14 bases or longer 7 i Gee 65 Tm 81 5 16 6 log Na a r OT In the second formula the assumption is made that Na is always 0 1 Molar In each of these formulas A number of A bases G number of G bases etc Single synthesis cycle Cycle entry field Name of synthesis cycle Besides entering a cycle name into this field further information about the cycle should be provided so that the operator completing the order can correctly assign an ABI Cycle or other cycle Multiple synthesis cyc
101. ocedure 4 34 editing a sequence 4 8 end procedure creating editing 4 33 use in Synthesis Order 3 8 End Procedure View editing 4 32 Ending a OligoNet Session 2 10 Enter Password command 3 21 Export command 3 14 export files names and icons 3 14 F File commands Import Export 3 14 New Synthesis Order 3 5 Open 3 9 Open Synthesizer 3 10 Save a Copy In 3 12 File menu list of commands 3 3 list of tasks 3 4 3 15 files types imported 3 14 Find command 3 16 Find Same command 3 17 functions creating 5 6 printing 4 42 without step times 436 H help getting 1 7 I Import command 3 14 IMPORTANT 1 5 initiating synthesis at the synthesizer 3 19 installation MELCard 2 5 Installing OligoNet 2 4 installing OligoNet 2 4 interruping a cycle 4 14 Interrupt command 3 20 L legal bases 4 6 listing of standard CE and RNA cycles 5 5 M Macintosh interconnection with synthesizer 2 4 standard commands 3 16 system requirements 2 3 manual info about 1 4 MELCard installation 2 5 MELCard version required 2 11 mixed bases 4 6 Monitor Synthesis view 413 Monitor view fields 4 13 multiple synthesis cycles Set Up Synthesis View 4 12 Synthesis Order form 3 8 Appen IlI 3 multi zone network 2 8 N name changing the synthesizer 3 22 naming asequence 4 6 network installation 2 4 new sequence creating 4 6 New Synthesis Order 3 5 normal bottle replacement 420 Note 1 5 June 2002 Applied Biosystems June 2
102. oduction of big oligos where the increasing length of the oli go could be compensated for by changes in cycle step times A multiple cycle synthesis can be set up using either a Synthesis Order or a Synthesizer Window In a Synthesis Order the multiple cycle option is select ed by clicking the Multiple check box In a Synthesizer Window the mul tiple cycle option is selected in the Set up Synthesis view by selecting Multiple from the Cycle pop up menu Either of these actions adds a new area to the window similar to that shown in Figure 5 1 Note The new area for the Synthesis Order is like Figure 5 1 but does not show columns and does not initially list a cycle either in the ta ble or in the Cycle entry field When more than one column is used all must have identical base numbers because all are syn thesized in parallel using only one cycle The base numbers listed for each cycle in the multiple cycle option correspond to the inter nucleotide linkages to be added by that cycle counting from the 3 end BASES CYCLE coL 1 cOL2 COL 3 COL 4 Insert Cycle Cycle 2 2 um CE v Delete Eurie Number of bases fi Figure 5 1 Set Up View Multiple Cycle Option Example of Multiple Cycle Synthesis This example illustrates how to produce a hybrid normal phosphorothioate DNA oligo using the following mixed 18 mer sequence 5 TsCA CAG TCT GAT CTC GAT 3 This sequence contains phosphorothioate linkages after
103. ollowing Instrument model ROM_VERSION Text following ROM Version SYNTHESIZER Text in popup menu Instrument SEQ NAME Contents of Sequence name text box SEQ_TEXT Contents of Sequence text box COMMENTS Contents of Comments text box CYCLE Contents of Cycle text box Appendix IN Using OligoNet Order June 2002 Applied Biosystems June 2002 END_PROC DMT MULTICYCLES Contents of End procedure text box Either ON or OFF depending on state of radio buttons Number of multiple cycles or 0 if Multiple check box is unchecked The following fields appear only as needed CYCLE1 BASES1 CYCLE2 BASES2 etc Name of first multiple cycle Number of bases for first multiple cycle Name of second multiple cycle Number of bases for second multiple cycle etc As long as the version number does not change you may rely on the order of fields as given above Appendix IN Using OligoNet Order Appendix III 5 Applied Biosystems June 2002 Index A Advanced Feature Column and sequence fields 3 6 explanation of convention 1 5 AGCT 5678 button 4 8 alarms bottle 4 16 trityl 4 18 ambiguity characters 4 7 ambiguity conventions Appen I 1 application memory setting default 3 10 Applied Biosystems supplied procedures bottle change 434 shutdown 4 38 auto dilute calibration 4 36 delivery of different volumes 4 36 time equation for delivery 4 37 Auto D
104. one of the phosphoramidite reservoirs has not been accessed within 12 hours ABI Begin is presented in the initial Edit Begin Procedure view as shown in Figure 4 25 and is not editable as indicated by the lock icon EDES sunthesizer Synthesizer 1 EN Choose function Edit Begin Procedure v Procedure 1 ABI Begin w Lopy from Name ABI Begin iasert Beiere a g STEP FUNCTION NAME NUM TIME ACTIVE SAFE fime 6 8 gt Begin 06 TEE Functien 86 End Block to Col Waste Port Waste Bottle Block Vent mri tyi Ahemece K Safe es ONG fictive 6 0 08 OreoOsort 0o 0s Tet to Haste 18 to Haste Block Flush End 5 2 2 2 2 2 2 2 2 2 0 0 oooooo0oo0ooco9 cae97 Figure 4 25 The Edit Begin Procedure View This view also provides you with two locations for creating your own begin procedures The procedure labeled user may contain a begin procedure entered at the synthesizer but can be edited in OligoNet The procedure labeled Mac is intended to be downloaded into a location reserved for this purpose in the synthesizer 4 Editing the Synthesizer Window June 2002 Applied Biosystems Creating a New Begin Procedure You create your own begin procedure in the same way that a new cycle is cre ated using the Edit Cycle view If you need more help than provided here because you are unfamiliar with editing this type of view refer to the more detailed procedure provide
