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ABI PRISM® 3100 Genetic Analyzer User`s Manual

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1. Step Action 1 Push down on the polymer reserve syringe to move bubbles through to the lower right of the block Push slowly or tap to minimize the amount of polymer used 2 Push down slowly on the array fill syringe to move bubbles down the channel The bubbles will collect where the channels join Bubbles collect here 5 Polymer block tube 0 3 a Hold down the anode buffer pin valve and simultaneously push down on the array fill syringe to build pressure in the channels b Release the buffer pin valve while still pressing down on the array fill syringe to expel bubbles into the polymer block tube y 4 Repeat step 3 as necessary IMPORTANT Make sure all air bubbles are pushed out of the tubing assembly into the lower buffer reservoir before proceeding There should be no bubbles in the tubing or channel of the lower polymer block Section Autosampler Calibration When to Calibrate the Autosampler Calibrating the Autosampler Calibrate the autosampler only as needed Symptoms of autosampler alignment problems may include Poor injection for a small number of capillaries Low signal strength No evidence of sample To calibrate the autosampler Step Action 1 From the Tools menu select Calibrate Autosampler 3100 Data Collection Software Version 1 0 File View Instrument Bye Service Help Cele Az Pla
2. Save itas a Change the plate tab delimited text file name in Notepad using the Export command Close the file In 3100 Data saving changes Collection software as a tab delimited import the text file filename plt file rong pe For instructions for each method see the pages listed in the following table Method See Page Method See Page 1 6 19 4 6 28 2 3 31 and 3 36 5 6 29 3 6 24 6 6 30 6 4 Working with Plate Records About the Plate Record Fields Introduction Definitions and options for the following plate record fields are provided in this section Dye Sets Dye sets 4 Mobility files Run modules Analysis modules DNA fragments are detected and identified by the fluorescent dyes with which they are chemically labeled Dyes are purchased and used as dye sets which are optimized for particular applications When a dye is bound to a DNA fragment it changes the rate at which the fragment migrates during electrophoresis When DNA fragments that are labeled with different dyes are electrophoresed together the fragments do not migrate with equal spacing because different dyes change the migration rate to different extents Without correction this would lead to an uneven separation of peaks in the electropherogram The data needed to perform mobility shift correction are contained in mobility files see Mobility Files below Dye Sets Provided
3. Support Files File nam2 Fa Files of type Jan Files x Cancel Navigate to the folder in which you want to save the run module file Note Due to software limitations you cannot select a folder on the desktop Double click the destination folder so that its contents are displayed in the pane In the File name box type a name for the file Click OK This creates a run module in the specified folder Comments This message confirms Export complete a successful export About Importing a Module Importing a Run Module File The data in the exported file is copied to the donor database to re create the original run module This is known as importing the module The re created run module has the same name as the original except for a unique number added by the software The number is based on the date This prevents conflicts with the original run module in the donor database Note You cannot read a run module file because it is written in code To import a run module file Step Action 1 Go to the computer to which you want to transfer the run module 2 Click the Module Editor button on the toolbar to open the Module Editor dialog box 3 In the Modules group box click Import This opens a browser dialog box Select file x Look in fo abi x al Ez e 3100 E Gene can E SegAnal E Shared T Support Files E Genefcan36_POP4D2
4. Cancel Prey Next Fie 3 Follow the directions given in the wizard to replace or install an array Maintenance 8 15 Capillary Array Maintenance Caring for the Follow these guidelines to properly care for the capillary array Capillary Array 4 Wear gloves and handle the capillary array gently Do not touch the detection cell If it is dirty see Cleaning the Detection Cell on page 8 14 Keep the ends of the capillary array wet at all times Always loosen the capillary array nut before pulling out the upper polymer block Do not overtighten the capillary array nut Cleaning the To clean the capillary array Capillary Array Step Action 1 Flush the capillary array with fresh polymer as instructed in the Installing and Removing the Capillary Array on page 8 15 Clean off any polymer buildup crystals on the instrument including the capillary electrodes and the stripper plate with deionized water and lint free tissue Note When cleaning the capillary electrodes be careful not to bend them out of position If the electrodes do get bent follow the procedure Checking Capillary Alignment Using the Capillary Ruler on page 8 16 IMPORTANT Never use organic solvents to clean the instrument Clean the detection cell as instructed on page 8 14 Filling the Capillary To fill the capillary array with polymer using manual control commands A
5. Section Checking the Available Space and Deleting Records In This Section The following topics are covered in this section Topic See Page Checking the Available Hard Drive Space 3 16 Checking the Available Database Space 3 17 Deleting Records from the Database 3 17 Introduction Before a run or batch of runs check the available space to ensure there is sufficient space to store the data you will create Every week delete records in the database The sections that follow tell you How to check the available hard drive space on drive D for the extracted sample files How to check the available space in the instrument database on drive E for the raw data Where to find the procedures for deleting database records Performing a Run 3 15 Checking the Available Hard Drive Space Checking Hard To check the hard drive for space for sample files Drive Space Step Action 1 Double click the My Computer icon on the desktop to view the drives 2 Right click on a the D drive and select Properties A My Computer Op x Fils Edit View Help B My Conputer z Sl alte Bs m 3 Floppy 4 ra Os C Open Explore Find Sharing Format Create Shortcut Propertes The Properties dialog box opens displaying the used and free space 3100Files D Properties i 1x General Tools Sharing Security Label 3100Files Type Locd Disk
6. 0 0 ee cece eee ene ene 3 8 Working with Plate Assemblies 0 2c eee cc cence eens 3 9 Section Starting the 3100 System 0 ccc ccc cece een ene ee eenees 3 11 Starting the Computer soc i ec nse deere bg decd Genet bags ede de Mande ead 3 12 Starting the Instrument e seces nort ie ee ee cee ee eect e net eeenee 3 13 Starting the 3100 Data Collection Software 00 00 eee eee ee 3 14 Section Checking the Available Space and Deleting Records 3 15 Checking the Available Hard Drive Space 0 0 0 cece eee eee 3 16 Checking the Available Database Space 0 00 cece cee eee 3 17 Deleting Records from the Database 00 0 eee ee eee 3 17 Section Preparing the Instrument 0 c ccc cee c eet e ee eeees 3 19 Instrument Setups acid etd ent ait wea S eM GEENA ea oss 3 20 Preparing Buffer and Filling the Reservoirs 0 0 00 e cece cee ee eee 3 22 Calibrating the Instrument 2 0 0 0 cece ee eee nee 3 24 Placing the Plate onto the Autosampler 0 0 cece cee cee eee 3 25 Section Setting Up the Software 0 0 0 ccc ccc ccc cece nent n eee eeees 3 27 Setting Software Preferences cesigo uipa cece ee eee ee eee ence ee enee 3 28 About Plate Records 260 0 sc dhat eters ged noe e aae a be ie See ete eee ads 3 30 Creating a Plate Record for GeneScan Analysis 0 00 00 e eee eee nee 3 31 Creating a Plate Record for DNA Sequencing
7. 0 0 eee eee eee ee 4 46 numSpectralBins Parameter 1 0 0 00 ccc eeeeen ene 4 46 Parameters Specific to sequenceStandard dataType 0 0 0 cece eee eee ee 4 47 startptOffset Parameter 2 2 cece eee eens 4 48 maxScansAnalyzed Parameter 1 0 0 0 cee ce eee eee eee ee 4 49 startptRange Parameter eiiie eip iis aera ee SA eats oe alia ae 4 49 minRankQ Parameter oe n ee eee tenn nee eens 4 50 Software OVENI W e aa ates Meats aatancd a a US slate ota al cep adler ER sothangue EE 5 1 Section About the 3100 Software 0 0 ccc ccc ccc cence een e een eeeeeees 5 3 ABI PRISM 3100 Genetic Analyzer Software CD ROMS 00000000 e eee 5 4 3100 Genetic Analyzer Software Suite 0 0 cee eee 5 5 Types and Locations of Files 00 0 tar nire ce teen eens 5 9 Section Setting the Format for the Displayed Dye Colors 00e0eees 5 11 Using the Edit Dye Display Information Dialog Box 00000000005 5 12 Using the Set Color Dialog Box 2 0 ccc ene 5 13 Section Controlling the Instrument Using Manual Control 4 5 15 Manual Control Commands 0 0c cece ee eee e teen eee 5 16 Using Manual Control Commands 0 0 0 eee 5 17 Section Working with Run Modules 0 0 ccc cence een n ence ee neeees 5 19 Viewing a Run Module 2 i n a E eee A 5 20 Editing or Creating a Run Module 0 0 0 5 21 Run Mod le Parameters ois a
8. Check for bubbles and remove if present Bubbles can cause polymer to fill the jar Detection window pops out while replacing the capillary array Replacing the window in the correct orientation is difficult Tightening of the array ferrule knob at the gel block causes high tension Loosen the array ferrule knob to allow the secure placement of the window Retighten and close the detection door Detection window stuck It is difficult to remove when changing the capillary array To loosen the detection window a Undo the array ferrule knob and pull the polymer block towards you to first notch b Remove the capillary comb from the holder in oven c Hold both sides of the capillary array around the detection window area and apply gentle pressure equally on both sides d Release 9 8 Troubleshooting Data Flow Overview In This Appendix The following topics are covered in this appendix Topic See Page About Data Flow A 2 Organization of the CCD A 3 Incident Fluorescence A 4 Frame Data A 5 Multicomponenting A 6 Configuring Data Flow A 7 Mobility Shift Correction for DNA Sequencing A 8 Data Flow A 1 About Data Flow Introduction To successfully operate and troubleshoot the ABI PRISM 3100 Genetic Analyzer it helps to have a basic understanding of how data is collected and processed prior to analysis A summary of the data flow is shown below
9. Data Collection Summar Fluorescence image Frame data y on the CCD Binary data that is a mathematical representation of the fluorescence image on the CCD One set of frame data is collected per time point with thousands of time points in a run ABIF sample file 16 unanalyzed files stored on computer workstation hard drive Accessory data ai Stored in the ABI Prism Decis 1on 3100 Data Collection Software point database e g plate infor BioLIMS data sets mation EPT data run events Auto Extractor Data processing 16 unanalyzed data sets on e Data summation e Multicomponenting another networked computer Auto Extractor entir e Data reorganization Processed frame data Run data stored in the instrument database A 2 Data Flow Organization of the CCD Pixelated Array Spatial and Spectral Dimensions Bins As the dye labeled DNA fragments pass through the laser beam the fluorescence emitted is focused onto the CCD by the spectrograph The CCD is a silicon chip that is divided into a two dimensional array composed of thousands of electrically insulated pixels Each pixel stores an amount of electrical charge proportional to the intensity of light striking it The spatial and spectral dimensions of the array are as follows Axis Dimension X axis Spatial Y axis Spectral When the fluorescence data is read from the CCD the charges from 3 pixels in the spatial dime
10. 1 650 638 5981 BioInformatics includes BioLIMS BioMerge 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 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 1 800 542 2369 U S only or 1 781 271 0045 1 781 275 8581 Applied Biosystems MDS Sciex 1 800 952 4716 1 650 638 6223 Outside North Americ
11. Command Category Electrophoresis Send Command Command Name Value Range Set power supply v 0n Comment Turn the electrophoresis power supply on or off Select a Command Category from the drop down list Select a Command Name Note To check a command s function read the Comment box Enter or select a Value Click Send Command Note Some tasks require that you send more than one manual control command For example to heat the oven to 50 C you first send a command to turn on the oven and then you send a command to set the temperature Software 5 17 5 18 Software Section Working with Run Modules In This Section The following topics are covered in this section Topic See Page Viewing a Run Module 5 20 Editing or Creating a Run Module 5 21 Run Module Parameters 5 22 Transferring Run Modules Between Computers 5 23 Introduction The run module specifies the conditions for how the sample is run Examples include Duration of the run Run temperature Injection time Software 5 19 Viewing a Run Module Viewing a Run To view a run module Module Step Action 1 From the Tools menu select Module Editor or click the Module Editor button on the toolbar BI eo 3100 Data Collection Software Version 1 0 File View Instrument i Service Help cs gt T Plate Editor Polymer izard Install Capilla
12. POP polymers are stable on the instrument for 7 days Store any remaining ABI PRISM 3100 POP polymer at 2 to 8 C until the expiration date printed on the jar Note Excessively hot environments may shorten the working life of the polymer As the generator of potentially hazardous waste it is your responsibility to perform the actions listed below Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure the health and safety of all personnel in your laboratory Ensure that the instrument waste is stored transferred transported and disposed of according to all local state provincial or national regulations Note Radioactive or biohazardous materials may require special handling and disposal limitations may apply Injection Solution Overview The injection solution is a fluid that is used to Denature separate the DNA strands Resuspend DNA samples before starting a sample run Resuspend calibration standards during the preparation of a calibration or sample run Maintain the electrical connection between the polymer in the capillaries and the injection wells in the electrophoresis chamber by acting as an electrolyte necessary for electrophoresis Hi Di Formamide_ The injection solution recommended for use with the 3100 Genetic Analyzer is Hi Di Formamide P N 4311320 or formamide of equivalent qu
13. Step Action 1 Quit the 3100 Data Collection software 2 Ensure OrbixWeb Daemon is running 3 Right click the Start button and select Explore This opens Windows NT Explorer 4 Navigate to the Bin folder in the following directory D AppliedBio abi 3100 Bin 5 Double click the Reextractor exe icon This opens the 3100 Reextractor box 6 If the 3100 Reextractor box was opened before one or more runs were completed click Refresh to update the displayed runs 7 In the 3100 Reextractor box select the run that you want to re extract The Extract button is now enabled Refresh Extract 7 6 System Management and Networking To re extract processed frame data continued Step Action 8 Click Extract This opens the Specify Extraction Directory dialog box Please specify a directory which will be created in the Data Extractor directory Prun_C285_1 999 10 01_4 Cancel Type the name that you want to give the folder in which the re extracted sample files will be saved Note You cannot specify the location of this folder It will be created in the existing Data Extractor folder in the following directory D AppliedBio abi 3100 Data Extractor 9 To analyze these sample files open them in either ABI PRISM DNA Sequencing Analysis software or ABI PRISM GeneScan Analysis software System Management and Networking 7 7 Deleting Processed Frame Data The Cleanup Dat
14. 8 12 Maintenance The waste produced by the ABI PRISM 3100 Genetic Analyzer is not classified as hazardous however local regulatory personnel should be contacted before dispensing the waste into a sanitary sewer system Waste should be disposed of in accordance with all local state and federal regulations See also the ABI PRISM 3100 Genetic Analyzer Site Preparation and Safety Guide The 3100 Genetic Analyzer generates nonhazardous waste composed of polymer buffer and water Pure formulated ABI PRISM 3100 POP polymer should not be dispensed into the sewer system because the concentration of its component reagents means that it is classified as hazardous waste If you have old polymer that you want to dispose of arrange to have it removed to an appropriate waste treatment facility by a local waste hauler Tests conducted in accordance with the guidelines of the Environmental Protection Agency show that 3100 POP polymer is not acutely toxic For more information consult the MSDS for POP polymer Section Capillary Array In This Section The following topics are covered in this section Topic See Page Before Installing a Previously Used Capillary Array 8 14 Installing and Removing the Capillary Array 8 15 Capillary Array Maintenance 8 16 Storing a Capillary Array on the Instrument 8 17 Storing a Capillary Array off the Instrument 8 17 Maintenance 8 13 Before Installing a Previously Used Capil
15. Action 1 Click the Module Editor button on the toolbar to open the Module Editor dialog box BI Select a run module to use as a template Edit the parameter values that you want to change IMPORTANT Only whole numbers are accepted IMPORTANT Be sure that all values are red Values in black are not saved 4 Click Save As to create a new run module Enter a unique descriptive name and click OK Enter Name of New Module fry_new_module OK Cancel Note Save cannot be applied to default run modules 5 When you are finished click the Close button X to exit the Module Editor Software 5 21 Run Module Parameters Introduction You can change the module parameters listed below when creating run modules The parameters are listed in the order in which they appear in the run module editor Modifiable Run Module Parameters 5 22 Software Note Not all parameters are visible in the run module editor for all supplied sequencing and GeneScan run method files The following table lists the user modifiable run module parameters Parameter Comment Run The temperature of the electrophoresis chamber during the run The Temperature speed of electrophoretic migration decreases as the electrophoresis temperature decreases Cap FillVolume The time set for the array fill syringe to pump polymer into the capillaries IMPORTANT If this value is decreased from that in the supplied
16. Select a dye set from among the following options For Select Dye Set Dyes in Set DNA sequencing E dR6G dR110 dTAMRA dROX Fragment analysis with ABI PRISM D 6 FAM HEX NED ROX Linkage Mapping Sets IMPORTANT If you select the wrong dye set you will have to re run your samples You cannot correct this after the run because multicomponenting is applied before run data storage Working with Plate Records 6 5 Mobility Files Note Mobility files are identical to the dye set primer files used on other ABI PRISM genetic analyzers A mobility file is a software file containing the data that is used to compensate for differences in the electrophoretic mobilities of DNA fragments caused by labeling with different dyes see Dye Sets above Mobility files are for DNA sequencing only Mobility files are different for different dye sets and instrument types Mobility Files Provided The following mobility files are provided with the 3100 software and stored in the following directory D AppliedBio Abi Shared Analysis Basecaller Mobility Mobility File DNA Sequencing Chemistry DP3100POP6 BD 21M13 v1 mob BigDye Primer chemistry using the 21m13 primer DP3100POP6 BD M13Rev v1 mob BigDye Primer chemistry using the reverse primer DT3100POP6 BD v2 mob BigDye Terminator chemistry DT3100POP6 dRhod v1 mob dRhodamine Terminator chemistry Note New versions of these mobility files may b
17. Select the required options then click the Close button in the top right corner of the dialog box A Sequencing Analysis alert box appears wll Sequencing Analysis Save changes to the Seq4 document untitled 3 before closing Don t Save Cancel Click Save The Save this document as dialog box appears Save this document as 21x Save in asenso al cl a FSFfile fsf File name MEE Swe Save as type Factura Settings FSF z Cancel In the File name box type the name you want to use for the Factura settings file Note Do not use any of the following characters in the file name lt gt 1 Do not uses spaces Make sure that the file will be saved to the following directory D AppliedBio abi Shared Analysis Factura Settings Creating a New To create a new sequencing analysis module Sequencing Analysis Module Step Action 1 From the File menu point to New and select Seq AZ Settings This opens the untitled dialog box Basecaller Basecaller Type Basecaller 3100 hd T Write Seq Fies r Sequence File Format ABI C FASTA Factura Settings Fie Don t Fectuize w From the Basecaller Type drop down list select a basecaller Either Select the name of the basecaller settings file that you just created from the Basecaller Settings drop down list or Use the default settings Select Write Seq Files
18. and therefore develop customized applications for the ABI PRISM 3100 Genetic Analyzer OrbixWeb 3 2 Professional Edition provides database management services between the 3100 Data Collection software Auto Extractor and the Oracle database OrbixWeb v 3 2 Professional Edition has no user interface however it must always be running when the 3100 Data Collection software or Auto Extractor are running Software 5 7 Orbix Desktop Persistence Powertier Java Runtime Environment Adobe Acrobat Reader Oracle Database GeneScan Analysis Software DNA Sequencing Analysis Software Additional Information 5 8 Software Orbix Desktop 2 3 software is middleware that is used by the 3100 Data Collection software and Auto Extractor Persistence Powertier 4 321 is an application server that allows the 3100 Data Collection software to interact with the instrument database Java Runtime Environment 1 1 7b is software that enables the 3100 Data Collection software to run Adobe Acrobat Reader is a program that allows you to read electronic documents saved in the portable document format PDF The Oracle instrument database stores the following types of information Processed but unanalyzed fluorescence data which is collected from the CCD Plate records which contain information about plates and their samples Run schedules which are lists of runs automatically assigned by the software Run log and error log data 310
19. 6 7 8 9 See ececcooos 61 Please verify 67 Please verify 59 Please verify 58 Please verify 59 Please verify 58 Please verify Capillary 14 Matrix produced Capillary 15 Matrix produced Capillary 16 Matrix produced ananan onun ht wt ut Go Go Go Go GO tO Spatial and Spectral Calibrations 4 37 Spectral Calibration Parameter Files Introduction Locating Parameter Files List of Parameter Spectral calibration parameter files are text files that contain the run parameters used for spectral calibrations You can edit the parameters of an existing parameter file to create your own parameter file Spectral calibration parameters files are stored in the following directory D AppliedBio abi Support Files Data Collection Support Files Calibration Data Spectral Calibration Param Files The spectral calibration parameter files included with the 3100 software are listed Files Supplied below Witha Source of Dyes for conditionBounds Calibration Parameter File Name Application Check Matrix standard for the desired MtxStd AnyDyeSet par Sequencing with any dye set No dye set DS 30 matrix standard MtxStd GeneScan SetD par Fragment analysis with Yes Dye Set D DS 01 matrix standard MtxStd Sequencing SetE par Sequencing with Dye Set E Yes Sequencing sample from any SeqStd AnyDyeSet par Sequencing with any dye set No dye set Sequencing sample from Dye SeqStd Sequencing SetE
20. 866 40 10 48 22 866 40 20 Warszawa Portugal Lisboa 351 0 22 605 33 14 351 0 22 605 33 15 Russia Moskva 7 095 935 8888 7 095 564 8787 South East Europe Zagreb Croatia 385 1 34 91 927 385 1 34 91 840 Spain Tres Cantos 34 0 91 806 1210 34 0 91 806 1206 Sweden Stockholm 46 0 8 619 4400 46 0 8 619 4401 Switzerland Rotkreuz 41 0 41 799 7777 41 0 41 790 0676 The Netherlands Nieuwerkerk a d IJssel 31 0 180 331400 31 0 180 331409 United Kingdom Warrington Cheshire 44 0 1925 825650 44 0 1925 282502 All other countries not listed 44 0 1925 282481 44 0 1925 282509 Warrington UK Japan Japan Hacchobori Chuo Ku Tokyo 81 3 5566 6230 81 3 5566 6507 Technical Support B 3 Telephone Fax Region Dial Dial Latin America Del A Obregon Mexico 305 670 4350 305 670 4349 To Reach Technical We strongly encourage you to visit our Web site for answers to frequently asked Support Through questions and for more information about our products You can also order technical the Internet documents or an index of available documents and have them faxed or e mailed to you through our site The Applied Biosystems Web site address is http www appliedbiosystems com techsupp To submit technical questions from North America or Europe Step Action 1 Access the Applied
21. Deleting Records from the Database 3 17 Section Preparing the Instrument 3 19 Instrument Setup 3 20 Preparing Buffer and Filling the Reservoirs 3 22 Calibrating the Instrument 3 24 Placing the Plate onto the Autosampler 3 25 Section Setting Up the Software 3 27 Setting Software Preferences 3 28 About Plate Records 3 30 Creating a Plate Record for GeneScan Analysis 3 31 Creating a Plate Record for DNA Sequencing Analysis 3 36 Linking and Unlinking a Plate 3 41 Section Running the Instrument 3 45 About Run Scheduling 3 46 Controlling the Run 3 47 Performing a Run 3 1 3 2 Performing a Run Topic continued See Page Run Times 3 48 Section Monitoring a Run 3 49 Run View Page 3 50 Status View Page 3 51 Array View Page 3 53 Capillary View Page 3 56 Instrument Status Monitor 3 57 Section Working with Data 3 59 Recovering Data if Autoextraction Fails 3 60 Viewing Raw Data from a Completed Run in the Data Collection Software 3 61 Viewing Analyzed GeneScan Data 3 62 Viewing Analyzed DNA Sequencing Data 3 63 Archiving Data 3 64 Section Introduction In This Section The following topics are covered in this section Topic See Page Summary of Procedures 3 4 Planning Your Runs 3 5 Performing a Run 3 3 Summary of Procedures Flowchart ofa This flowchart provides an overview of the steps required to perform a run on the Typical Run ABI PRisM
22. E HE g ESS o A A a E ESEB 2 E create EN Mae OOOO0O0O0O a 0000000000000000 w ov lt fe 3 EJ OO000000 gl SsessessEsER GN sz n p a Ee a E Ze g E a E jas g 255 gt a FOOOOOOO0QO A 0000000000000000 Lla S geL z o O e o0o00000000000000 Oo oO og laj lpOOOOOOC0OO 0000000000000000 oo x Ecg 2 o z 0000000000000000 DE T SOS c22 gt OQOOOO0O0O00O 0000000000000000 2a x p Oo o 0000000000000000 fa 5 2 Q 2 2 aed Wid dhedotiea deal i ces eae Fas q A e lt O A wu O T e e 5 os a Lie o 29 Nn a Sam F S F MN 4 20 Spatial and Spectral Calibrations Preparing the Plate and Instrument Creating a Plate Record Follow the instructions in Chapter 3 Performing a Run to Assemble the plates page 3 9 Check and refill the fluids on the instrument page 3 22 Place the plate on the autosampler page 3 25 To create a plate record for the denatured matrix standards Step Action 1 Within the Plate View page of the 3100 Data Collection software click New This opens the Plate Editor dialog box 2 In the Plate Editor dialog box a Name the plate b Select Spectral Calibration c Make sure that the appropriate plate size is selected Plate Editor x Plate Name SpectralCalibration Application C Sequencing C GeneScan Spectral Calibration Plate Type 96 Wel v Comments Finish
23. El Select the matrix to use ACA 4 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Intensity vs Bin Number 3000 4000 5000 Intensity vs Scan Number Capillary Number 1 Condition Number 3 39 Q Value 0 992 Data Source Override all capillaries Override cap 1 Confirm that the correct capillary appears next to Capillary Number and then click the appropriate button Overriding with To override a spectral calibration profile with previously collected data Previously Collected Data Step Action 1 From the File menu select Override Spectral Calibration The Select the dye set to display dialog box appears E Select the dye set to display x pO OK Cancel 4 30 Spatial and Spectral Calibrations To override a spectral calibration profile with previously collected data continued Step Action 2 From the drop down lists select the appropriate Dye Set and then click OK The current spectral profile is displayed Fa Spectral Calibration Profile for E ID ttt tt 1 2 3 4 5 6 7 8 9 10 41 12 13 14 15 16 17 18 19 20 Intensity vs Bin Number 3000 4000 5000 Intensity vs Scan Number Capillary Number 1 GD a Condition Number 3 39 Q Value 0 992 Data Source Override matrix from another source From capillary _ From data fle ee cae 3 Use the slider bar to select the capillary with the profile to be overridden Click From data file Locate and s
24. File system NTFS Hl Used space 1 644 572 672 bytes 1 53GB JT Free space 5 305 786 338 bytes 4 94GB Capacity 6 950 359 040 bytes 6 47GB Diive D I Compress D 3 Estimate how much free space you need by using the information provided below Approximate Space File Type Required Per File kB Analyzed sample file for DNA sequencing 250 Analyzed sample file for fragment analysis 500 Unanalyzed sample file 100 a The values provided are estimates only The actual file size depends on the run module selected 4 If there is insufficient space a Archive the sample files to another volume b Delete the original files from the drive 3 16 Performing a Run Checking the Available Database Space Checking Database Note The instrument database automatically expands from 2 9 GB depending on the Space amount of data that needs to be stored To check the database space Step Action 1 Run the Diskspace utility For instructions see Checking Database Space The Diskspace Utility on page 7 5 If the used space is more than 8 GB purge the database of some or all data See the procedure references below Deleting Records from the Database Cleanup Database Utility Reference Deleting an Individual Plate Record Reference See page 7 8 to run the Cleanup Database utility Once per week or When the used space is more than 8 GB or
25. Handling Instrument Waste 8 12 Section Capillary Array 8 13 Before Installing a Previously Used Capillary Array 8 14 Installing and Removing the Capillary Array 8 15 Capillary Array Maintenance 8 16 Storing a Capillary Array on the Instrument 8 17 Storing a Capillary Array off the Instrument 8 17 Section Syringes 8 19 Syringe Maintenance 8 20 Cleaning and Inspecting Syringes 8 21 Priming and Filling Syringes 8 22 Installing and Removing Syringes 8 23 Section Polymer Blocks 8 25 Removing the Polymer Blocks 8 26 Cleaning the Polymer Blocks 8 27 Removing Air Bubbles from the Upper Polymer Block 8 28 Section Autosampler Calibration 8 29 Maintenance 8 1 8 2 Maintenance Section Instrument Maintenance In This Section The following topics are covered in this section Topic See Page Maintenance Task Lists 8 4 Routine Cleaning 8 5 Moving and Leveling the Instrument 8 6 Resetting the Instrument 8 7 Shutting Down the Instrument 8 8 Maintenance 8 3 Maintenance Task Lists Overview This section lists common tasks required to maintain your 3100 Genetic Analyzer in good working condition The tasks are divided into tables based on how often you should perform each task IMPORTANT Wear gloves any time you handle the capillary array glass syringes septa or buffer reservoirs Daily Tasks Perform these tasks at least once per day Maintenance Task Frequency See Page Ensure the reservoir se
26. Q Value on page 4 42 Condition number from 3 5 for sequencing or 4 7 for fragment analysis See Condition Number C Value on page 4 44 6 Click Cancel to close the dialog box For a Closer Look To zoom in on a portion of either graph press SHIFT and drag the mouse 6000 4000 Intensity vs Scan Number 5000 To reset the view press R 4 26 Spatial and Spectral Calibrations Examining Profiles To examine the matrices used to process a previous run Used for Previous Runs Step Action 1 From the Tools menu select Display Spectral Calibration 3100 Data Collection Software Version 1 0 Question 2 Click Previous Run From the drop down list select the profile to be displayed and then click OK Spatial and Spectral Calibrations 4 27 Overriding a Spectral Calibration Profile Introduction You can override unsatisfactory spectral calibration profiles in the Data Collection software The profiles can be overridden for individual capillaries one at a time or for all capillaries at once However we do not recommend applying a matrix from a single capillary to all 16 capillaries You can override a profile with a good quality profile that was collected either From another capillary during the same calibration run stored as tmp files or From previously collected data after the capillary array was last moved or replaced sto
27. Reextractor uses the run data in the instrument database to make a new file If an ABIF sample file becomes corrupt or if you accidently delete a file that you want you can use the Re extraction utility to replace the sample file Directions for using the Re extraction utility start on page 7 6 The Cleanup Database utility CleanupDB deletes some of the information stored in the instrument database to make room for new run data Directions for using the Cleanup Database utility start on page 7 8 The New Method Import utility NewMethodImport imports the data contained in method files into the instrument database The utility is used to install new versions of methods sent out by Applied Biosystems after your 3100 Genetic Analyzer is installed Directions for running the New Method Import utility start on page 7 10 The Remove Run Modules utility RemoveRunModules removes all modules and associated information from the instrument database Use this utility to quickly delete all old modules before importing new ones Directions for running the Remove Run Modules utility start on page 7 11 The Initialize Database utility InitDB completely erases and reinitializes the instrument database Use this utility only when instructed to do so by an Applied Biosystems representative Directions for running the Initialize Database utility start on page 7 12 The ABI Sample File Toolkit is an option that can be used to read ABIF sample files
28. ale ee xcs B Run_demo_3100_2000 07 05_21 log a Tet_700_A12_02 sa a Tet_700_B11_03 sa Tet_700_B12_04 sa a Tet_700_C11_05 sa a Tet 700 C12 06 sa a Tet_700_D11_O7 fsa a Tet_700_D12_08fsa aa et_ UU_E11_UYsa Tet_700_E12_10 sa sa Tet_70_F11_11 Isa fxg Tet_700_H12_16fsa 17 abject s 3 65MB The default name of the run folder is Run_ lt lnstrument name gt _ lt date gt _ lt runlD gt An example of a run folder name is shown below C Run_3100_Rabbit 2000 06 09_3 L M oL Run number for the day Year month day Instrument name Viewing Analyzed DNA Sequencing Data Introduction After a run has been extracted to sample files you can use the DNA Sequencing Analysis software to view the electropherogram data both raw and analyzed Refer to the ABI PRISM DNA Sequencing Analysis Software v 3 6 NT User s Manual P N 4308924 for details on viewing and analyzing GeneScan data Locating Sample When arun is finished the analyzed sample files are extracted into a run folder along Files with a run log in the following directory D AppliedBio abi 3100 DataExtractor ExtractedRuns An example of the run folder and its contents is shown below amp D perkin elmer abi 31 00 D ataE xtractor ExtactedRuns Run_demo_3100_2000 05 23_1 i E File Edit View Help Text Document 623 00 12 20 PM A 148KB AB1 File 6 23 00 12 19 PM A 146KB AB1 File 6 23 00 12 20 PM A 147KB AB1 File 6
29. irritation Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Use for research and development purposes only IMPORTANT Wear gloves while performing the following procedure and any other time you handle the capillary array glass syringes septa or buffer reservoirs To prepare the array fill syringe for use Step Action 1 Draw a small volume of room temperature polymer into a clean array fill syringe 2 Pull up the plunger to the 250 uL mark 3 Invert the syringe about six times to coat the walls with polymer Discard this polymer into aqueous waste Note Priming the syringe ensures that the running polymer is at the intended concentration and not diluted by residual water 4 To prevent air bubbles gently and slowly aspirate the polymer into the syringe until the desired volume has been reached 5 Point the syringe up and slightly press the plunger to purge any air Installing and Removing Syringes Installing Syringes To install the syringes Step Action 1 Follow the procedures to remove clean and dry the upper polymer block starting on page 8 26 Place the polymer reserve syringe tip in the left port on the top of the upper polymer block and screw the syringe tip clockwise into the polymer block IMPORTANT Always hold the syringe by the metal sleeve not the glass when screwing the syringe into th