105. only the bottles selected on the synthesizer Auto Dilute menu will actually be diluted 4 38 4 Editing the Synthesizer Window June 2002 Applied Biosystems June 2002 Edit Shutdown Procedure View The Shutdown procedure prepares the synthesizer for long term storage storage for periods of one month or longer It removes all reagents in the delivery lines and washes and dries all chemical pathways ABI Shut the ABI shutdown procedure provided with the synthesizer is pre sented in the initial Edit Shutdown Procedure view as shown in Figure 4 30 Two user shutdown procedure locations are available The procedure labeled user may contain a shutdown procedure entered at the synthesizer IOE synthesizer Synthesizer 1 Saaz Choose function Edit Shutdown Procedure w Procedure 1 ABI Shut v Lopy from Delete Name ABI Shut a STEP FUNCTION NAME NUM TIME ACTIVE SAFE Te G dag 7 2 gt Block Flush 1 15 0 Yes Functen 186 3 18 to Waste 64 15 0 Yes begin 4 Block Flush 1 15 0 Yes Ert 5 18 to Column 42 30 0 Yes Block to Col 6 Block Flush 1 30 0 Yes Waste Port 7 Flush to Colect 5 15 0 Yes Waste Bottle 8 Waste Port 109 Yes Block Vent 9 18 to Column 42 30 0 Yes mi tyi Ahem z 10 gt Trityl Flush 3 30 0 Yes 11 gt Waste Bottle 110 Yes Safe es ONG 12 18 to Column 42 60 0 Yes A A 13 Reverse Flush 2 60 0 Yes Ac Hues A H 6 O QO 8 14 gt 18 to Colu
106. ore base positions all of the bottle positions are active as a default by clicking the appropriate check box in the lower left corner of the view Note Active Step This column allows you to specify that a particular step be active only for specified bases For example you could add an extra detritylation step to the cycle allowing you to adjust for the length of detritylation required by some bases Bases C and T require a longer detritylation than A and G By adding a step active only for CT56787 the step would not be applied to bases A and G When an A or G is being detritylated the program sees the step as not active for it and bypasses the step If you are using 5 as A then you may want to change that step to be active 4 Editing the Synthesizer Window 4 27 Applied Biosystems 4 28 SSS synthesizer Synthesizer SSS Cycle 11 1 0 um CE copy v Name 1 0 um CE copy FUNCTION NAME NUM TIME ACTIVE Begin 18 to Waste 34 Tet to Column StTet to Column 10 to Collect 11 to Column 12 to Column Cap to Column Safe Yes O No Active K6 N K8 KRARGHCHT HS 18 to Column Reverse Flush Block Flush Phos Prep Column 1 On Block Vent Tet to Haste B Tet to Column Tet to Column AGCTS678 AGCTS678 AGCTS678 AGCTS678 AGCTS678 AGCTS67S Tet to Column B Tet to Column Push to Column Column 1 Off AGCTS67S AGCTS67S Figure 4 23 Edit Cycle View after Selection of Function
107. original bottle 3 enter 7 as the new bottle e For original bottle 4 enter 8 as the new bottle b For bottles 5 through 8 as original positions e For original bottle 5 enter 1 as the new bottle e For original bottle 6 enter 2 as the new bottle e For original bottle 7 enter 3 as the new bottle e For original bottle 8 enter 4 as the new bottle 4 Click the After cycles entry field and enter the number of cycles after which you want to change bottles Customized Bottle Assignment Replacement In some cases however you may want to change the original bottle assign ments For example you might want to synthesize sequences labeled with letter bases on positions 5 through 8 where 5 A 6 G 7 C and 8 T In this case in the first column of the table assign position 5 to A position 6 to G position 7 to C and position 8 to T This assignment is done as in the proce dure above except that you click the Original bottle entry field in step 2 to assign original bottle positions After assigning original bottle positions you can then assign the remaining bottle positions for replacement during synthesis Note If you have made custom bottle assignments for synthesis and later change bottles back to their original states don t forget to change replacement bottles using the Base Source view 4 Editing the Synthesizer Window 421 Applied Biosystems 4 22 Edit Cycle View The Edit Cycle view allows you to pro
108. r 1 copy Cancel Figure 3 8 Save a Copy Dialog Box When you click on Save a status indicator is presented while saving is under way Send Copy to Synthesizer The Send Copy to Synthesizer command is used to send a copy of a Synthe sizer window previously saved using Save a Copy In to a Synthesizer This command is like the Send to Synthesizer command except that it provides a dialog box similar to that shown in Figure 3 9 allowing synthesizer informa tion obtained from one synthesizer and saved to a file on the Macintosh to be transferred to any available synthesizer This command is useful for restoring all information and synthesizer set tings to a previous backup version The combination of Save a Copy In and Send Copy to Synthesizer allows many users to share one synthesizer and still have completely individual custom setups Note For multi zone networks the dialog box also provides a zone list to allow selection of the zone Copy to Synthesizer Select a Synthesizer Synthesizer 1 Cancel Figure 3 9 Copy to Synthesizer Dialog Box 3 Overview of Commands and Capabilities 3 13 Applied Biosystems 3 14 Import Export 3 E The Import and Export commands are available while editing sequences cycles and procedures in a Sequence window The name of the commands change to reflect the name of the Synthesizer window edit view which is cur rently acti