30. par Sequencing with Dye Set E Yes Set E BigDye and preferred method dRhodamine chemistry Parameter Files Are Editable Editing a Spectral Calibration File You can use one of the spectral calibration parameter files supplied with the 3100 software or create your own using a supplied file as a template There are two reasons to set your own spectral calibration parameters To fine tune the conditions of a sequencing calibration run with Dye Set E ora fragment analysis calibration run with Dye Set D To use a four color dye set other than Dye Set D or Dye Set E In other words to use the sequenceStandard type of calibration rather than the matrixStandard type To edit a spectral calibration parameter file Step Action 1 Locate the parameters folder in the following directory D AppliedBio abi Support Files Data Collection Support Files Calibration Data Spectral Calibration Param Files 2 Select the file to edit and open it in WordPad or Notepad accessory application 4 38 Spatial and Spectral Calibrations To edit a spectral calibration parameter file continued Step Action 3 Edit the parameter values as desired See Spectral Calibration Parameters on page 4 40 for the list of parameters and their acceptable values 4 From the File menu select Save As Save the file with a unique name and a par extension in the same directory IMPORTANT D
31. type the number s that you want to use into the text boxes The Create a set button becomes Save this set as Note Ns means bases that could not be assigned an identity Click Save this set as This opens an unnamed dialog box a Save this set as Cancel Save a Type a name for the basecaller settings file into the text box b Click Save In the Preferences dialog box click OK This saves the basecaller settings Software 5 31 Factura Settings File 5 32 Creating a New To create a new Factura settings file Software Step Action 1 From the File menu point to New and select Factura Settings This opens the FSFfile fsf dialog box ail FSFfile fsf x W Revert Sample Fles To original Base Calls M Identify Vector Sequence Maximum permited gaps fpo Minimum match setween vector amp sequence FO I Szarch tor short inserts M Ichore leacing N s T Log search statistics Identify Confidence Range Keep the range from fi to pao Identify Ambiguity Remove bases irom the beginning and end until no more than fi ambiguities remain out of fo basas Reject remaining region if has morethan F anbiguity Identify IUB Heterozyqous Bases Do IC if ratio of 2nd highest peak to righestis gt O ID as a stop if ratio of 3rd highest te 2ndis gt fo I Update edted bases I Export Sample Fil s Clear Bange To New Text File M White Results Back To File
32. 0 0 0c cc cece nce c nce c cece ence nee eeenees 2 17 Supported Dye Sets vs ir ies andere tas el Pew SRN A eed Sy 2 18 Labeling Chemistries i neson Goce eae sce eee We ei BS Ge i ea ea diene 2 19 Polymers e iee een Silas et fone eth sida 28s ad aie Si na eo ees on eel alec al aes 2 20 Injection Solution s es wn ott s seks eg Sis a Shes See wee de eae ee 2 21 Section Electrophoresis Overview ccc cece cence ence ene e nee eeeeees 2 23 Capillary Arrays ests eas to eg hob eg eee Daas es eed at Ba Shoal Sa 2 24 Electrophoresis Seea bd ee die Gog we eed aig deed doh Hd ee ES ee ES 2 25 Electrophoresis Circuit nssr irge peed ale lhe SES 4B bd Shag lele ona widen ale eae ewe Gee 2 26 Section Fluorescence Detection Overview cee ce cence cence eee enee 2 27 TntroducnOn se ee eee etd Sal bad oe eed hie Sta ed ee eae ds 2 28 TEASER aces ace hin eb RW GME a A aa a EERE yb EER a A T Greate eng EE EEA 2 29 Transmission Grating 0 0 kc ec N E E E N EOE eee 2 29 CCD Camera seert New ea WER She a etn ne edo OS ees Gaia oe es 2 29 3 Performing a Run OVELVIEW aoe dd dupane aes deine Oot athe eo Gt Hb eee whee 3 1 S CHON Introduci n sisser siraman ni cade Se ette teed dae sed AE E Raa 3 3 Summary of Procedures 0 0 0 cece cece ete eens 3 4 Planning Your Runs 3 1 c0 dca ees be bebe aud ecb bape dodo e eee age abe 3 5 Section Working with Samples and Plate Assemblies 0 000 ccc wcenees 3 7 Sample Preparation
33. 23 00 12 19 PM 151KB AB1 File 6 23 00 12 20 PM 147KB AB1 File 6 23 00 12 19 PM 153KB AB1 File 6 23 00 12 20 PM A 152KB AB1 File 6 23 00 12 19 PM 147KB AB1 File 6 23 00 12 20 PM E01 151KB AB1 File 6 23 00 12 19 PM A H sample_E02_1 O ab1 147KB AB1 File 6 23 00 12 20 PM A a sample_FU1_11 ab17 154KB ABT File bf23 UU 12 19 PM A H sample_F02_1 2 ab1 152KB ABI File 6 23 00 12 20 PM A lll sample_G01_13 ab1 152KD ADI Tile 623 00 12 20 PM A H sample_GO2_1 4 ab1 147KB ABI File 623 00 12 20 PM A E sample _H01_15 ab1 147KB AB1 File 6 23 00 12 20 PM E sample _H02_1 6 ab1 152KB AB1 File 6 23 00 12 20 PM 17 objects 2 32MB Run Folder Default The default name of the run folder is Name Run_ lt nstrument name gt _ lt date gt _ lt runID gt An example of a run folder name is shown below GR una 00_R abbit_2000 06 09_3 Run number for the day Year month day Instrument name Performing a Run 3 63 Archiving Data 3 64 Introduction Installing a SCSI Storage Device Performing a Run There are many options for archiving your data You could for example copy the data to another networked computer and from there use any archiving system such as an external SCSI storage device We do not recommend that you add a SCSI storage device to the computer workstation However if you need to temporarily install one follow the procedure below IMPORTANT Do not install a SCSI device on the computer works
34. 24 00 12 33 PM 4 5 cbject s 1 44KB 3 Double click the supplied parameter file that is appropriate for the type of calibration run you are performing see the explanation given in each parameter file This opens the file Note The file for the matrixStandard dataType and Dye Set E will be used in this example FA MtxStd S equencing SelE par WordPad File Edit View Insert Format Heb Cia Sle al Hiel amp E Mtxetds Sequencing SetE par This is the parameter file for ye Set E dataType matrixStandard This field must be given in ALL parameter files numDyes 4 writeLummybyes 1 numSpectralBins 20 wing 0 95 tonditionBounds 3 0 5 0 minRarkQ 4 4 Edit the values for minQ and or conditionBounds as appropriate Note Under normal circumstances do not change the default value for the conditionBounds parameter See page 4 44 From the File menu select Save As In the File name text box type a name for the new spectral parameter file 4 34 Spatial and Spectral Calibrations To create a spectral calibration parameter file for matrixStandard dataType continued Step Action 7 Click Save Note This saves the file in the ParamFiles folder 8 Then follow the Performing a Spectral Calibration If you are performinga Using Default Processing Parameters on calibration run for page 4 18 but DNA sequencing select the p
35. 5 5 3100 Data Collection Main Menus Submenus Submenus Software Menus Import Plate Override Spectral Calibration Exit Plate View Array View Instrument Status Monitor Preferences BioLIMS Project Info 3100 Data Collection Run software window with tabs e Plate View Skip to Next Run e Run View e Status View e Array View Pause Set Color e Capillary View Stop Display Run Data Data Collection Data Analysis Manual Control Display EPT Data Data Aquisition Extract Data into Sample Files C Module Editor gt Autosampler Calibration Wizard Perform Spatial Calibration Display Spatial Calibration Display Spectral Calibration Verify Instrument Send SCPI Command Run Service Module About 3100 Data Collection 5 6 Software Auto Extractor Diskspace Utility Re extraction Utility Cleanup Database Utility New Method Import Utility Remove Run Modules Utility Initialize Database Utility ABI Sample File Toolkit OrbixWeb Auto Extractor is used by the Data Collection software to automatically extract and analyze the data after each run The Diskspace utility lists the amount of space that the database uses the amount that is free for use and the percent filled Directions for using the Diskspace utility start on page 7 5 The Re extraction utility
36. 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 s HITACHI Applied Biosystems is committed to providing the world s leading technology and information for life scientists Printed in the USA 06 2010 Part Number 4315834C
37. 9 6 to 9 7 customer support See technical support C value 4 44 Index 1 D data archiving 3 64 hiding for specific dyes 5 12 none in capillaries 9 5 recovering 3 60 re extraction utility 7 6 spatial calibration 4 5 storage 7 4 viewing analyzed GeneScan data 3 62 viewing analyzed sequencing data 3 63 viewing raw data 3 61 Data Collection software 5 5 5 6 starting 3 14 won t launch 9 2 Data Delay Time run module parameter 5 22 data flow overview 7 14 database BioLIMS See BioLIMS database checking space using Diskspace utility 7 5 deleting records 3 17 LIMS See LIMS database reinitializing 7 12 removing run modules using utility 7 11 dataType parameter 4 40 4 41 debug log 5 55 deleting from plate import table 6 21 plate records 6 37 processed frame data from database 7 8 detection cell cleaning 8 14 developer s toolkit 5 7 directories list 5 9 Diskspace utility 7 5 display colors changing using HSV 5 13 5 14 Display Run Data command 3 61 displayed dye colors 5 12 to 5 14 documentation 1 4 Documents on Demand B 4 documents list C 3 dRhodamine terminators 2 19 dye colors changing 5 12 changing the name or color intensity 5 12 See also displayed dye colors dye sets 6 5 composition 2 18 selecting for GeneScan 3 33 selecting for sequencing 3 37 E Edit Dye Display Information dialog box 5 12 electrophoresis discussed 2 23 to 2 26 elevated baseline 9 6 e mail address technical support B 1 emission spectrum A 4
38. Every 400 500 runs Delete individual plate records when you want to free database space without deleting all of the records see page 6 39 Performing a Run 3 17 3 18 Performing a Run Section Preparing the Instrument In This Section The following topics are covered in this section Topic See Page Instrument Setup 3 20 Preparing Buffer and Filling the Reservoirs 3 22 Calibrating the Instrument 3 24 Placing the Plate onto the Autosampler 3 25 Performing a Run 3 19 Instrument Setup Using Manual While you are setting up the instrument you may find manual control useful For Control example you can use manual control commands to move the syringe plungers up and down open or close the pin valve and turn on the oven before starting your run For a complete list of the commands and instructions for using them see Controlling the Instrument Using Manual Control on page 5 15 Attaching the To attach the polymer blocks to the instrument Polymer Blocks Step Action 1 If necessary clean the polymer blocks and the tubing as instructed on page 8 27 2 Push the upper polymer block onto the two guide pins on the instrument 3 Install the lower polymer block Ensure the block is pushed all the way against the instrument 4 Connect the tubing between the two blocks a Insert one ferrule into the upper polymer block and rotate clockwise until finger tight b Inse
39. Import one plt file to create one plate record Simultaneously import multiple plt files to create multiple plate records Import one plt file to create one plate record and simultaneously link it to its plate Repeat Repeat Place plates if not done yet Link one plate plate record Repeat 6 32 Working with Plate Records Simultaneously Importing and Linking a Plate Record Introduction You can speed up the process of importing a tab delimited text file plt file to create a plate record and link the plate record to its plate by performing the two procedures together This decreases the number of read write operations performed by the instrument database so it only takes about 2 min to import and link a 384 well plate The disadvantage of using this procedure is that you can import and link only one plate record at a time Simultaneously Importing and Linking a Plate Step Action Record 1 Place the plates that you want to link onto the plate deck 2 In the Plate Setup page of the 3100 Data Collection software click Import This opens an unnamed browser dialog box To import a plate record and link it to its plate in a single procedure ee 5 fo DBPurgeUtility File name ER Files of type Plate Import Files pit x Cancel Navigate to the directory location of the plate file plt file that you want to import and link and then select the pl
40. International 4311692 Epson Stylus 900 Color Printer for use at 100 120 volts 4311623 Description Part Number 96 well plate kit 4316471 384 well plate kit 4316472 Description Part Number ABI PRISM 3100 GeneScan Analysis Software Module Kit 4317379 ABI Prism 3100 DNA Sequencing Analysis Software Module Kit 4317380 Description Part Number ABI PRISM 3100 POP 6 polymer 4316357 ABI Prism 3100 capillary array 50 cm 4315930 ABI PRISM 3100 capillary array 36 cm 4315931 Genetic Analyzer Buffer with EDTA 10X 402824 Matrix Standard Set DS 01 dROX dTAMRA dR6G dR110 4315974 ABI Prism BigDye Terminator Sequencing Standards Kit 4304154 Hi Di Formamide 25 mL bottle 4311320 Part Numbers C 1 GeneScan Reagents and Consumables Instrument Consumables Instrument Spare Parts C 2 Part Numbers Description Part Number ABI PRISM 3100 POP 4 polymer 4316355 ABI PRism 3100 capillary array 36 cm 4315931 ABI PRISM 10X buffer with EDTA 402824 Matrix Standard Set DS 30 6FAM HEX NED ROX 4316100 ABI PRISM 3100 GeneScan Installation Standard DS 30 4316144 Hi Di Formamide 25 mL bottle 4311320 Description Part Number 96 well plate septa 4315933 MicroAmp Optical 96 well Reaction Plates N801 0560 384 well plate septa 4315934 MicroAmp 384 well Reacti
41. Making Buffer for a Single Run Storing the Buffer When to Replace the Buffer Filling the Water and Cathode Buffer Reservoirs 3 22 Performing a Run The following materials are required to prepare 3100 1X running buffer ABI PRism 3100 10X Running Buffer with EDTA P N 402824 Quality deionized water To prepare 50 mL of 1X running buffer Step Action 1 Add 5 0 mL of 10X running buffer into a graduated cylinder 2 Add deionized water to bring the total volume up to 50 mL 3 Mix well The 3100 1X running buffer can be stored at 2 8 C for up to 1 month Replace the 1X running buffer in the anode buffer reservoir and the cathode buffer reservoir daily or before each batch of runs IMPORTANT Failing to replace buffer may lead to loss of resolution and data quality IMPORTANT Replenishing buffer and placing the plate requires that the autosampler be in the forward position with the capillary tips removed from the buffer solution Do not leave the autosampler in this position for an extended time because the capillaries can dry out IMPORTANT Wear gloves while performing the following procedure and any other time you handle the capillary array glass syringes septa or buffer reservoirs To fill the water and cathode buffer reservoirs Step Action 1 Close the instrument doors 2 Press the Tray button on the outside of the instrument to bring the autosampler to the forward posi
42. Options After the data has been processed the data is Stored in the instrument database as processed frame data Converted to either ABIF sample files or BioLIMS data sets depending on the Data Analysis preferences settings Processed Frame After multicomponenting the data is stored as groups of binary streams in the Data instrument database This data is called processed frame data to distinguish it from other data stored in the database such as plate records and EPT data Data Flow A 7 Mobility Shift Correction for DNA Sequencing Introduction Background Mobility Files A 8 Data Flow Mobility shift correction is an additional data processing step that occurs when performing DNA sequencing The correction is carried out during autoanalysis when the basecaller software is assigning bases to the collected and processed data When a dye is bound to a DNA fragment it changes the rate at which the fragment migrates during electrophoresis When DNA fragments that are labeled with different dyes are electrophoresed together the fragments do not migrate with equal spacing because different dyes change the migration rate to different extents Without correction this would lead to an uneven separation of peaks in the electropherogram The data needed to perform mobility shift correction are contained in mobility files Mobility files are different for different dye sets and instrument types When creating a plate record you s
43. Pending window Create plate records in advance of placing the plates on the instrument A plate record cannot be created while a run is in progress The next two sections cover the most common method for creating a plate record There is a separate procedure for each analysis application Note There are several other methods for creating a plate record See Chapter 6 Working with Plate Records For information on creating plate records for spectral calibration runs see Creating a Plate Record on page 4 21 Creating a Plate Record for GeneScan Analysis Entering Plate Note You cannot create a plate record while a run is in progress Record Information To enter plate record information Step Action 1 Click the Plate View tab on the 3100 Data Collection software window to go to the Plate View page Plate View tab Plate View Run View Status View Array View Capillary View 2 On the Plate View page click New Or click the Plate Editor button on the toolbar EEE EEE nee The Plate Editor dialog box appears Plate Editor x Plate Name my _plate_record Application C Sequencing GeneScan C Spectral Calibration Plate Type os wen z Comments This is an example plate record 3 Use the Plate Editor dialog box to name your plate and to specify the application and plate type Entering comments is optional IMPORTANT When naming the plate you can use letters numbers and t
44. Plate File Using a Provided Template 6 24 Creating a Plate File from a New Spreadsheet 6 28 Creating a Plate File from a Custom Spreadsheet Template 6 29 Creating a Plate File from an Edited Plate Record 6 30 Section Importing Plate Files and Linking Plate Records 6 31 About Importing Tab Delimited Text Files and Linking Plate Records 6 32 Simultaneously Importing and Linking a Plate Record 6 33 Sequentially Importing and Linking a Plate Record 6 35 Section Deleting Plate Records and Run Data 6 37 Introduction 6 38 Cleanup Database Utility 6 38 Deleting Individual Plate Records 6 39 Working with Plate Records 6 1 6 2 Working with Plate Records Section Introduction In This Section The following topics are covered in this section Topic See Page About Creating Plate Records 6 4 About the Plate Record Fields 6 5 Working with Plate Records 6 3 About Creating Plate Records Introduction The instrument database stores information about the plates and the samples they contain as data tables named plate records Each plate placed on the instrument requires a plate record Note A plate record is analogous to the Sample Sheet used with the ABI PRism 377 DNA Sequencer and an Injection List used with the ABI PRism 310 Genetic Analyzer When to Create Create plate records in advance of placing the plates on the instrument Piue Records Data that will be imported for the creation of plate records can be prep
45. Recovering Data if Autoextraction Fails 3 60 Viewing Raw Data from a Completed Run in the Data Collection Software 3 61 Viewing Analyzed GeneScan Data 3 62 Viewing Analyzed DNA Sequencing Data 3 63 Archiving Data 3 64 Performing a Run 3 59 Recovering Data if Autoextraction Fails Introduction Runs that are stopped before completion display the status Completed in the run table on the Run View page The auto extractor should automatically extract data from stopped runs If autoextraction fails use the Extract data into sample files command as described below Recovering Data To recover data from a stopped run from a Stopped Run Step Action 1 From the Instrument menu point to Data Acquisition and select Extract data into sample files Fie View Toos Service Help a E a Ron Ship to MEXE kan Plate view pause Array View Capillary View Sto Set Color Applicat Gore Ful Status ta Camplete Display Runt Dala Display EPT Data Fytrart data intn sample fles Pendin SSEL Fate Name 2 Look for the message Sample Files Successfully Extracted on the Status bar Note The extracted data is unanalyzed Use the analysis software to analyze the sample files 3 60 Performing a Run Viewing Raw Data from a Completed Run in the Data Collection Software Introduction Raw data is data that has been multicomponented corrected for spectral overlap but mobility co
46. Reposition one or more of the red crosses To move a cross change the value in the Capillary Position box and then click outside of that box c Override the data with data from a previous run see page 4 28 If the calibration continues to provide unsatisfactory results see If the Calibration Fails on page 4 9 Click OK to close the Perform Spatial Calibration window The Question dialog box opens Q Save spatial calibration data Ifyou choose Yes spatial calibration data will be saved in the database and sent to the instrument o 4 8 Spatial and Spectral Calibrations To view the spatial calibration results and save the data continued Step Action 4 To Then save this calibration data to the Click Yes 3100 Data Collection Software database delete this data and use datafroma a Click No previous run b Proceed to Overriding the Current Spatial Calibration Map on page 4 12 If the Calibration If the calibration failed or if you do not like the appearance of the passed calibration Fails profile try one or more of the following corrective actions Repeat the calibration Fill the capillaries with polymer and then repeat the calibration Clean the detection cell and then repeat the calibration see page 8 14 Reposition the array window in the detection cell and then repeat the calibration Spatial
47. added to the plate record using the plate editor after the tab delimited text file has been imported Note If you are importing data from a LIMS database there cannot be any errors in the data because there is no opportunity to review it and the import will fail see page 6 19 Working with Plate Records 6 11 Using Spreadsheets to Create Tab Delimited Text Files Introduction You can enter plate record data into any spreadsheet program that can save files as tab delimited text files You can create spreadsheets in a program that uses the Macintosh operating system however you must then convert the files into Microsoft Windows format files Example of An example of a spreadsheet prepared in Microsoft Excel for samples intended for Sequencing DNA sequencing is shown below Spreadsheet For an explanation of the labels see page 6 14 Version number Plate header Seq96_FullPlate SQ 96 Vell Column header Well Sample Name Dye Set Mobility File BioLIMS Project Run Modul Analysis E Al DT3100POPE BDW 3100 Project StdSeq50_BC 3100 Sample data E DT3100POPE BDW 3100 Project StdSeqS0_BC 3100 L E DT3100POPE BDW 3100 Project StdSeq50_BC 3100 T KI Dy Example of When the above spreadsheet is converted to a tab delimited text file and opened with Sequencing the Notepad program it looks like the example below Tab Delimited Text File FullPlate_SeqWell_96 Notepad Of x File Ed
48. and stored in the database After the run this data is considered by the algorithm as it performs its analysis AutoStart Point The first data frames typically have no fluorescence data and are used by the algorithm to collect baseline information The data frame at which the first fluorescence is detected is called the Autostart point startptOffset Value When the startptOffset value is set to zero the Autostart point is also the first frame used by the algorithm for analysis When the startptOffset parameter is set to a higher value the first analyzed frame is one that was collected later If you want to prevent early dye signals from being used by the algorithm you can increase the startptOffset value You might for example do this to prevent data for a primer peak from being used A typical starting point offset is 200 frames 4 48 Spatial and Spectral Calibrations maxScansAnalyzed Parameter Introduction Purpose How to Use If you use the sequenceStandard dataType parameter you can select a value for the maximum number of scans analyzed maxScansAnalyzed parameter The maxScansAnalyzed parameter sets the number of data frames that are analyzed by the spectral calibration algorithm If the quality of the spectral data being collected is high this value can be lowered to speed up the calibration procedure and conserve instrument database space However if you set this value too low there may be insufficient good re
49. and Spectral Calibrations 4 9 Displaying a Spatial Calibration Profile Introduction By performing the procedure below you can display the spatial calibration profile for the current capillary array or the profile that was used for a previous run Note With this procedure you can view spatial calibration data but you cannot change which data is set as the current map Displaying a Spatial To display a spatial calibration profile Calibration Profile 4 10 Spatial and Spectral Calibrations Step Action 1 From the Tools menu select Display Spatial Calibration This opens the Question dialog box sth Question x Display Spatial Calibration for Current array Previous run Cancel If you want to display the profile for Then the current array Click Current Array This opens the Spatial Calibration Profile box for the current calibration data Note The title bar is now displayed as Current Spatial Calibrations a previous run a Click Previous run b Select the desired run in the Select the source to display dialog box c Click OK Note For information about the profile see Evaluating a Spatial Calibration Profile on page 4 11 Evaluating a Spatial Calibration Profile Evaluation Criteria While viewing the calibration profile in the Details dialog box use the following criteria to evaluate the data Peak Attribute Cr
50. and drip trays with lint free tissue dampened with water 5 Close the instrument doors 6 Shut down the computer and turn off the instrument 7 Wash the syringes polymer blocks and reservoirs with warm water Rinse with deionized water IMPORTANT Make sure all parts are completely dry before long term storage Section Fluids and Waste In This Section The following topics are covered in this section Topic See Page Buffer 8 10 Polymer 8 10 Handling Instrument Waste 8 12 Maintenance 8 9 Buffer When to Change the Buffer Making Buffer for a Single Run Storing Buffer Polymer Storing Polymer When to Change the Polymer Adding and Changing the Polymer 8 10 Maintenance We recommend that you change the buffer before each run or at least every 24 hours To prepare 50 mL of 1X 3100 running buffer Step Action 1 Add 5 0 mL of 10X 3100 running buffer into a graduated cylinder 2 Add deionized water to bring the total volume up to 50 mL 3 Mix well The 1X 3100 running buffer can be stored at 2 8 C for up to 1 month Store any remaining ABI PRISM 3100 POP polymer at 2 to 8 C until the expiration date printed on the jar Note Excessively hot environments may shorten the working life of the polymer We recommend that you change the polymer weekly The polymer is good at 25 C for about 7 days 7 NOP NGREIO S CHEMICAL HAZARD POP
51. calc Fz lenin o lt Microsoft Excel Files xI xls xla Navigate to the Plate Import Files folder in the following directory D AppliedBio Abi Support Files Data Collection Support Files Plate Import Files Notice that no files are displayed This is because there are no Microsoft Excel files in this folder In the Files of type list box select All Files Select the plate file plt file template you want to use and click Open The Text Import Wizard dialog box opens Text Import Wizard Step 1 of 3 Windows ANSI p CO M Tr L ca eak Emh T A 5 a a A rT Working with Plate Records 6 25 6 26 To create a plate record using a template continued Step Action Click The file is displayed as a spreadsheet Finish lt i FullPlate_Seqwell_96 Seque FulSQ Well Al std B1 std c1 std D1 std El std F1 std G1 std H1 std A2 std B2 std C2 std D2 std E2 std F2 std G2 std H2 std A3 std B3 std 96 Well Sample N Dye Set Immmmmmmmmmmmmmmm mm Mobility Fi Comment BioLIMS P Sample Tr Run Modul Analysis Module T3100PC T3100PC OPC OPC OPC 0PC DPC 0PC DPC DPC DPC DPC T3100PC T3100PC ddddddddda g s pe le T3100PC T3100PC T3100PC nanmanna 0000000000 0 00 0o00 00O 3100_Pro 3100_Pro 3100_Proj 3100_Pra 3100_Proj 3100_Proj 3100_P
52. can cause painful and sometimes permanent back injury Use proper lifting techniques when lifting or moving the instrument Two or three people are required to lift the instrument depending upon instrument weight To prepare for moving the instrument Step Action 1 Remove the following components from the instrument Any plate assemblies from the autosampler Water and buffer reservoirs from the autosampler Capillary array For instruction see page 8 15 Syringes from the upper polymer block For instruction see page 8 23 Upper polymer block For instruction see page 8 26 Anode buffer reservoir gt gt gt gt gt o Lower polymer block For instruction see page 8 26 Switch off the breaker on the back of the instrument Disconnect the power cord and the Ethernet cable While moving the instrument avoid any shock or vibration Leveling the To level the instrument Instrument Step Action 1 Place the bubble level on the autosampler deck 2 Turn the instrument legs to level the instrument To move the instrument corner Turn the leg up right clockwise down left counterclockwise 8 6 Maintenance Resetting the Instrument Introduction Reset the instrument when There is a fatal error as indicated by the red status light The instrument does not respond to the ABI PRism Data Collection software T
53. chemistry DP3100POP6 BD 21M13 v1 mob using the 21m13 primer BigDye Primer chemistry using the DP3100POP6 BD M13Rev v1 mob reverse primer ABI PRISM BigDye Terminator DT3100POP6 BD v2 mob chemistry ABI Prism dRhodamine Terminator DT3100POP6 dRhod v1 mob chemistry Enter a BioLIMS project IMPORTANT A BioLIMS project is required for every sample even if a BioLIMS database is not used a Click in the BioLIMS Project cell for Well A1 b Select a project name from the drop down list BioLIMS Project no selection lt no selection 3100 Project IMPORTANT You must enter a BioLIMS project Note To set up a BioLIMS project see page 5 48 c To assign the same project name to each sample in the plate record Click the column header to select the whole column Press CTRL D The Project Name for every sample in the plate record is now the same Note Press CTRL D whenever a field is the same for all samples in the plate record To enter sample information and save the plate record continued Step Action 5 For each sample select the appropriate Run Module from the drop down list Run Module sno selection z actions RapidSeq36_POP6DefautModule SteSeq50 POP6DefauttModule Note If you need to view or edit a run module file see page 5 20 The following table shows the run module to select based on your run type Analysi
54. event viewer blank 9 2 Excel See Microsoft Excel Index 2 exporting run modules to file 5 24 extensions filename 5 9 extracting data See auto extractor F Factura settings file creating 5 32 FASTA format for Seq files 5 33 file types 5 9 ABIF sample 7 4 basecaller settings bcp 5 30 GeneScan analysis modules gsp 5 43 portable document format pdf 5 8 rundata 7 4 run module modexp 5 23 5 25 sequencing analysis module saz 5 33 size standard szs 5 40 spatial calibration log log 4 5 spectral calibration mcl 4 36 spectral calibration log log 4 37 tab delimited text files plate records 6 9 to 6 18 filename extensions 5 9 Fill Down command 3 32 3 38 firmware 5 5 fluorescence detection discussed 2 27 to 2 29 fluorescence display 3 54 fluorescence pattern on CCD camera A 4 fragment analysis chemistry types supported 2 18 data analysis 3 62 3 63 frame data A 5 See also processed frame data G GeneScan analysis modules creating 5 43 to 5 45 analysis modules discussed 5 37 to 5 45 analysis modules selecting 3 34 analysis modules viewing 5 38 dye sets 3 33 polymer 2 20 preparing matrix standards 4 19 run modules 3 33 runtimes 3 48 viewing analyzed data 3 62 GeneScan Analysis Software 5 8 gsp files See GeneScan analysis modules H hard drive 2 14 hard drive space checking 3 16 hardware overview 2 9 to 2 12 help See technical support B 1 Hi Di formamide 2 21 HSV color system 5 14 I i
55. for 0 1 seconds can burn the retina and leave permanent blind spots Never look directly into the laser beam or allow a reflection of the beam to enter your eyes Transmission Grating Overview CCD Camera Overview The transmission grating is a grooved disk that spectrally separates the fluorescence emitted light from the dye labeled DNA fragments After the light is spectrally separated it is focused onto the charge coupled device CCD camera The CCD camera includes a rectangular silicon chip that converts the incident fluorescence light into digital information This digital information data will be processed by the 3100 Data Collection software A description of the role of the CCD camera in data processing starts on page A 3 System Overview 2 29 Performing a Run Overview In This Chapter The following topics are covered in this chapter Topic See Page Section Introduction 3 3 Summary of Procedures 3 4 Planning Your Runs 3 5 Section Working with Samples and Plate Assemblies 3 7 Sample Preparation 3 8 Working with Plate Assemblies 3 9 Section Starting the 3100 System 3 11 Starting the Computer 3 12 Starting the Instrument 3 13 Starting the 3100 Data Collection Software 3 14 Section Checking the Available Space and Deleting Records 3 15 Checking the Available Hard Drive Space 3 16 Checking the Available Database Space 3 17
56. for a single plate record however but you can import many tab delimited text files at once This may make it faster than the procedure on page 6 33 if you have a lot of files to import To import one or more tab delimited text files to create plate records Step Action 1 In the Plate View page of the 3100 Data Collection software click Import This opens an untitled browser dialog box a x Look in ea Bin eB fe E3 B DBPurgeutility File name OK Files oftype Piate Import Files pi E Cancel 2 Navigate to the directory location of the plate file s plt that you want to import and link If you want to create Then a single plate record a Select the plt file b Click OK This opens the Link to dialog box c Select the letter that corresponds to the plate position more than one plate record a Click the Up One Level button b Select a folder of plt files All plt files are imported and appear in the pending plate records table ready to be linked 3 Click OK This imports the plt file s and creates one or more plate records Note If there is missing information in the file you may be warned by an information box This may happen for example if you make a typing error or list a module that no longer exists Depending on the problem the warning may accompany rejection of the entire plate record However in some circumstances the data will b
57. if you want a Seq file created this saves the sequence as a text file Select either ABI or FASTA in the Sequence File Format group box Select FASTA only if you intend to export the data to a program that accepts FASTA files If you do Then not want to use Factura leave Factura Settings File as Don t Facturize software want to use Factura software select a Factura settings file from the drop down list Don t Facturize Factura Settings File Software 5 33 Saving the To save the sequencing analysis module Sequencing Analysis 5 34 Software Module Step Action 1 Click the Close button A Sequencing Analysis alert box appears ll Sequencing Analysis 3 6 1 A Save changes to the Sec docurrent untited before closing Dorit Save Cancel 2 Click Save The Save this document as dialog box opens Save in a Params z Sl c a BC 31C0_SeqDHFtOft saz fa BC 31CORR_SeqOMFIOf saz a BC POPSLR_SeqDffFtOflsaz a BC POPG_SeqOfFtOtt sez Save as type saz Setting saz OOo Cancel 3 In the File name text box type a name for the analysis module Note Do not use any of the following characters in the file name lt gt Do not uses spaces 4 Make sure that the file will be saved to the following directory D AppliedBio abi Shared Analysis Basecaller Params 5 Click Save This creates an analysis module with
58. importing 6 31 plate import table 6 21 plt plate record files 6 23 to 6 36 plate records creating for GeneScan 3 31 to 3 35 creating for sequencing 3 36 to 3 40 creating overview of procedures 6 4 deleting 6 37 discussed 3 30 importing tab delimited text files 6 31 linking and unlinking 3 41 tab delimited text files 6 9 Plate View tab 3 31 3 36 3 41 plates assembling 3 9 placing onto autosampler 3 25 unlinking from plate records 3 43 polymer changing 8 10 discussed 2 20 disposing 8 12 polymer blocks air bubbles 8 28 cleaning 8 27 removing 8 26 polymer reserve syringe See also syringe volume and function 8 20 POP See polymer Pre Run Time run module parameter 5 22 Pre Run Voltage run module parameter 5 22 preferences setting 3 28 processed frame data A 7 deleting 7 8 size of 7 4 processed frame data storing 7 4 Project Name field in plate record 3 32 3 38 pull up pull down peaks 4 42 Q Q value 4 42 R raw data viewing 3 61 recovering data from a stopped run 3 60 red status light 9 2 Index 4 re extracting processed frame data 7 6 reinitializing the database 7 12 remote extraction software 7 18 RemoveRunModules bat file 7 5 7 11 removing air bubbles 8 28 reservoirs filling 3 22 positions on the autosampler 3 23 reset button location 2 10 resetting the instrument 8 7 resolution loss 9 6 RGB color system 5 13 run elevated baseline 9 6 elevated current 9 7 fast migration time 9 7 fluctuating cur