109. r Front Reagent Valve Block RRVB stands for Rear Reagent Valve Block RVB stands for Regulator Valve Block 2 Type in a name of your choosing in the NAME entry field You cannot highlight the NAME entry field for entry until you choose a user function One at a time select each valve open action you desire to add to your new function and then click Add to place the valve number and description in the function listing area If you decide to remove a valve from your function select the valve in the function listing and then click Remove 4 Editing the Synthesizer Window June 2002 Applied Biosystems Printing Functions As shown in Figure 4 32 the print dialog box for the Edit Function view allows two main options printing of function text or printing of the current Function view window image If you choose the Text option you can choose to print a single selected function system or user or choose to print all user functions If you choose to print all user functions the program will only print those that have non empty valve lists User functions are number 200 224 This produces a blank page if you haven t defined at least one user function Note If you select an individual function system or user a printout will be produced even when the system function lists INVALID FNC TION for function name and the User function is empty In these cases the printout will list the number of the function INVALID
110. r needs to be saved to a file A hard copy of the order should be printed for a record or to accompany the Synthesis order file in placing the order for a synthesis When a Synthesis Order prepared by OligoNet Order is opened by the oper ator in OligoNet the instructions provided in Section 3 apply for complet ing the Synthesis Order and sending it to a synthesizer Format of Synthesis Order Files Users who have programming skills and are interested in writing their own custom database or spreadsheet programs to manipulate the Synthesis Order files will find the following information useful The Order file is a plain ASCII text file having the Macintosh file type TEXT The file contains repeated entries of the form lt field_name gt lt tab gt lt field gt lt tab gt followed at the end of the file by a newline character Field names do not contain any embedded spaces Fields may be empty and may contain any printable character tab or newline may not appear within a field The first field name is always SYNTH_JOB_FILE and is followed by the ver sion number of the file format The current version described here is 1 The fields for this format are as follows SYNTH_JOB_FILE 1 RUN_DATE Contents of Run date text box RUN_ID Contents of Run ID text box CUSTOMER Contents of Customer text box USER_FIELD1 USER_FIELD2 Contents of 1 text box Contents of 2 text box MODEL Text f
111. s 304 for the example of 7 win dows When you next open OligoNet you will be able to open an additional win dow for each increment of 140K you set beyond the default value of 1024K Appendix II Resetting OligoNet Memory Appendix II 1 Applied Biosystems June 2002 Appendix II Using OligoNet Order Description of OligoNet Order OligoNet Order is the companion program provided with OligoNet and is intended to be used only for filling out Synthesis Orders It has no other pur pose since it has no connection with synthesizers on an AppleTalk network OligoNet Order is provided for distribution to people requesting DNA for use in order entry This appendix is written to address the needs of custom ers filling out the Synthesis Order When the application is first opened a Synthesis Order like that shown in Figure Appen II l appears Synthesis Order Untitled 1 a End procedure DMT 0n OMT Off Figure Appen Ill 1 Initial Synthesis Order in OligoNet Order The only default information on a Synthesis Order on OligoNet Order is the Run date provided by the Macintosh All of the fields which rely on com munication with a synthesizer are grayed out These include Instrument ROM Version and Instrument model Later when the form is opened in OligoNet these fields are filled in by communication with a synthesizer Appendix IN Using OligoNet Order Appendix
112. s and Functions Backing Up the Synthesizer Setup Since using the Save a Copy in command saves all the sequences cycles procedures and functions stored in your 392 394 synthesizer to a Macin tosh file in a single operation if you have made no changes since opening the Synthesizer window this feature should be used for backup of the en tire instrument setup because the process is time consuming and because you cannot restore just a single sequence or cycle The Send Copy to Syn thesizer command is then used to restore information to the synthesizer by overwriting any new sequences cycles procedures or functions entered since saving the original Synthesizer window Backing up the contents of your synthesizer as you would your hard disk is recommended on a regular basis One such backup copy should be kept and backup should be done on a periodic basis such as once a week or once a month Besides being used for backup the Save command is useful if you create a lot of custom cycles procedures or if you have any custom functions The command is particularly useful with custom functions since this is the only way to save such functions Importing and Exporting Sequences The Import Sequence command is used to read in files of various formats to Synthesis Orders and Synthesizer windows Acceptable formats include Model 373A DNA Sequencer files SeqEd files INHERIT sequence files and plain text files such as those produ
113. s 3 3 Applied Biosystems 3 4 Besides providing access to several standard Apple File menu commands which are not described here Close Save Save As Page Setup Print the File menu contains commands for the following tasks Creating a new Synthesis Order Opening a saved Synthesis Order or saved Synthesizer file Open com mand Opening communication with a synthesizer dialog box like that shown in Figure 3 5 Open Synthesizer command Sending synthesis setup information to a synthesizer using an open Syn thesizer window Send to Synthesizer command Sending synthesis setup information to a non active synthesizer using a Synthesizer file Send Copy to Synthesizer command Saving a copy of an open Synthesizer window Save a Copy In com mand Save an open Synthesis Order Save command and Save As commands Importing and exporting sequences from an open Synthesizer window 3 Overview of Commands and Capabilities June 2002 Applied Biosystems June 2002 New Synthesis Order 3 N The New Synthesis Order command is used both to document synthesis order information received from a customer and transfer the information to the synthesizer Synthesis information includes a listing of the sequence and the name of the cycle end procedure and DMT state to be used for synthe sis The Synthesizer Order looks like Figure 3 2 when the command is used while one or more Synthesizer windows are open and contains information