59. in a microcentrifuge 6 Heat the standard tube at 95 C for 5 min to denature the DNA 7 Immediately place the tubes on ice for 2 min DNA Sequencing The best samples to choose for making a matrix have approximately 25 each of A Preparing BigDye C G and T A good example of this is the BigDye Terminator Sequencing Standard or Sequencing Sample PGem To prepare the BigDye Terminator standard for Dye Set E Matrices Action Resuspend a tube of BigDye Terminator Sequencing Standard P N 4304154 with 170 pL of Hi Di formamide CHEMICAL HAZARD Formamide is harmful if absorbed through the skin and may cause irritation to the eyes skin and respiratory tract It may cause damage to the central nervous system and the male and female reproductive systems and is a possible birth defect hazard Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Vortex thoroughly Spin the mixture briefly in a microcentrifuge Heat the standard tube at 95 C for 5 min to denature the DNA for Dye Set E Matrices Step 1 2 3 4 5 Immediately place the tubes on ice for 2 min Fragment Analysis To prepare the Matrix Standards for Dye Set D Matrices Action Thaw and mix thoroughly the four DS 30 P N 4316100 matrix standard tubes Spin the tubes briefly in a microcentrifuge Prepare the Matrix Standard Set D
60. may cause eye skin and respiratory tract irritation Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Use for research and development purposes only To put fresh polymer on the instrument Step Action 1 From the Tools menu select Change Polymer Wizard E 13100 Data Collection Software Fie View Instrument EWES Service Help HEA j Plate Editor rods Faresi Module Editor Plate View Run View Instrument Condition Change Polymer Wizard Install Capillary Array Wizard Autosampler Calibration Vizard Perform Spatial Calibration Display Spatial Calibration Display Spectral Calibration To put fresh polymer on the instrument continued Step Action 2 This opens a warning message Q The currently installed capillary array is 50cm If this is acceptable continue with the polymer change If this is not acceptable please click cancel install your preferred capillary array and restart this wizard Cancel 3 If the length of the array actually on the instrument is the same as the length given in the warning message click OK to begin the Change Polymer Wizard 4 Follow the directions given in the wizard to put fresh polymer on the instrument Maintenance 8 11 Handling Instrument Waste 3100 Genetic Analyzer Waste Composition Pure Formulated 3100 POP Waste
61. performed The capillary window displays the signal intensity by capillary number The fluorescence emission spectrum displays the real time fluorescence emission spectrum of the dye labeled fragments from the capillary selected The spectrum is plotted against the CCD bin number instead of wavelength 180 E S a E E A E a 2 4 6 8 aft Os Pelco E a A i Intensity vs Spectral Bin Note This window is updated each time you select a different capillary in the Capillary Display window during data collection An electropherogram is a graph of relative dye concentration against time plotted for each dye The data displayed has been multicomponented The relative dye concentration is determined by applying chemometric algorithms to the collected fluorescence data There are two plots for each dye The plots represent the upper and lower confidence limits associated with the measured fluorescence intensity 69 3 69 6 70 Intensity vs Run Time mins Total Intensity The total intensity graph is a graph of the total intensity detected for each capillary Graph 3780 2930 1980 1080 180 2 46 3 10 412 44 416 Intensity vs Cas Number Note This window works only during data collection This window is updated each time you select a different capillary in the Capillary Display window during data collection Performing a Run 3 55 Capillary View Page Function Feat
62. problems may indicate that a new capillary array is required Poor sizing precision or allele calling Poor resolution and or decreased signal intensity IMPORTANT Wear gloves while performing the following procedure and any other time you handle the capillary array glass syringes septa or buffer reservoirs P RO CHEMICAL HAZARD POP polymer may cause eye skin and respiratory tract irritation Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Use for research and development purposes only To replace a capillary array or to install a capillary array on an instrument Step Action 1 Close the oven and instrument doors and then press the Tray button 2 From the Tools menu select Install Capillary Array Wizard 13100 Data Collection Software Version 1 0 File View Instrument GME Service Help Hele mal Plate Editor Module Editor ee Run View Change Poymer Wizard Install Capillary Array Vizard Astosampler Calibracion Vizard Perform Sratial Calikration Display Spatial Calibration Display Spectral Calibration Pending Plate Reco Rate Name This opens the wizard Install Replace Capillary Anay Wizard x 1 Toinstall an array delermine what to do with the currently installed array C Removethe array for storage C Remove and discard the array C Install only Thereis nn array on the instrumert
63. procedure after cleaning the polymer reserve syringe or before the polymer in the syringe is 1 week old P RO CHEMICAL HAZARD POP polymer may cause eye skin and respiratory tract irritation Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Use for research and development purposes only IMPORTANT Wear gloves while performing the following procedure and any other time you handle the capillary array glass syringes septa or buffer reservoirs To prepare the polymer reserve syringe for use Step Action 1 Draw approximately 0 3 mL of room temperature polymer into a clean polymer reserve syringe Pull up the plunger to the 5 mL mark 3 Invert the syringe about six times to coat the walls with polymer Discard this polymer into aqueous waste Note Priming the syringe ensures that the running polymer is at the intended concentration and not diluted by residual water 4 Fill the polymer reserve syringe with a maximum of 4 5 mL of polymer IMPORTANT Avoid introducing air bubbles into the polymer by keeping the syringe tip just submerged in the polymer while aspirating gently 5 Remove any air bubbles by inverting the syringe and pushing a small amount of polymer out of the tip Note Do not return the unused portion of the polymer to the bottle 7 SO NGREL N CHEMICAL HAZARD POP polymer may cause eye skin and respiratory tract
64. run the same sample up to five times using different combinations of analysis modules and run modules as follows Same run module but different analysis module Same analysis module but different run module Different run module and analysis module Same run module and analysis module replicate run Note Make sure that you have enough sample for the number or runs you specify Multiple runs of the same sample are set up in the plate record or tab delimited text files imported to create a plate record To perform more than one run with the same sample add additional pairs of run modules and analysis modules to the tab delimited text file as shown in the examples below Example One A Sample Running with More Than One Run Module Here is part of a spreadsheet showing data for a sample that will be run with three different run modules with the same analysis module Run Module Analysis Module runmod005 5 analmod35 runmod008 1 analmod35 runmod010 1 analmod35 The Run Module and Analysis Module column headings are used only once The run modules and analysis modules are grouped in pairs with the run module always placed to the left of its paired analysis module Example Two A Sample Running with More Than One Analysis Module Here is part of a spreadsheet showing data for a sample that will be run with three different analysis modules but with the same run module Run Module Analysis Module runmod005 3 analmod22 runmodO05
65. spatial calibration problems The log file is stored in the same directory as the spatial calibration files with the following file name format SpatialCal instrumentname Runaate time log Spatial and Spectral Calibrations 4 5 Performing a Spatial Calibration Performing a Spatial To perform a spatial calibration Calibration Step Action 1 From the Tools menu select Perform Spatial Calibration The following dialog box appears E Perform Spatial Calibration x Spatial calibration progress Click on the Start button to initiate spatial calibration Start Cancel P Fill capillaries Select the Fill capillaries check box if the Capillaries have no polymer i e a new capillary array or Polymer in the capillaries has been used in a run Note You need not fill the capillaries each time you perform a spatial calibration Click Start The calibration takes approximately 2 min without filling the capillaries 6 min with filling the capillaries 4 6 Spatial and Spectral Calibrations To perform a spatial calibration continued Step Action 4 If the calibration Then succeeded the following dialog box appears F Perform Spatial Calibration Ea Spatial calibration progress Spatial calibration was successful Fill capillaries Details OK Cancel a Click Details to view the Spatial Calibration Profile window b Co
66. startptRange has no difference 2000 3000 Frame number is less than lower bound of range so lower bound is used 4200 4000 Frame number is higher than lower bound of range so upper bound is used Spatial and Spectral Calibrations 4 49 minRankQ Parameter Introduction The minRankQ parameter is used as an internal check on spectral purity Spectral purity is measured by a mathematical metric called rank Pure peaks will have rankQ values close to 1 whereas peaks consisting of mixtures of two or more dyes will have lower rankQ values closer to 0 Peaks with rankQ values less than the value of minRankQ are rejected For this instrument minRankQ is set to 0 4 4 50 Spatial and Spectral Calibrations Software Overview In This Chapter The following topics are covered in this chapter Topic See Page Section About the 3100 Software 5 3 ABI Prism 3100 Genetic Analyzer Software CD ROMs 5 4 3100 Genetic Analyzer Software Suite 5 5 Types and Locations of Files 5 9 Section Setting the Format for the Displayed Dye Colors 5 11 Using the Edit Dye Display Information Dialog Box 5 12 Using the Set Color Dialog Box 5 13 Section Controlling the Instrument Using Manual Control 5 15 Manual Control Commands 5 16 Using Manual Control Commands 5 17 Section Working with Run Modules 5 19 Viewing a Run Module 5 20 Editing or C
67. stored for a dye set Spectral Calibration Action Profile for a Dye Set Step 1 From the Tools menu select Display Spectral Calibration bg Service Help Plate Editor Module Editor Change Polymer Vizard Install Capillary Array Wizard Autosampler Calibration Vizard Perform Spatial Calibration Display Spatial Calibration tral Calibration The Question dialog box appears 7 Display matrices from Dye set Previous run Cancel Click Dye set This opens the Select the source to display dialog box E Select the source to display x Drop down list of dye sets D v OK Cancel From the drop down list select the dye set for the matrices that you want to examine Spatial and Spectral Calibrations 4 25 To display a current spectral calibration profile stored for a dye set continued Step Action 4 Click OK This opens the Matrices for dye set box if Matrices for dye set D E n eee e a a a aaaea aaa 1 2 3 4 5 6 7 8 9 10 14 12 13 14 15 16 17 18 19 20 Intensity vs Bin Number 3000 4000 5000 Intensity vs Scan Number k E Q Value 0 994 Capillary Number 1 Condition Number 5 18 Data Source ok Cea 5 Use the arrow buttons or the slider to review the data for each capillary For a good quality calibration each capillary should have a Q value above 0 95 See
68. template To create a custom spreadsheet template Step Action 1 Use the directions starting on page 6 23 to create a plate file plt file that contains the basic information that you need for a plate record Open the plt file in a spreadsheet program Save the spreadsheet as a read only file to ensure that it does not get overridden To create a plate record from a custom spreadsheet template Step Action 1 Open the spreadsheet that you just created to use as a template 2 Save the spreadsheet under a different name making sure that it is not read only as above Edit the plate and sample data in the spreadsheet according to the specific plate and samples you are using Save the spreadsheet as a tab delimited text file giving it the plt extension If needed repeat steps 1 4 to create other tab delimited text files Follow the directions starting on page 6 32 for importing a tab delimited text file to create a plate record Working with Plate Records 6 29 Creating a Plate File from an Edited Plate Record Introduction To save time when preparing plate records you can save the data entered into the plate editor table as a tab delimited text file After changing the plate name the file can be re imported Alternatively it can be saved as a read only file and used as a template Creating a Plate File To create a plate file from an edited pla
69. the ABI PRISM 3100 Data Collection software Plate Editar File Edit Plate Name SEM Wel Sample Neme Dyes Color into 3 Beginning the Run The operator places the plates on the instrument and starts the run The autosampler automatically moves the sample plate into position to be sampled by the 16 capillaries 4 Electrophoresis Molecules from the samples are electrophoretically injected into thin fused silica capillaries that have been filled with polymer Electrophoresis of all samples begins at the same time when a voltage is applied across all capillaries The DNA fragments migrate towards the other end of the capillaries with the shorter fragments moving faster than the longer fragments 5 Excitation and Detection As the fragments enter the detection cell they move through the path of a laser beam The laser light causes the dye on the fragments to fluoresce The fluorescence is captured by a charge coupled device CCD camera 2 6 System Overview Stage Description Diagram Data Collection The CCD camera converts the fluorescence information into electronic information which is then transferred to the computer workstation for processing by the 3100 Data Collection software Data Processing After the data is processed it is stored in the instrument database and displayed as an electrop
70. the toolbar to begin the run Run Times The following table lists the spectral calibration run times Capillary Array Length Approximate Run Time Application cm min Fragment analysis 36 30 DNA sequencing 36 40 50 65 Spatial and Spectral Calibrations 4 23 Spectral Calibration At the end of the run while the data is being analyzed the Spectral Calibration Result Result Box dialog box opens to indicate which capillaries have passed and which have failed The example below for Dye Set E shows one failed capillary which is represented by an X and 15 passed capillaries which are represented by a dot r Spectral Calibration Result x Found 15 possible spectra for dye set E Please view and edit the spectra 0K Failed capillary X Passed capillary To acknowledge the completed calibration run Step Action 1 In the Spectral Calibration Result dialog box click OK IMPORTANT Review and evaluate the spectral calibration profile for each capillary even if the Spectral Calibration Results box indicated that they all passed See Displaying a Spectral Calibration Profile on page 4 25 When a Capillary If a capillary fails it is automatically assigned the spectral profile of its nearest passing Fails capillary to the left If there are no passing capillaries to the left it will be assigned the profile of the nearest passing capillary
71. these effects on your back legs eyes and upper extremities neck shoulder arms wrists hands and fingers design your workstation to promote neutral or relaxed working positions This includes working in an environment where heating air conditioning ventilation and lighting are set correctly See the guidelines below MUSCULOSKELETAL AND REPETITIVE MOTION HAZARD These hazards are caused by the following potential risk factors which include but are not limited to repetitive motion awkward posture forceful exertion holding static unhealthy positions contact pressure and other workstation environmental factors Use a seating position that provides the optimum combination of comfort accessibility to the keyboard and freedom from fatigue causing stresses and pressures The bulk of the person s weight should be supported by the buttocks not the thighs Feet should be flat on the floor and the weight of the legs should be supported by the floor not the thighs Introduction and Safety 1 7 Electric Shock Lifting Moving 1 8 Introduction and Safety Lumbar support should be provided to maintain the proper concave curve of the spine Place the keyboard on a surface that provides The proper height to position the forearms horizontally and upper arms vertically Support for the forearms and hands to avoid muscle fatigue in the upper arms Position the viewing screen to the height that all
72. to failure to follow the recommended maintenance procedures b modified or repaired by a party other than Applied Biosystems or c used in a manner not in accordance with the instructions contained in the Instrument User s Manual This Warranty does not cover the customer installable accessories or customer installable consumable parts for the Instrument that are listed in the Instrument User s Manual Those items are covered by their own warranties Applied Biosystems obligation under this Warranty is limited to repairs or replacements that Applied Biosystems deems necessary to correct those failures of the Instrument to meet the Specifications of which Applied Biosystems is notified prior to expiration of the Warranty Period All repairs and replacements under this Warranty will be performed by Applied Biosystems on site at the Customer s location at Applied Biosystems sole expense No agent employee or representative of Applied Biosystems has any authority to bind Applied Biosystems to any affirmation representation or warranty concerning the Instrument that is not contained in Applied Biosystems printed product literature or this Warranty Statement Any such affirmation representation or warranty made by any agent employee or representative of Applied Biosystems will not be binding on Applied Biosystems Limited Warranty Statement D 1 Applied Biosystems shall not be liable for any incidental special or consequential loss damag
73. 0 Data Collection software preference settings gt gt gt ad Electrophoresis modules run modules and calibration modules EPT data This manual describes how the database is used by the 3100 software Consult an Oracle database administrator for more information about administering the database If you purchased the GeneScan option GeneScan Analysis software will be installed on the hard drive of your computer workstation This software is used to Review the fragment analysis profile and size data Reanalyze the data If you purchased the sequencing option DNA Sequencing Analysis software will be installed on the hard drive of your computer workstation This software is used to Review basecalled sequences Reanalyze the basecalled sequence Additional information about the ABI PRISM 3100 Genetic Analyzer software can be found in the readme files and release notes on the software CD ROMs Types and Locations of Files Introduction The ABI PRiSM 3100 Genetic Analyzer software includes many different files and folders Some of these are created to store run data and calibration data Others are required to run the software IMPORTANT Never move or delete any file or folder unless specifically directed to do so by an Applied Biosystems representative or the 3100 Genetic Analyzer documentation Doing this could render the software inoperable Filename Extensions You can recognize certain file types by the
74. 00 Genetic Analyzer 1 2 To Get Started Quickly 1 3 Additional Documentation 1 4 Safety 1 5 Introduction and Safety 1 1 ABI PRISM 3100 Genetic Analyzer Definition The ABI PRism 3100 Genetic Analyzer is an automated capillary electrophoresis system that can separate detect and analyze up to 16 capillaries of fluorescently labeled DNA fragments in one run System Components 1 2 Introduction and Safety The 3100 Genetic Analyzer system includes the following components ABI PRISM 3100 Genetic Analyzer Computer workstation with Microsoft Windows NT operating system ABI PRISsM 3100 Genetic Analyzer software ABI Prism DNA Sequencing Analysis or ABI PRism GeneScan Analysis software Capillary array Reagent consumables To Get Started Quickly Important Safety Information What You Should Know Getting Started Quickly Before using the 3100 Genetic Analyzer read the safety information starting on page 1 5 and in the ABI PRISM 3100 Genetic Analyzer Site Preparation and Safety Guide P N 4315835 This manual is written for principle investigators and laboratory staff who are planning to operate and maintain a 3100 Genetic Analyzer Before attempting the procedures in this manual you should be familiar with the following topics Windows NT operating system General techniques for handling DNA samples and preparing them for electrophoresis Detailed information about prepa
75. 1 Ensure that the Oven door is closed and locked Instrument doors are closed Note If the doors are open during power up the red failure light will illuminate 2 Ensure that the Computer is powered on see Starting the Computer on page 3 12 4 Microsoft Windows NT operating system has loaded Note The computer must be on and running the Windows NT operating system because the instrument must copy the firmware from the computer 3 Turn on the instrument by pressing the on off button on the front of the instrument Status lights Press the on off button to start the instrument Note While the instrument is booting up and performing self checks the yellow status light will blink 4 Ensure the green status light is on and constant before proceeding Note Ifthe green light does not come on start the ABI PRISM 3100 Data Collection software and look at the event log If this is not helpful call a Technical Support representative See Appendix B Technical Support Performing a Run 3 13 Starting the 3100 Data Collection Software Before You Begin Before starting the Data Collection software Step Action 1 Ensure the ABI PRiSmM 3100 Genetic Analyzer is powered on and that the green status light is on solid not flashing 2 Ensure OrbixWeb Daemon is running by finding its button on the Windows NT taskbar A Start E OrbikWeb Daemon If OrbixWeb Daemon is not running go to the Start menu p
76. 3 51 stopping a run 3 47 storage device installing 3 64 syringes cleaning and inspecting 8 21 controlling See manual control commands installing and removing 8 23 leaking 9 7 overview 8 20 priming and filling 8 22 system administration privileges computer 7 16 system management options 7 14 to 7 15 Szs size standard files creating 5 40 to 5 43 T tab delimited text files See text files tab delimited technical support B 1 to B 5 e mail address B 1 Internet address B 4 telephone fax North America B 2 temperature electrophoresis 2 25 templates location 6 24 text files tab delimited plate records 6 9 See also seq sequence text files toolbar 3 47 total intensity graph 3 55 transmission grating discussed 2 29 tray See plates Index 5 U unlinking a plate 3 43 username 3 12 7 16 utilities Cleanup Database 7 8 Diskspace 7 5 Initialize Database 7 12 New Method Import 7 10 Re Extraction 7 6 Remove Run Modules 7 11 V viewing run schedule 3 50 WwW warning laser 2 29 warnings 1 5 warranty D 1 waste 8 12 Windows NT Security dialog box 7 16 wizards Autosampler Calibration 8 29 Change Polymer 8 10 Install Capillary Array 8 15 write seq files option 5 33 writeDummyDyes parameter 4 46 WWW address Applied Biosystems B 4 Documents on Demand B 4 Y yellow capillary in Array View 4 24 Index 6 Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free 1 800 345
77. 3 analmod10 runmod005 3 analmodO6 The Run Module and Analysis Module column headings are used only once The run modules and analysis modules are grouped in pairs with the run module always placed to the left of its paired analysis module 6 18 Working with Plate Records Section Creating a Plate Record by Importing LIMS Data In This Section The following topics are covered in this section Topic See Page Data Transfer 6 20 Plate Import Table 6 21 Introduction This section provides an overview of transferring data from a laboratory information management system LIMS to the plate import table and a description of the format in which the LIMS data must be written This section does not describe the detailed procedure which is beyond the scope of this manual Note To import LIMS data you must know how to import binary data BLOBS into an Oracle database Working with Plate Records 6 19 Data Transfer Advantages of Importing Data from a LIMS Database Automatic Data Transfer Configuring the 3100 Data Collection Software for LIMS Import Data transferred from a LIMS database creates plate records that are identical to plate records created from tab delimited text files The advantages of using a LIMS database over tab delimited text files are The sample data is already entered into a LIMS database Therefore the data can be assembled quickly into the format required for import Trans
78. 3100 Genetic Analyzer plate assemblies Turn on computer Turn on instrument Prepare samples and Launch ABI Prism 3100 Data Collection software Enough space on computer for sample data Run Cleanup DB utility Calculate space required Check available space Setup instrument e Prepare syringes e Install capillary array e Add change polymer e Fill reservoirs A No Spatial calibration done ae Perform calibration Display and check calibration Spectral calibration done Perform calibration Display and check calibration Place plate s on autosampler View set software preferences Create plate record s Link plate s to plate record s Start and monitor run s View and archive data GR1939 3 4 Performing a Run Planning Your Runs Decision Table Decisions to Make The main decisions you will need to make when preparing for a run are listed below Decision Comments Analysis application Either ABI PRISM GeneScan Analysis software for fragment analysis ABI PRism DNA Sequencing Analysis software for sequencing or Rapid DNA Sequencing Analysis Type of polymer Either ABI PRism 3100 POP 4 for fragment analysis ABI PRISM 3100 POP 6 for DNA sequencing or rapid DNA sequencing Length of capillary array Either 36 cm array for fragm
79. 5 Moving and Leveling the Instrument 0 0 eee eee eee 8 6 Resetting the Instrument 21 0 0 0 eee eee eens 8 7 Shutting Down the Instrument 0 0 0 eee eee ee 8 8 Section Fluids Gud Waste sree ciosa As Rs REEDS ARES CO Me Ok OOS SE 8 9 Buffer ra cee sien ba te oat eee Phe Labtec abe weds Eda es 8 10 POlyiletis oe deog etade psn pn bed aage des cede sae E ieee alulsersiaiteecte aly yc geass 8 10 Handling Instrument Waste 0 0 cece eee nee 8 12 Section Capillary Array 0 0 0 cc cc ccc cee eee ee enna ee ee eee 8 13 Before Installing a Previously Used Capillary Array 00 0 0000000002 eee 8 14 Installing and Removing the Capillary Array 0 0 00 eee eee 8 15 Capillary Array Maintenance 0 0 cence tenes 8 16 Storing a Capillary Array on the Instrument 0 0 0 0 eee eee eee eee 8 17 Storing a Capillary Array off the Instrument 0 00 0 eee eee eee ee 8 17 SECHNON SYTINGES o VEEE EEN EEEN Eke Read Se Ae ERA PES 8 19 Syringe Maintenance seas sik oe Sek eee eA nh WS as PO oak eI oa Wee oo Bae Mea 8 20 Cleaning and Inspecting Syringes 0 0 0 cee eens 8 21 Priming and Filling Syringes 1 2 0 cece een ee 8 22 Installing and Removing Syringes 0 0 0 0 cece eee eee ee 8 23 Section Polymer Blocks ro edict eee EGR OS OBO eS Ba Sad RN 8 25 Removing the Polymer Blocks 0 00 c cece cc eee eee ee 8 26 Cleaning the P
80. 5 28 Sequencing Analysis software 5 8 directory path for SeqA exe 5 28 sequencing chemistry types supported 2 18 Set Color command 5 12 to 5 14 shut down 8 8 signal too high 9 5 size standard szs files creating 5 40 to 5 43 software list of applications 5 4 overview of suite 5 5 setting preferences 3 28 setup 3 27 to 3 43 spatial calibration discussed 4 3 to 4 13 displaying 4 10 evaluating profiles 4 11 failed 4 9 log files 4 5 overriding 4 12 performing 4 6 persistently bad results 9 3 procedure 4 6 purpose A 4 unusual peaks 9 3 when required 3 6 3 24 4 4 spatial dimension on CCD camera A 3 spatial maps 4 5 spectral calibration 4 15 to 4 32 displaying a profile 4 25 displaying profiles for past runs 4 27 failure 4 24 file 4 36 fine tuning 4 34 log files 4 37 matrices 4 36 matrix A 6 no signal 9 4 overriding profiles 4 28 parameter files 4 38 parameters 4 40 performing for sequencing 4 18 to 4 23 preparing standards for 4 18 4 19 procedure 4 18 purpose of A 4 run times 4 23 setting parameters 4 40 viewing the matrix 4 36 when required 3 6 3 24 spectral dimension on CCD camera A 3 spectral overlap A 6 spectrograph 2 29 spreadsheet programs creating plate records 6 12 6 29 standards loading 4 20 starting instrument 3 13 run 3 47 spectral calibration run 4 23 startptOffset parameter 4 40 4 48 startptRange parameter 4 40 4 49 status lights 2 10 on instrument startup 3 13 Status View page discussed
81. ABI PRISM 3100 Genetic Analyzer User s Manual AS appisd ns HITACHI Copyright 2001 2010 Applied Biosystems For Research Use Only Not for use in diagnostic procedures NOTICE TO PURCHASER This instrument Serial No is Authorized for use in DNA sequencing and fragment analysis This Authorization is included in the purchase price of this instrument and corresponds to the up front fee component of a license under process claims of U S patents and under all process claims for DNA sequence and fragment analysis of U S patents now or hereafter owned or licensable by Applied Biosystems for which Authorization is required and under corresponding process claims is foreign counterparts of the foregoing for which an Authorization is required The running royalty component of licenses may be purchased from Applied Biosystems or obtained by using Authorized reagents pur chased from Authorized supplier in accordance wit the label rights accompanying such reagents Purchase of this instrument does not itself convey to the purchaser a complete license or right to perform the above processes This instrument is also licensed under U S patents and apparatus and system claims in foreign counterparts thereof No rights are granted expressly by implication or by estoppel under composition claims or under other process or system claims owned or licensable by Applied Biosystems For more information regarding licenses please contact the Director of Licensin
82. Analysis 00 0 00005 3 36 Linking and Unlinking a Plate 2 0 0 eee eens 3 41 Section Running the Instrument 000 cc ccc ce ene e eee eeeees 3 45 About Run Scheduling 1 0 0 0 ee cece eee eee eee 3 46 Controlling the RUN gt i44 esse amp seek ao eee tee os Kee ee ees Le bP ie bah ee 3 47 Run Dimes verde ia wakes si ee ee ae nee eA ae ih ee eek ak conde eens 3 48 Section Monitoring a RUN 1 0 ccc cee cence eee eeeees 3 49 Run Mew Pago di renna lett of eta ee oleae bce arhs hae aw aye kee 3 50 Status View Paget aie wcs sashes eae ye ese GAP ieee ey EE wy ey ey ey EAS 3 51 Array View Page cerconta etnies baie Rise baie heed ot ta ay eee 4 3 53 Capillary View Page 2 220000 2 esi RA bce ste bGaa aad BAG aoe le deine es 3 56 Instrument Status Monitor 2 02 eect eee eee 3 57 Section Working with Data 0 0 ccc ccc ccc cen een nen ee eee eeees 3 59 Recovering Data if Autoextraction Fails 0 0 0 3 60 Viewing Raw Data from a Completed Run in the Data Collection Software 3 61 Viewing Analyzed GeneScan Data 0 eee eee 3 62 Viewing Analyzed DNA Sequencing Data 0 0 eee eee 3 63 Archiving Data pecen sidene iaa ee Ghee EE wh eres Ved nb heeds eee 3 64 Spatial and Spectral Calibrations OVE eW ihe hese en E aa ea eas E Suelo alaarsicg God aves ROE A EAE 4 1 Section Spatial Calibration 0 0 0 0 cc ccc ccc eee eee eee e teen eens 4 3 About Spatial Calibrat
83. 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 To Obtain Free 24 hour access to Applied Biosystems technical documents including MSDSs Documents on iS available by fax or e mail or by download from our Web site Demand To order documents Then by index number a Access the Applied Biosystems Technical Support Web site at http www 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 a From the U S or Canada call 1 800 487 6809 or delivery from outside the U S and Canada call 1 858 712 0317 b Follow the voice instructions to order the documents you want Note There is a limit of five documents per request B 4 Technical Support To order documents Then through the a Access the Applied Biosy
84. Cancel d Click Finish This opens the Plate Editor spreadsheet Spatial and Spectral Calibrations 4 21 To create a plate record for the denatured matrix standards continued Step Action 3 Complete the Plate Editor spreadsheet for the wells you have loaded For Perform the following Dye Set E a Type a name for the samples Matrix standard b Select Dye Set E c Select the run module depending on your capillary array size 36 cm Spect36_POP6DefaultModule 50 cm Spect50_POP6DefaultModule d Select the spectral parameter MtxStd Sequencing SetE par Click OK Dye Set E Type a name for the samples Select Dye Set E Select the run module depending on your capillary array size 36 cm Spect36_POP6DefaultModule 50 cm Spect50_POP6DefaultModule d Select the spectral parameter SeqStd Sequencing SetE par Click OK BigDye sample 9 Oo 9 0 Dye Set D Type a name for the samples Select Dye Set D Select the run module Spect36_POP4DefaultModule d Select the spectral parameter MtxStd GeneScan SetD par e Click OK oO oO YM oO IMPORTANT Make sure the correct spectral parameter file has been selected for the type of dyes you are running Selecting the incorrect parameter file will cause the spectral calibration to fail This creates a plate record for the calibration run in the database After a few secon
85. Clean and rinse the reservoir with deionized water and then rinse with buffer 2 3 4 Fill the reservoir to the fill line with fresh 1X running buffer about 9 mL c Fill line GR1878 Performing a Run 3 23 To fill the anode buffer reservoir to the fill line with 1X running buffer continued Step Action 5 Put the anode buffer reservoir on the instrument Note The meniscus should line up with the fill line 6 If the reservoir fills with fluid repeat this procedure to discard and replace the running buffer Note The reservoir could fill during bubble clearing Calibrating the Instrument When to Perform a Spatial Calibration When to Perform a Spectral Calibration 3 24 Performing a Run If necessary perform a spatial calibration A spatial calibration must be performed after each time you Install a capillary array Replace a capillary array with a new one Temporarily remove the capillary array from the detection block For instructions see Spatial Calibration on page 4 3 If necessary perform a spectral calibration A spectral calibration must be performed Whenever you use a new dye set on the instrument After the laser has been realigned by a service engineer After the CCD camera has been realigned by a service engineer If you begin to see pull up and or pull down peaks consiste
86. Dye preparing sequencing sample 4 19 primers 2 19 terminators 2 19 bins A 3 BioLIMS database option discussed 7 15 working with 5 47 to 5 55 BLOB 6 22 buffer discussed 3 22 C c value See condition number camera CCD See CCD camera capillary alignment 8 16 status 3 54 capillary array 2 24 checking alignment of capillaries 8 16 filling See manual control commands installing and removing 8 14 maintenance 8 16 poor performance 9 7 storing off the instrument 8 17 Capillary View page discussed 3 56 capillary to CCD pixel mapping A 4 CCD camera 2 29 A 3 emission spectrum A 4 pixel arrangement A 3 CDs list 3100 software 5 4 chemistry dye sets 2 18 labels 2 19 overview 2 17 to 2 21 types supported 2 18 chemometric method A 6 cleaning instrument routine 8 5 Cleanup Database utility 7 8 colors displayed dye See displayed dye colors commands manual control 5 16 components of 3100 system 2 4 computer checking hard drive space 3 16 checking logon user name 7 16 frozen 9 2 hard drive partitions 2 14 installing an internal SCSI device 3 64 name finding 7 17 network domain finding 7 17 networked computer requirements 7 18 networking 7 14 to 7 17 requirements 2 14 start up and logon 3 12 system administration privileges 7 16 condition number 4 44 to 4 45 conditionBounds parameter 4 40 4 44 to 4 45 confidence bands electropherogram display A 6 confidence limits A 6 Control Panel window 7 17 current troubleshooting