114. s Orders 2 Follow the Checklist of OligoNet Synthesizer Use on page 2 12 The Pre syn thesis Tasks checklist in Section 5 may be useful in setting up your syn thesizer 3 Refer to Sections 3 and 4 as needed in using OligoNet Section 3 is a reference for using Menu bar commands and Section 4 is a reference for editing a Synthesis Window Special Text Usage User Attention Five User Attention formats are used in the text of this man ual They are designed to draw your attention to safety issues or to issues rel evant to proper operation of the instrument Each one requires a particular level of observation or action as follows Note Used to call attention to information IMPORTANT _ Indicates information that is necessary for proper instrument oper ation Caution Damage to the instrument could result if you do not comply with this information WARNING Physical injury to the user or other persons could result if these precautions are not inplemented Advanced Feature A paragraph or other material marked with this term is not needed for normal operation but may be of interest to advanced users Advanced features are denoted in the body of the manual by the term Advanced Feature in bold at the beginning of a paragraph 1 Introduction 1 5 Applied Biosystems Special Program Symbols Two special symbols may occur in a Synthesizer Window view This symbol may appear and flash at the top of any Synt
115. sic knowledge of Macintosh operation OligoNet enables you to perform remotely most of the operations available at the synthesizer Besides creating a large library of sequences you can edit or create your own versions of the cycles functions and procedures used to perform syntheses at a synthesizer Then you set up the synthesizer and transfer to it all the information needed to perform your synthesis You can then start the synthesis at the synthesizer and monitor the progress of the synthesis at your Macintosh By providing you with the capability of entering sequences cycles functions and procedures at your Macintosh OligoNet provides you with more than one entry method for sequence and other synthesis information DNA sequences obtained from 373 sample files INHERIT sequence files and other database manipulation programs can also be directly transferred to the synthesizer This minimizes errors and gives you mass storage capability If you operate a core facility that receives DNA synthesis orders OligoNet can simplify your task Customers can electronically send their orders to your computer Using OligoNet you can directly transfer a customer s sequence to the instrument for synthesis When you enter the New Synthesis Order for a customer s synthesis in OligoNet this document serves as a syn thesis log allowing you to keep exact records of such transactions Ifyou have questions or need assistance while using OligoNet pleas
116. stem Administrator for aid in setting up a multi synthesizer network for your site Se Terminator Figure 5 4 Simple AppleTalk Network 5 Using OligoNet June 2002 Applied Biosystems June 2002 Organizing and Processing Synthesis Order Files To give you some idea of how you might set up your file system to support OligoNet and process Synthesis Orders the file structure shown in Figure 5 5 and the procedure presented in Processing Synthesis Orders are offered together as an example You can adopt this organization and process or modify them as you desire to meet your needs Setting Up Your File System As you ll notice upon examining Figure 5 5 the key elements in the example file system are the To Do and the Done folders The To Do folder con tains unprocessed Synthesis Orders and the Done folder contains complet ed Orders The Synthesis Order file names are assigned by the Run ID parameter when creating the form and should contain both a name and number component so that orders can be uniquely identified both by cus tomer name and order of processing OligoNet Folder Alice001 Alice002 Alice003 Alice004 D D D D D lice005 D D D D Jim003 Jim004 Jim00S lice009 Alice006 AliceO07 Alice008 Jim001 Figure 5 5 Example File Organization 5 Using OligoNet 5 15 Applied Biosystems 5 16 Processing Synthesis Orders Since ea
117. synthesizer Prepare to Start Interrupt Synchronize Clocks Enter Password Change Name Figure 3 14 The Synthesizer Menu The Synthesizer menu allows you to prepare to start the synthesizer from your Macintosh interrupt a synthesis in progress or perform three tasks from your Macintosh which affect communication between the application and the synthesizer Prepare to Start The Prepare to Start command is used after a synthesis setup and all other information needed for synthesis is ready and you want to perform synthesis Like the Send to Synthesizer command this command sends the synthesis setup and all other information needed for synthesis to the synthesizer In addition this command initiates the starting process in the synthesizer As part of this process the synthesizer checks all reagent bottles to determine usage using bottle alarm information entered during the last bottle change Synthesis is started in a two step process 1 use the Prepare to Start com mand at the Macintosh and then 2 continue the start process at the synthe sizer If one or more synthesizer bottles contain insufficient reagent to complete the synthesis a message like that shown in Figure 3 15 will be dis played at the synthesizer Bottle 9 will interrupt Cancel Start gt in 18 cycles Next Alarm gt Change Bottle gt Total of 2 alarms Continue Start gt Figure 3 15 392 394 Message Indicating Insufficient Reagent