87. GeneScan analysis module gsp file Analysis Modules Step Action 1 Start the GeneScan Analysis software You may have a program icon for the GeneScan Analysis software on the Start menu or a shortcut icon on your desktop If not you can find the application GeneScan exe in the following directory D AppliedBio abi GeneScan Bin 2 From the File menu select Open 3 Select the Analysis Parameters icon all x Open Existing TA BA o BE Project Sample Foalysis Size Parameters Standard 4 Select the analysis module you want to view or edit The analysis modules are stored in the following directory D AppliedBio abi Shared Analysis Sizecaller Params Look in Params 7 amp cl ia GS3504nalysis asp GS400CubicAnalysis gsp a GS400HDAnalysis gsp 8 GS4000rd2Analysis gsp a GS5004nalysis gsp File name Files of type Analysis Params File GSP Cancel 5 Click Open This opens the analysis module 5 38 Software To view or edit a GeneScan analysis module gsp file continued Step Action 6 If you want you can make changes to the analysis module For more information about the parameters see the ABI PRISM GeneScan Analysis Software v 3 5 NT User s Manual P N 4308923 Analysis Range Size Call Range Full Range Full Range This Range Data Points This Range Base Pairs po is fi2000 fioo Data Processing Size Calling Method C 2nd
88. LIMS database This requires Specifying a BioLIMS project in the plate s sample sheet Setting BioLIMS preferences for the plate Specifying a To specify a BioLIMS project in the plate s sample sheet BioLIMS Project Step Action 1 From the Tools menu select Plate Editor The Plate Editor window appears Plate Editor Lx Place Name Apalication Sequencing GeneScan C Spectral Calibration Plae Type fewer gt Comments Finish Cancel 2 Fill in the window items as follows Item Action Plate Name Type the plate name Application Click on the appropriate application Plate Type Choose the appropriate type from the drop down list Comments Type comments if desired 5 50 Software To specify a BioLIMS project in the plate s sample sheet continued Step Action 3 Click Finish The Plate Editor spreadsheet appears with the plate name you assigned in step 2 Plate Editar File Edit Plate Name ESEE Run Modul Fill in the spreadsheet as appropriate making sure to choose a BioLIMS project To do this click on the BioLIMS Project column for each well and choose a project from the drop down list IMPORTANT If you do not choose a BioLIMS project your samples will not be extracted into the BioLIMS database successfully Note If you need more i
89. Log Files The spatial map is a simple numerical table that defines the number of pixels at the center of the fluorescence from each capillary in the spatial dimension of the CCD For information on the organization of the CCD and frame data refer to Appendix A Data Flow Only one spatial map at a time is stored in the database on drive E The term current spatial map refers to the spatial map that is currently stored in the instrument database You can replace override the current spatial map stored in the instrument database with a spatial map stored in a spatial calibration file For the procedure see page 4 12 The maps are stored as text files in the SpatialCalLogs folder in the following directory D AppliedBio abi 3100 DataCollection SpatialCalLogs Spatial maps are also saved to the 3100 Calibration file and to the firmware Spatial calibration files are stored on drive D Each spatial calibration file contains one spatial map from either the current or previous calibrations Spatial calibration files have the following file name format SpatialCal instrumentname Rundate time scl A spatial calibration log file is created during a spatial calibration It contains a summary of the data collected during the spatial calibration run including the pixel positions assigned to each capillary The log file is a text file that can be opened and viewed in the Notepad accessory It can be useful for troubleshooting
90. MPORTANT Never allow more than about 400 or 500 runs to reside in the database This could lead to indiscriminate loss of data System Management and Networking 7 5 Re Extracting Processed Frame Data The Re Extraction Utility Function File Name and Directory Used Only for Extracted Runs Re Extraction Into a BioLIMS Database Re Extracting On a Networked Computer Re Extracting Processed Frame Data The Re extraction utility makes new ABIF sample files from run data stored in the database Note This utility cannot be used to replace a sample file once the run data in the instrument database has been deleted The Re extraction utility file is named Reextractor exe and is located in the following directory D AppliedBio abi 31 00 Bin The Re extraction utility works only for runs that have the status Extracted You will not be able to re extract failed runs acquired runs or runs that failed to be extracted The Re extraction utility cannot re extract directly to a BioLIMS database To re extract into BioLIMS use Reextractor exe to extract from the instrument database to ABIF sample files Then use the S2DB utility to upload the sample files into the BioLIMS database If you are re extracting data on a networked computer the computer must meet specific requirements and have certain software loaded See Requirements for a Networked Computer on page 7 18 To re extract processed frame data
91. NT Damage to the array tips will occur if the plate retainer and septa strip holes do not align correctly The plate retainer holes must align with the holes in the septa strip Section Starting the 3100 System In This Section The following topics are covered in this section Topic See Page Starting the Computer 3 12 Starting the Instrument 3 13 Starting the 3100 Data Collection Software 3 14 Performing a Run 3 11 Starting the Computer Starting the IMPORTANT You must start the computer workstation before starting the ABI PRISM 3100 Computer Genetic Analyzer Workstation To start the computer workstation Step Action 1 Turn on the monitor 2 Power on the computer The computer boots and then the Begin Logon dialog box appears 3 Press CTRL ALT DELETE and enter the user name and password Note The default user name for the workstation is 3100User Do not change this user name There is no default password If you would like to use a password your system administrator can create one If the computer is connected to a network you do not need to log on to the network before starting the instrument OrbixWeb Daemon will launch automatically If it does not launch repeat steps 1 3 3 12 Performing a Run Starting the Instrument Starting the To start the 3100 Genetic Analyzer Instrument Step Action
92. ORTANT The name of the run module must be typed correctly If the name is typed incorrectly the plate will be imported but the run module will not be entered in the plate record Analysis Module Specifies the analysis module used to run the sample Sequencing analysis modules have the file format filename saz IMPORTANT This cell or token must always be last from left to right IMPORTANT You must always select an analysis module if you want the data to be extracted and analyzed IMPORTANT The name of the analysis module must be typed correctly If the name is typed incorrectly the plate will be imported but the analysis module will not be entered in the plate record Note The Dye Set Comment and Project Name tokens can be presented in any order Working with Plate Records 6 15 Column Header for The column header for GeneScan analysis contains up to 10 cells or tokens that GeneScan Analysis provide headings for the columns that will contain the sample data The order in which the column cells or tokens are listed is important and is noted below Cell or Token Function Well Position Identifies the well in which the sample is located e g A1 G6 O18 etc For 96 well plates the well positions are A H and 1 12 For 384 well plates the well positions are A P and 1 24 IMPORTANT This cell or token must always be first from left to right Sample Name Identifies the sample The sample n
93. Order Least Squares paella Options C 3rd Order Least Squares vee Cubic Spline Interpolati Light ubic Spline Interpolation Hess Local Southern Method Global Southern Method Peak Detection Baselining Peak Amplitude Thresholds BaseLine Window Size 251 Pts Auto Analysis Only Size Standard Min Peak Half Width Pts Polynomial Degree B Peak Window Size fia Pts Slope Threshold for po Peak Start Slope Threshold for fg Peak End 7 the analysis module and you If you have made changes to Then want to save the changes From the File menu select Save As assign a unique name and then click OK or From the File menu select Save to save the changes to the current analysis module IMPORTANT The analysis modules must be stored in the following folder D AppliedBio abi Shared Analysis Sizecaller Params do not want to save the changes Click the Close button to close the window Software 5 39 Creating a GeneScan Analysis Module Before Beginning Creating a Size Standard File 5 40 Software Before creating a GeneScan analysis module you may need to create a custom size standard file You will need to create a custom file for performing Denaturing runs but not using GS350 400HD or GS500 Non denaturing runs using applications such as SSCP Runs using one of the Applied Biosystems internal lane standards but significantly altering collection time or analysi
94. Plate Record About the Procedure Plate Records for Spectral Runs 3 30 Performing a Run A plate record is similar to a sample sheet or an injection list that you may have used with other ABI PRism instruments Plate records are data tables in the instrument database that store information about the plates and the samples they contain Specifically a plate record contains the following information Plate name type and owner Position of the sample on the plate well number Sample name Dye color of size standard if present GeneScan analysis only Mobility file DNA sequencing analysis only Comments about the plate and about individual samples Dye set information BioLIMS project this entry is mandatory even when BioLIMS is not used gt gt oo oo oo o o The name of the run module run modules specify information about how samples are run The name of the analysis module analysis modules specify how raw data is autoanalyzed at the end of the run A plate record must be created for each plate of samples for the following types of runs Fragment analysis DNA sequencing Spectral calibrations Note For fragment analysis and sequencing runs there is no need to re create a plate record for a plate that has failed Simply edit the plate record to add a run module and an analysis module column to the rows that need to be re run This will move the existing plate record from the Processed window to the
95. Project1 GeneScan36_POP4b1Defaulthodu std 3166 Project1 GeneScan36_POP4b1Defaulthodu std 3166 Project1 GeneScan36_POP4b1Defaulthodu std 3166_Project1 GeneScan36_POP4b1DefaultModu std 3166_Project1 GeneScan36_POP4b1DefaultModu d EWA Empty Cells or Tab delimited text files may be imported with empty tokens Missing data can be tokens added using the plate editor IMPORTANT A space character entered by pressing the space bar must be entered between tab stops in a tab delimited text file as a place marker for missing information A space character must be entered into each blank cell of a spreadsheet before converting it to a tab delimited text file IMPORTANT Do not leave whole empty rows with the exception of the Well Location row ina spreadsheet or tab delimited text file that is intended for import as illustrated by the example below Do this Do not do this A B A B 1 0 N 2 PlateName0 5Q _2 PlateName0 5Q 3 Well Sample Name 3 Well Sample Name WA cell_sample 01 4 Al cell_sample 01 El cell_sample 03 E cell_sample 03 leci cell_sample 04 6 c1 cell_sample 04 EAF cell_sample 05 1 REI BRF 1 cell_sample 05 Typing Accuracy It is extremely important to be accurate when typing information into a spreadsheet and Error Messages tab delimited text file or LIMS database that will eventually be imported into the 3100 Data Collection software When the 3100 Data Collection software is importing data from a te
96. S 30 for Dye Set D by combining the following in a labeled 1 5 mL microcentrifuge tube Reagent Volume uL 6FAM 1 25 HEX 1 25 NED 1 25 ROX 1 25 Hi Di Formamide P N 4311320 195 Final Volume 200 CHEMICAL HAZARD Formamide is harmful if absorbed through the skin and may cause irritation to the eyes skin and respiratory tract It may cause damage to the central nervous system and the male and female reproductive systems and is a possible birth defect hazard Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Vortex thoroughly Spin the mixture briefly in a microcentrifuge Heat the standard tube at 95 C for 5 min to denature the DNA Preparing the Matrix Standards SteP for Dye Set D 1 Matrices 2 3 4 5 6 7 Immediately place the tubes on ice for 2 min Spatial and Spectral Calibrations 4 19 Loading the To load the standards 4 oO g a g T c5 a JAA ose S25 L38 58 5e 2 g vo o5 OE z x 5L aL os 5 o 22S po 2 5 e x gt coo nt OD f Oo g 55 5 2 2 g E3 8 8 s g C28 OF OG 5 o Sooo 5 z LAA gt N xe g T 5 g 2 2 gej o D c ao oo e gil So gej Ww a x Pe oO n c Q a 2 om 2 By oO oe ares EP lictetctevetetetel aed Cosel E z AT e333 Fem Gh sessesseesssssss B
97. S project IMPORTANT A BioLIMS project is required for every sample even if a BioLIMS database is not used a Click in the BioLIMS Project cell for Well A1 b Select a project name from the drop down list _BioLIMS Project Jeno selection sno selection 3100 Projectt IMPORTANT You must enter a BioLIMS Note To set up a BioLIMS project see page 5 48 c To assign the same project name to each sample in the plate record Click the column header to select the whole column Press CTRL D Note Press CTRL D whenever a field is the same for all samples in the plate record To enter sample information and save the plate record continued Step Action 4 For each sample select the appropriate Dye Set from the drop down list For GeneScan analysis select Dye Set D Dye Set IMPORTANT Be sure to select the correct dye set for your run s Data collected with the incorrect dye set selected cannot be saved and the runs will have to be repeated because multicomponenting is applied during collection 5 For each sample select the appropriate Run Module from the drop down list Run Module 1 no seection GeneScan36_POP4DefautModule The following table shows the run module to select based on your run type Analysis Type Run Module GeneScan GeneScan36_POP4DefaultModule Note If you need to view or edit a run module file see page 5 20 Note If you select diff
98. Site 2 Reservoir Site 1 positions El i Delete Delete button Each row in the table provides information about a scheduled run A run can be selected by single clicking on a row Note Although individual runs can be deleted the order in which the runs are scheduled cannot be altered For more information on run scheduling see page 3 46 This grid displays the capillaries in use during a run and the name of the sample that will be injected into a specific capillary Each cell in the grid represents a specific capillary Once a run has started the cells representing capillaries in use will turn blue Placing the cursor over an individual cell will display the name of the sample to be injected in that capillary The plate images provide a visual representation of the physical sample layout for a selected run The Delete button removes a run from the list of scheduled runs First select the run in the Run Schedule window on the left and then click the Delete button Note The Delete button does not delete the samples from the plate record The samples can be run later if desired Status View Page Function Click the Status View tab to monitor the status of the instrument during a run Features This is an example of the Status View page Instrument Condition group box Status bar T o J Waiting for Oven to Stabilize at Run Temperature Actual value 3100 Data Collection Software x Fie View Instrument
99. Spectral Calibrations 4 41 minQ Parameter Introduction About Pull Up and Pull Down Peaks Cause of Pull Up and Pull Down Peaks Q Value The minQ parameter is used to set the tolerance for pull up pull down peaks A pull up peak is a small peak of another color that appears under a main dye peak in an electropherogram A pull down peak is the same except it appears below the baseline under a main dye peak Pull up Pull down peaks Pull up and pull down peaks are caused by Overloading the calibration standards Differences in the shapes of the dye peaks recorded during a spectral calibration run compared to those in a theoretically perfect spectrum The imperfections drag the intensity of the processed fluorescence data from a neighboring dye either up or down The 3100 Data Collection software calculates a value named Q which is a measure of the consistency between the final matrix and the data from which it was computed When the Q value is 1 0 the fit is perfect providing an ideal matrix with no detected pull up pull down peaks The minQ value sets the minimum allowable Q value for a passing capillary After a spectral calibration run the software calculates the Q value for each capillary used in the run If this number is less than the minQ value set in the selected parameter file the capillary will fail and the matrix will be automatically overridden by one from another capillary Note The methods
100. Step Action 4 od BC 3100_SeqOffFtOff saz x Basecaller Basecaller Type Basecaller 3100 v Basecaller Settings Default Settings w P Write Seq Files Sequence File Format ABI FASTA If you want you can edit the settings Basecaller Type can be Basecaller 3100 for standard sequencing or Basecaller 3100RR for rapid run sequencing The files are located in the following directory D AppliedBio abi Shared Analysis Basecaller Params Basecaller Settings are specified in the Preferences dialog box accessed from the Edit menu Ifthe Write Seq Files box is selected text files of the basecalled sequence are written in either ABI or FASTA formats Ifa Factura Settings File is selected Factura processing will be applied during analysis To view or edit a Factura settings file From the File menu point to Open and select Factura Settings The files are located in the following directory D AppliedBio abi Shared Analysis Factura If you have made changes to the analysis module and you Then want to save the changes click Save As to create a new analysis module Enter a unique descriptive name and click OK don t want to save the changes click the Close button to close the window Software 5 29 Creating a Sequencing Analysis Module Procedure Overview Creating a sequencing analysis module requires Creating a basecaller settings file Cre