118. systems MDS Sciex 1 800 952 4716 1 650 638 6223 Outside North America Region Telephone Dial Fax Dial Africa and the Middle East Africa English Speaking and West Asia Fairlands South Africa 27 11 478 0411 27 11 478 0349 South Africa Johannesburg 27 11 478 0411 27 11 478 0349 Middle Eastern Countries and North Africa Monza Italia 39 0 39 8389 481 39 0 39 8389 493 Eastern As ia China Oceania Australia Scoresby Victoria 61 3 9730 8600 61 3 9730 8799 China Beijing 86 10 64106608 86 10 64106617 Hong Kong 852 2756 6928 852 2756 6968 Korea Seoul 82 2 593 6470 6471 82 2 593 6472 Malaysia Petaling Jaya 60 3 758 8268 60 3 754 9043 Singapore 65 896 2168 65 896 2147 Taiwan Taipei Hsien 886 2 22358 2838 886 2 2358 2839 Thailand Bangkok 66 2 719 6405 66 2 319 9788 Europe Austria Wien 43 0 1 867 35 750 43 0 1 867 35 75 11 Belgium 32 0 2 712 5555 32 0 2 712 5516 Czech Republic and Slovakia Praha 420261 222 164 420 2 61 222 168 Denmark Naerum 45 45 58 60 00 45 45 58 60 01 Finland Espoo 358 0 9 251 24 250 358 0 9 251 24 243 France Paris 33 0 1 69 59 85 85 33 0 1 69 59 85 00 Germany Weiterstadt 49 0 6150 101 0 49 0 6150 101 101 Warszawa Hungary Budapest 36 0 1 270 8398 36 0 1 270 8288 Italy Milano
119. t Menu Undo Redo Can t Undo Cut Copy Paste Clear Select All Show Clipboard Read Selection Find Find Same Replace Replace Same Use Sounds Synthesizer Prepare to Start Interrupt Synchronize Clocks Enter Password Change Name Order Menu Send Order to Synthesizer Send All Orders Send Order to Synthesizer Send All Orders Copy from Synthesizer Window Menu 3 3 3 5 3 10 3 11 3 12 3 13 3 14 3 15 3 16 3 16 3 16 3 17 3 17 3 17 3 17 3 18 3 18 3 20 3 20 3 21 3 22 3 22 3 23 3 23 3 25 3 26 3 26 June 2002 Applied Biosystems June 2002 Editing the Synthesizer Window About the Synthesizer Window Communication View Edit Sequence View Naming a Sequence Creating a New Sequence Importing a Sequence Copying a Sequence Editing a Sequence Set up Synthesis View Single Cycle Synthesis Setup Multiple Cycle Synthesis Setup Monitor Synthesis View Explanation of Monitor View Fields Interrupting a Cycle at a Safe Step Bottle Alarms View Trityl Monitor View Regular Monitor View Raw Data View Base Source View Normal Bottle Replacement Customized Bottle Assignment Replacement Edit Cycle View Creating a New Cycle Editing Existing User Cycles Edit Begin Procedure View Creating a New Begin Procedure Edit End Procedure View Creating a New End Procedure Edit Bottle Procedure View General Instructions for Creating User defined Bottle Change Procedures Special Instructions for Creating Auto Dil
120. t a synthesizer or first a zone and then a synthesizer from the dialog box and click OK a Synthesizer window like that shown in Figure 3 6 appears 3 Overview of Commands and Capabilities June 2002 Applied Biosystems June 2002 SS synthesizer Synthesizer Choose function Instrument 394 8 Rev 2 00 Model 394 Base positions 8 Columns 4 ROM Version 200 Trityl Monitor Available Remote control from Macintosh With password Reading amp editing allowed Without password Reading amp editing allowed Figure 3 6 Open Synthesizer window A complete description of all the views available in a Synthesizer window and the entries that can be made in these views is presented in Section 4 Send to Synthesizer The Send to Synthesizer command is used to send any changes made in a Synthesizer window to a synthesizer Changes which may be sent include new or modified versions of the following e Sequences e Synthesis Setup e Bottle Alarms e Trityl Monitor Setup when the synthesizer is equipped with AutoAnal ysis e Base Source e Cycles e Begin Procedures e End Procedures e Bottle Procedures e Shutdown Procedure e Functions e Power Fail setting e Time Date setting 3 Overview of Commands and Capabilities 3 11 Applied Biosystems 3 12 e Relay Configuration If a sequence cycle or procedure is actively in use by the synthesizer you will not be allowed to change
121. tablishing communications with OligoNet For each additional synthesizer added to the network the default name increments by one For example the second synthesizer establishing communications is called Synthesizer 2 8 Click OK to open communication with Synthesizer 1 the default name assigned to your synthesizer If the Use Sounds command in the Edit menu is checked you will hear some beeping You will then see a Syn thesizer window open like that shown in Figure 2 9 Besides seeing a Synthesizer window open when you click OK you will see the Menu bar change to appear as shown in Figure 2 8 All of the Synthesizer commands on the Menu bar are grayed out until you open a Synthesizer window File Edit Synthesizer rder Window Figure 2 8 Menu Bar 2 8 2 Installation and Start Up June 2002 Applied Biosystems June 2002 Choose function Communication vj Instrument 394 8 Rev 2 00 Model 394 Base positions 8 Columns 4 ROM Version 200 Trityl Monitor Available Remote control from Macintosh With password Reading amp editing allowed Without password Reading amp editing allowed Figure 2 9 Synthesizer 1 Communication View The presentation of the Communications view for your synthesizer indicates that communications has been established This view will show the model number of your instrument the version of software the number of base positions and the number of columns with which it is
122. tep in creating or editing a bottle change procedure is to choose the user supplied procedures from the Procedures pop up menu If you need help because you are unfamiliar with editing this type of view refer to the more detailed procedure provided for Creating a New Cycle Editing Existing User Cycles on page 4 23 Note The most likely changes you will make to a bottle change proce dure are 1 insert one or more new steps 2 delete one or more steps and 3 change the time or active base status set for an existing procedure step The general procedure is as follows 1 Select an empty user bottle change procedure and then copy the desired non programmable bottle change procedure 2 Insert any additional steps needed to add functions to your procedure This is done by selecting the step above the desired new step inserting a blank step line selecting the desired function and changing the time for the new step if desired Note Time values can not be entered for some steps steps with blank values Except for the possible exception of the Time field and the Active field all of the controls which are used to modify a bottle change procedure Insert Delete Time Function and Safe are open for entry as soon as a step is selected 3 Delete any existing step that you don t want 4 Type in a new name if you want a name other than the copy name pro vided as a default une 2002 4 Editing the Synthesizer Window
123. ting in a core laboratory environment When a Synthesizer window is first created or opened it looks like Figure 4 1 for synthesizers equipped with AutoAnaly sis the Trityl Monitor will be available Synthesizer Synthesizer 1 Choose function Instrument 394 8 Rew 2 00 Model 394 Base positions Ea Columns 4 ROM Version 200 Trityl Monitor Available Remote control from Macintosh With password Reading amp editing allowed Without password Reading amp editing allowed Figure 4 1 The Communication View of a Synthesizer Window A separate view exists for each line listed on the Choose Function pop up menu as shown in Figure 4 2 This menu is used to navigate between views in the Synthesizer window 4 Editing the Synthesizer Window 4 3 Applied Biosystems 4 4 Communication View The Communication view is the initial Synthesizer window view that appears when you use the Open Synthesizer command The window also appears when you choose a synthesizer upon starting the application As you can see it provides you with the basic information about the hardware on a par ticular synthesizer such as Model either 392 or 394 Base positions either 5 or 8 Columns either 2 or 4 ROM Version number Trityl Monitor AutoAnalysis installed or not installed and describes the present settings for Read Write permission If a password is required you will be pres
124. tion Start up the Macintosh and launch the application by double clicking the OligoNet icon This will present the application identifier window shown in Figure 2 6 briefly and then the Open Synthesizer dialog box shown in Figure 2 7 appears epi ene OligoNet 1991 1992 Applied Biosystems Inc This program was written with Mac Appi 1985 1991 Apple Computer Inc AN Rights Reserved Figure 2 6 OligoNet Identifier Window Any time you start OligoNet the start up window is displayed briefly and then the Open Synthesizer window appears The Open Synthesizer dialog box will contain names of synthesizers available on the network If this box is empty refer to the information later on in this section under Trouble shooting 2 Installation and Start Up 2 7 Applied Biosystems Open Synthesizer Select a Synthesizer Synthesizer 1 Cancel Figure 2 7 The Open Synthesizer Dialog Box Note If you have installed the synthesizer on a multi zone network you will see two lists when you start OligoNet the second list contains the names of zones on your network In this case first select the zone in which the synthesizer is installed and then select Synthe sizer 1 The default limit set for the number of Synthesizer Windows open at the same time is five If you need more see the instructions pro vided in Appendix II The name Synthesizer 1 is assigned to the first synthesizer es
125. to Active Note The second disk contains the OligoNet Order program This is a simplified version of OligoNet for filling out oligo order forms You do not need to install OligoNet Order If you decide you want to use it instructions are provided in Appendix III 2 Installation and Start Up June 2002 Applied Biosystems Program Cartridge Installation The Program Cartridge is easy to find on the rear of the instrument since it is located on the upper left edge as viewed from the rear and is oriented vertically 1 Turn off synthesizer power 2 Push in on old Program Cartridge until it clicks and then release so it pops out of its socket Remove the cartridge 3 Insert new Program Cartridge and push on it until it clicks into place Note If the Program Cartridge is inserted upside down it will not be firmly latched Make sure it clicks into place and is firmly latched after it has been inserted 4 Turn the power back on and make sure that the instrument boots up June 2002 2 Installation and Start Up 2 5 Applied Biosystems 2 6 Testing Your Installation Beginning an OligoNet Session The first step in checking out or beginning communication between Oligo Net on your Macintosh and the synthesizer or synthesizers on the network is to start one or more synthesizers as described below Setting Up Communication Turn the 392 394 power switch on This switch is located on the front of the instrume
126. tted access If you click None you will not have READ WRITE permission but either READ ONLY or NONE depending upon how permission has been set at the synthesizer when the password was first chosen Password Enter password for synthesizer Name Figure 3 20 The Password Dialog Box To obtain full permission for a protected Synthesizer window enter the pass word if you originally assigned it or obtained it from the owner if you need Write permission To set a password when none exists you must set the password from the syn thesizer This is done from the synthesizer Communications Display shown in Figure 3 21 which is accessed from Page 5 of the Main Menu Communications AppleTalk Name Synthesizer 1 Access with password READ WRITE without password READ WRITE Figure 3 21 392 394 Synthesizer Communications Display 3 Overview of Commands and Capabilities 3 21 Applied Biosystems e a u a Setting the password from the Communications Display is done as follows 1 Move the cursor to the READ WRITE field of the with and without password lines and then use the Next or Prev key to toggle through the available permission choices These include the following READ WRITE the default for both lines READ ONLY NONE 2 Move the cursor to the 4 character password entry field marked by parentheses and enter an alphanumeric password code of your choice The keypad is used
127. uals needing only the default synthesizer settings and at the same time supports individuals with special needs for custom Synthesizer files 5 Using OligoNet 5 13 Applied Biosystems Terminator 5 14 Setting Up a Multi synthesizer Network Once a number of synthesizers have been correctly installed as nodes in a network the only real requirement for using them is to assign unique names to the synthesizers using the Change Name command This is important since the name assigned to the synthesizers is used to uniquely identify them in both Synthesizer Windows and Synthesis Orders When synthesizers are first turned on the synthesizers themselves assign the names Synthesizer 1 Synthesizer 2 Synthesizer 3 etc according to the order in which they are powered up To find out which synthesizer is which use the front panel display on the synthesizer you want to identify On this display go to the page 5 of the Main Menu and then select Communi cations This brings up the Communications display like that shown in Fig ure 5 3 synthesizer name may differ Communications AppleTalk Name Synthesizer 1 Access with password READ WRITE without password READ WRITE Figure 5 3 Communications Display Figure 5 4 shows a simple AppleTalk network with a Macintosh 3 synthesiz ers and a printer It is beyond the scope of this manual to cover the variety of possible networks See your Sy
128. umber of addition cycles When an alarm is triggered the synthesizer interrupts the synthesis and displays a bottle alarm message To operate effectively the alarm must be set correctly If set too high the bottle could run dry causing the synthesis to fail The initial Bottle Alarms view is shown in Figure 4 12 UE sunthesizer Synthesizer 1 EN Choose function Bottle Alarms v BOTTLE CYCLES USED ALARM AT ALARM 0 lt gt Cycles used 2 0 a off 3 o o o kooo 4 o o off 5 o a off 6 o o off O Alarm on 7 o o off 8 o o off S a a hi Gee Alarm at cycle 11 o o off 12 o 0 off 14 o o off 15 o o off All alarms off 18 0 0 off Q Figure 4 12 The Initial Bottle Alarms View Synthesizer bottles are listed on the first column of the table in the view Notice that the first line of the table is selected and that the Alarm at cycle entry field is also selected The default value for the second column Cycles used is 0 During use if an alarm goes off and the bottle is changed using the Bottle Change procedure in the synthesizer the Cycles used value is automatically reset to 0 Entry is made as follows for each bottle alarm to be set 1 Select the table line to be edited by clicking it If you do not desire to set a value for the second column proceed to step 3 2 To seta value for the second column click the Cycles used entry field then enter the
129. us box is presented while your sequence is transferred to the location listed in the box and your synthesis setup Cycle and End Procedure names are loaded into the synthesis setup for column 1 You can see the synthesis setup transferred to the synthesizer by viewing the Set Up view in the Synthesizer window Note Using the Send Order to Synthesizer command fills in the Set up view for a Synthesizer window for that synthesizer Send All Orders The Send All Orders command is used when multiple open Synthesis Orders are to be sent at once This command works much like the Send Order to Synthesizer command but does not present a dialog box like that shown in Figure 3 24 Instead of relying on the information provided in the dialog box this command relies on the information provided in the fields behind the Column and the Seq No check boxes on each Synthesis Order see information under Advanced Feature on page 3 6 Like the Send Order command this command allows you to select both the column on which the order is to be placed and the sequence slot con taining the sequence to be used for synthesis when both entries are made on the Synthesis Order If you specify either the Column number or the Seq No but not both you obtain the same functionality provided by dialog box for the Send Order command By entering just the Seq No you specify that the sequence be copied into the specified slot location and the order is
130. using the mouse it is possible to choose a command by using the Command key equivalent listed on the menu by the command For example choose the Edit Sequence command by holding down the key and then pressing 1 Edit Sequence View 1 The Edit Sequence view of the Synthesizer window is used to select create copy or edit a sequence you desire to synthesize and initially appears as shown in the left side of Figure 4 3 The Sequence pop up menu shown in the right side of Figure 4 3 is used to select an existing sequence or select a sequence location to be used for creating a new sequence Once you have created or edited a sequence you can print out a copy send the sequence to the synthesizer or save the sequence either as part of the present Synthesizer window Save command or as a part of a new copy of a Synthesizer window As you can see 34 sequences may be created and stored in the Edit View of a Synthesizer window The term Mac used in the last four sequence names indicates that these locations are used in the synthesizer for sequences sent by Synthesis Order when no other sequence location is specified For this reason it is a good idea to avoid using the last four locations for permanent storage of sequences 1 Seq01 2 SeqO2 3 S eq03 4 Seq04 5 eq05 6 Seq06 7 SeqO 8 Seq08 9 eq09 ZUE synthesizer Synthesizer 1 Choose function Edit Sequence 1i v Sequence 1 Seq01
131. ute Procedures Edit Shutdown Procedure View Edit Function View General Description of View Procedure for Defining a User Function Printing Functions Power Fail View Set Time Date Format View Relay Configuration View 4 3 4 4 4 5 4 6 4 6 4 7 4 8 4 9 4 9 4 12 4 13 4 13 4 14 4 16 4 18 4 18 4 19 4 20 4 20 4 21 4 22 4 23 4 30 4 31 4 32 4 33 4 34 4 35 4 36 4 39 4 41 4 41 4 42 4 43 4 44 4 45 4 46 iii Applied Biosystems Using OligoNet Practical Use of OligoNet Entering Sequences Cycles and Procedures Creating Your Sequences Listing of Standard CE Sulfur and RNA Cycles Creating your own Cycles Procedures and Functions Pre synthesis Tasks Saving and Retrieving Sequences Cycles Procedures and Functions Backing Up the Synthesizer Setup Importing and Exporting Sequences Exporting and Importing Cycles and Procedures Performing Multiple Cycle Synthesis Example of Multiple Cycle Synthesis Sharing a Synthesizer with Many Macintoshes Setting Up a Multi synthesizer Network Organizing and Processing Synthesis Order Files Setting Up Your File System Processing Synthesis Orders Processing variations Appendix I Nucleotide Ambiguity Conventions Appendix II Resetting OligoNet Memory Appendix III Using OligoNet Order Index 5 3 5 4 5 4 5 5 5 6 5 7 5 8 5 8 5 8 5 9 5 10 5 10 5 12 5 14 5 15 5 15 5 16 5 17 June 2002 Applied Biosystems June 2002 1 Introduction This sect
132. v Procedure 33 Auto 1 09g v Name Auto 1 0g STEP FUNCTION NAME NUM TIME ACTIVE Sore 5 0 3 Block Flush 1 4 0 Yes Phos Prep 4 Block Vent 111 5 0 Yes Cap Prep 5 Flush to Bases 14 10 0 Yes Wait 6 18 to Bases 86 5 0 Yes Interrupt 7 Interrupt 104 Yes Sirt Datri tyi 8 Dilute A 170 Yes 5 Sayin 9 Dilute A 170 Yes 10 gt Dilute A 170 Yes Safer Yes ONG 11 gt Dilute A 170 Yes Fede 12 Bottle01 On 150 Yes AGES O mE oe 13 gt 18 to A 70 20 Yes O8 O amp Of OF O8 14 gt BottleOl Off 151 Yes Figure 4 28 ABI Auto Dilute Procedure Example An important detail of the standard ABI dilute procedures is shown in the figure above In the procedure shown in the figure Auto 1 0g you will notice that four steps are used to deliver acetonitrile to each bottle even though the actual dilution is carried out in one continuous delivery of ace tonitrile Each step represents the time required to deliver the standard vol ume of the phosphoramidite 0 25g The procedure for a 0 5g bottle will 4 Editing the Synthesizer Window June 2002 Applied Biosystems have two such steps for each delivery and the procedure for a 0 25g bottle will have one such step 1 The first step in developing your own procedure is to copy the informa tion on the Auto Dilute Calibration table from the synthesizer This is obtained as follows Note Each new synthesizer menu is accessed from the previous menu by usi
133. v Seq01 Clear Length 0 A 0 6 0 c 0 T 0 5 0 6 0 7 0 8 0 Name copa CAC gt CAG CAT S K GC cT GT gt nnn z nore AGC gt cACT gt AGT gt lt GCT gt AGCT gt gt lt 3 Rey Lorplement RELY lt gt 5678 Copy from Figure 4 3 The Edit Sequence View Sequence Pop up Menu 4 Editing the Synthesizer Window EK 10 Seqio 11 Seqit 12 Seqi2 13 Seql3 14 Seqi4 15 Segi5 16 Seql6 17 Seqi 18 Seqi8 19 Seqig9 20 Seq20 21 Seq 1 22 Seq2 23 eq23 24 Seq24 25 eq25 26 Seq 26 27 Seq27 28 eq28 29 Seq29 30 eq30 31 Seq31 Mac 32 eq32 Mac 33 eq33 Mac 34 Seq34 Mac 4 5 Applied Biosystems 4 6 Note If a vertical line appears in the Sequence name field this indicates that only the 10 characters of the sequence name appearing to the left of the line will be visible on the synthesizer display The entire name up to 31 characters is stored in the synthesizer but is not visible on the display Naming a Sequence Once you select a sequence name this name appears after Sequence on the first line of the window You can change the name if you desire by placing the insertion point in the Name field just below Initially the name in this field will be the same as the Sequence you selected from the pop up menu Note Sequence names must be unique A warning icon amp appears if a duplicate sequence n
134. ve for example Import sequence or Export Sequence when the Edit Sequence view is active The Export command is available in edit views for both locked and unlocked versions of edited views but the Import command becomes available only for unlocked versions of the views The Import Sequence version of the command can import the following types of files 373 sequence files INHERIT sequence files and plain text files such as those produced by word processors The Export Sequence com mand exports sequences in plain text format Using the Export Procedure command in all Edit views except Edit Sequence and Edit Function produces a specific type of OligoNet file Once exported each type of file can be imported into the same edit view from which it was exported The various file types and corresponding icons are shown in Figure 3 10 below Ls Sequence file icon E Bottle procedure 1 Seqit file icon i Eot01 AEI Ls Ls Cycle file icon B Shutdown procedure 5 file icon h Primer ABI Shut 1 L Begin procedure Ta file icon a ABI Begin End procedure file icon G m a oa co m Figure 3 10 OligoNet File Types with Icons 3 Overview of Commands and Capabilities June 2002 Applied Biosystems Edit Menu Edit beak Ende Find find tage Replace Hepiage tame Use Sounds Show Clipboard Figure 3 11 The Edit Menu Besides providing access to the standard Apple Edit menu commands listed

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