101. Tools Service Help m2 Mola me Plate View Run View Status View Array View Capillary View Instrument Condition Time Remaining this run 00 42 06 E Laser On 2 Run Time 00 44 24 E E Off EP Voltage EP Current 20 0 kY 800 0 pA E oven On W Front Doors Closed W Oven Door Closed W Autosampler Return Capillary Array Serial Number demo Oven Temp 65 0 foo 55 6 Capillary Array Usage 49 Events led Apr 12 16 58 24 PDT 2000 Set run status to STARTING ed Apr 12 16 58 24 PDT 2000 led Apr 12 16 58 23 PDT 2000 Sending run module to the instrument led Apr 12 16 37 15 PDT 2000 dis connect data communication port fed Apr 12 17 02 00 PDT 2000 RUN PARAMETERS command reply led Apr 12 17 02 03 PDT 2000 BEGIN command reply led Apr 12 17 02 36 PDT 2000 OVEN TEMPERATURE TOLERANCE 3 0 Oven temperature ti Errors Wed Apr 12 16 58 29 PDT 2000 Instrument offline Started a Gene Scan Run Run_demo_3100_2000 04 12_46 L Events box re e L Errors box Set value defined in the selected run module Instrument The color of the box provides a quick way to check the status of the item to the right Condition Group See the table below for a definition of each color Box Events Box A green box A red box A yellow box For indicates indicates indicates Laser Laser is off Laser is on Laser is idle EP Electrophoresis is off Ele
102. a 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 Asia 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 2719 6405 66 2 319 9788 Europe Austria Wien 43 0 1 867 35 75 0 43 0 1 867 35 75 11 Belgium 32 0 2 712 5555 32 0 2 712 5516 Czech Republic and Slovakia Praha 420 2 61 222 164 420 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 Hungary Budapest 36 0 1 270 8398 36 0 1 270 8288 Italy Milano 39 0 39 83891 39 0 39 838 9492 Norway Oslo 47 23 12 06 05 47 23 12 05 75 Poland Lithuania Latvia and Estonia 48 22
103. abase Utility Function The Cleanup Database utility deletes the processed frame data and all associated run information stored in the 3100 Data Collection software database This utility is used to make room for new run data The Cleanup Database utility deletes all of the Processed frame data Plate records and run data This utility does not delete the Electrophoresis modules automatically imported from the supplied method files Run modules that you have created Spatial and spectral calibration data obtained from the last calibration runs performed Instrument specific information such as the instrument name serial number user names dye set information etc File Name and The Cleanup Database utility is named CleanUpDB bat and is located in the following Directory directory D AppliedBio abi 3100 Bin When to Perform Run the Cleanup Database utility at least once a week and when the diskspace utility shows that the space used in association with diskspace cmd exceeds 8000 MB IMPORTANT Never run the Cleanup Database utility more than once a day because previously extracted sample files may be overwritten This can happen due to the format used for a run name Deleting Processed P NOLWAKOI The Cleanup Database utility deletes all run data and plate records in the Frame Data database Before running the utility be sure that all runs have been extracted from the database To delete processed frame data using th
104. acle for NT GeneScan Application Sequencing Analysis Application Contents 1 Introduction and Safety OVELVIOW et hei eked asthe he ks Pee aad ees Diet a a ee Ss A 1 1 ABI PRISM 3100 Genetic Analyzer 0 0 cee eee 1 2 To Get Started Quickly 5 ioc rini Sats sod a ea E A pened alk de ode Reanind aloes He ache a ees aes 1 3 Additional Documentation 1 0 0 0 0 e eben enn e eens 1 4 Saloy eek ek ie eee bee eee wip Me ak pees ee ce eh es vee eee ee ee 1 5 System Overview OVETVIEW ice ae a a FR ee i 4 ened Se See aa GG Od eh Teeth wedans 2 1 Section 3100 Instrument Overview 0 0 0 6 cc ccc cece eee eee eee eee eens 2 3 ABI PRISM 3100 System Components 0 2 0 02 cece cee eee eee 2 4 What the Instrument Does 0 cece enn eben een en ens 2 5 How the Instrument Works penaren a ccc cee eee eben Ea Ra 2 6 Section Instrument Hardware Overview 00 ccc cece cece cee e eee eeens 2 9 Front VIEW peder neha ed Rees ha wes BERR ea ee ee Rae BA EUR Eee oe alee 2 10 Front View with Doors Opens ici nac cig geek ak Sew wb EEE EEEa eb Sle ese a e e UTS 2 11 Back VIEW etira cies ta ed Se Poe 2 Hh eH E ee BO OE Oo R E 2 12 Section Computer and Software Overview 0 ccc cece cece nen cence 2 13 Computer Workstation ss iemest ale Ss ce ts Sees heads bald Hae aoe a a dae ae bee dogs 2 14 SOWAT Eea EA ee PE eae a ale I RD wea OO ee ne ete OV eae i i batt 2 15 Section Chemistry Overview
105. ality CHEMICAL HAZARD Formamide is harmful if absorbed through the skin and may cause irritation to the eyes skin and respiratory tract It may cause damage to the central nervous system and the male and female reproductive systems and is a possible birth defect hazard Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves System Overview 2 21 2 22 System Overview Section Electrophoresis Overview In This Section The following topics are covered in this section Topic See Page Capillary Array 2 24 Electrophoresis 2 25 Electrophoresis Circuit 2 26 System Overview 2 23 Capillary Array Overview The 3100 capillary array is a replaceable unit composed of 16 silica capillaries that when filled with polymer enable the separation of the fluorescently labeled DNA fragments by electrophoresis Diagram Combs Detection cell Loading bar Capillary sleeve Capillary electrodes Description Part Function Capillary sleeve Provides a seal along with the ferrule and array ferrule knob with the upper polymer block Capillary electrodes Hold the capillary ends in position Combs Separate the capillaries to maintain consistent positioning and heat distribution in the oven Detection cell Holds the capillaries in place for laser excitation Loading bar Supports the capillaries and provides a high v
106. ame you assign must not exceed 63 characters IMPORTANT When naming the sample you can use letters numbers and the following punctuation only _ Do not use spaces IMPORTANT You must limit the sample name to 63 characters 59 character filename and 4 character extension If you do the name may be truncated when exported from the 3100 Data Collection software IMPORTANT This cell or token must always be second from left to right Color Number Corresponds to a specific color button of the plate record Dye field Color Number Color Button 1 B 2 G 3 Y 4 R 5 O Standard Dye Represents the size standard color This should be the number 4 for all applications which corresponds to the red dye Selecting the number 4 in this field is equivalent to selecting the diamond in the R color box of the GeneScan Analysis software Dye Set Specifies the dye set used to label the samples It must match the names stored in the instrument database Color Info Enables you to identify the sample in GeneScan analysis software when you are examining samples by color if you enter the sample name in this optional field Color Comment Enables you to customize the output for downstream analysis Optional Project Name Designates the BioLIMS Genetic Information Management System Collection name into which this sample will be added Run Module Specifies the run m
107. analyzed by point algorithm algorithm i lt Baseline data Startpt lt Maximum Scans Analyzed i Data considered collected Olea by algorithm Data analyzed by algorithm Note that there are two separate processes involved Data collection which happens during the run Data analysis which occurs after the run by the spectral calibration algorithm using the data collected and stored in the instrument database Spatial and Spectral Calibrations 4 47 startptOffset Parameter Introduction If you use the sequenceStandard dataType parameter you can select a value for the startptOffset starting point offset parameter Purpose The startptOffset parameter gives you control over which data frame the spectral calibration algorithm examines first Data Delay Time When you click the Start Run button to begin a run time starts to be measured by the 3100 Data Collection software in data frames During the first part of a run there is no dye fluorescence because the DNA fragments have not yet migrated to the detection window Typically the software does not start to collect data immediately after sample injection to save instrument database space The time between injection and the start of data collection is called the data delay time and is one of the parameters that you can set when you create a run module Collected Data All of the data frames from the start to the end of data collection are collected
108. arameter file you just created instead of the default parameter file MtxStd Sequencing SetE par Fragment analysis select the parameter file you just created instead of the default parameter file MtxStd GeneScan SetD par Spatial and Spectral Calibrations 4 35 Spectral Calibration Matrices Introduction A spectral calibration matrix is a mathematical matrix that describes the fluorescence emission spectra of the four or five dyes being used For each capillary only one spectral calibration matrix can be stored in the instrument database At the end of a spectral calibration run the spectral calibration matrix in the database for each capillary is overwritten Locating Matrix For each matrix produced during the calibration run a separate spectral calibration file Files is stored in a folder named Spectral Cal Logs in the following directory D AppliedBio abi 3100 DataCollection Spectral Cal Logs SpectralCal amp D perkin elmer abi 3100 D ataCollection Spectral Cal Logs SpectralCal demo Ea File Edit View Help Pi SpectralCal demo_3100 Run_c x iS Xele x si paaa A A A A a Capi mel Cap03 mel Cap05 mcel Cap07 mel Cap08 mcel Cap12 mel File containing spectral matrix for capillary number 12 The naming conventions are given below Folder File Type Naming Format Spectral calibration folder SpectralCal instrumentname Runyear_date_time Spectral calibration file Capcapillaryn
109. are records which charges originated from which bins allowing the software to reconstruct the charge pattern into a digital format Data Summation At uniform time intervals the charges on the pixels within each bin are summed By summing data from bins that are adjacent in the spatial dimension of the CCD camera the number of data points is reduced The data matrix for each time point becomes 16 data points in the spatial dimension three pixels for each capillary by 20 data points in the spectral dimension 14 pixels per spectral bin This data matrix is named 16X20 data Note For more detailed information on the CCD camera pixels bins and the spatial and spectral dimensions see Organization of the CCD on page A 3 The 3100 Data Collection software organizes the electronic information into sets of binary frame data At this point these are unprocessed or raw frame data sets This is still 16X20 data i e each raw frame data set is 16 capillaries by 20 bins One raw frame data set is produced for every time point of data collected which can generate thousands of raw frame data sets during a single run Due to the large numbers produced the raw frame data sets are transient and never stored The raw frame data sets are processed to remove data from pixels outside the fluorescence area In the 3100 Data Collection software processing means multicomponenting This converts the data from 16 capillaries by 20 bins to16 ca
110. ared and stored on any networked computer or transferred from a computer on a disk Note Plate records cannot be created or linked while a run is in progress How to Create Plate There are numerous methods used to create plate records The most convenient Records method transfers data directly from a LIMS database Once set up in the Preferences dialog box of the ABI Prism 3100 Data Collection software and the LIMS program the transfer of data and creation of plate records is completely automatic requiring no operator intervention Plate records can be created using the methods described in the following diagram Method 1 Method 2 Method 3 Method 4 Method 5 Method 6 In 3100 Data Collection software use the plate editor to modify an existing plate record Make a new spreadsheet in any program Open the provided tab delimited text file template filename plt in Microsoft Excel Create a plate import record in a LIMS database Import automatically Open a spreadsheet template created by you in any compatible spread sheet program In 3100 Data Collection software input the plate and sample data directly into the plate editor Open the file as a Input the plate Modify the plate Spreadsheet and sample data and sample data Save as a tab delimited text file filename plt Modify the plate and sample data i tie Save as anew spreadsheet
111. ata to the BioLIMS database Leave the AutoAnalysis On check box blank analyzed data to the BioLIMS Check the AutoAnalysis On check box database 4 In the BioLIMS portion of the window check the Enables check box and type the appropriate information in the text fields Note The information below is used to connect to the BioLIMS database It is assigned to your system when the Oracle software is installed You may obtain the required information from the BioLIMS system administrator at your site Text Field Description Write your information here User Name This is your account name on the server Database Name This is the BioLIMS database schema name Password This is the password for your server account Server Name This is the server name of the BioLIMS database Note The server name is contained in the tnsnames ora file Software 5 53 5 54 Software To set BioLIMS preferences for the plate continued Step Action 5 In the Sample File Name Prefix Format portion of the window assign a sample file name format for the BioLIMS project To do this choose from the drop down list for each of the four identifiers IMPORTANT You must select Run ID for one of the identifiers In addition you may want to limit the format to two identifiers only if you choose more than two the sample name will be truncated in the BioLIMS database The recommende
112. ating a Factura settings file Creating a new sequencing analysis module Saving the sequencing analysis module For More For detailed information about the topics covered in this section see the ABI Prism Information DNA Sequencing Analysis Software v 3 6 NT User s Manual Creating a To create a basecaller settings file Basecaller Settings File Step Action 1 Quit the 3100 Data Collection software if it is running 2 Start the DNA Sequencing Analysis software The Sample Manager window opens inside the Sequencing Analysis window al Sample Manager Ol x PD store PI Pouse J I Cancer Remove MT open Fies Status Sample File Name Sample Name A FP Basecaller Spacing Basecaller Peak1 Settings Location olog jkr PJ PI 5 30 Software To create a basecaller settings file continued Step Action 3 To set a cutoff condition for the analysis from the Edit menu point to Preferences and select Basecaller Settings This opens the Preferences dialog box all Preferences Page Basecaller Settings v Basecaller Settings Default Settings W 7 3et endpoint at PCR stop point after Nsin bases Default Note The default setting has the cutoff conditions disabled In the Preferences dialog box click Create a set Check one or more of the Set endpoint check boxes as appropriate If you checked the second third or fourth check box
113. ating system with Service Pack 5 256 color display adapter card CD ROM drive For RAM MB Hard Disk Space MB Extraction only 64 80 Extraction and analysis 256 120 Ensure that the networked computer has sufficient hard disk space to hold as many sample files as desired One analyzed sample file is about 250 KB The following table lists the software that must be installed on the networked computer for remote extraction to work If Install data extraction is to be performed on this Auto Extractor computer re extraction is to be performed on this Reextractor exe computer Auto Extractor or 3100DBUtils exe are used All supporting shared files OrbixWeb Professional Edition Orbix Desktop 2 3 software Persistence Powertier 4 321 Oracle Client database not distributed with the 3100 software gt gt gt gt Note For directions on performing remote extraction see page 7 6 7 18 System Management and Networking Maintenance Overview In This Chapter The following topics are covered in this chapter Topic See Page Section Instrument Maintenance 8 3 Maintenance Task Lists 8 4 Routine Cleaning 8 5 Moving and Leveling the Instrument 8 6 Resetting the Instrument 8 7 Shutting Down the Instrument 8 8 Section Fluids and Waste 8 9 Buffer 8 10 Polymer 8 10
114. ation The maximum and minimum numbers should typically be set generously out on the tails of the distribution curve Spatial and Spectral Calibrations 4 45 numDyes and writeDummyDyes Parameters Introduction The numDyes and writeDummyDyes parameters are used together to provide information about the number of dyes being used for a spectral calibration The values of the two parameters must add up to five which is the maximum number of dyes that can be used With four dyes the numDyes value is set to 4 and the number left over is the value assigned to writeDummyDyes which is 1 numSpectralBins Parameter Introduction The numSpectralBins parameter defines the number of bins of data being collected in the spectral dimension of the CCD The 3100 instrument has 20 bins This parameter exists because no header information is provided in the raw color data files tmp so there is no mechanism for the algorithm to determine this value automatically 4 46 Spatial and Spectral Calibrations Parameters Specific to sequenceStandard dataType Parameters List The following parameters are used only when the sequenceStandard dataType is selected startptOffset maxScansAnalyzed startptRange Summary Diagram The following diagram summarizes the individual parameters that follow Start of End of run and Start of run data collection data collection i Delay time S i i K First frame Last frame f Autostart analyzed by
115. aturation chroma The purity of the color in a scale from gray to the most vivid version of the color Value intensity The relative lightness or darkness of a color in a range from black to white e g light red dark green etc To change the displayed dye color using the HSV system Step Action 1 Within the Edit Dye Display Information dialog box click the Color box of the color you want to change The Set Color dialog box is displayed Click the HSV tab if it is not already selected Hv RoB HSV tab Hue Saturation Value OK Cancel Reset 2 Click in the circle and drag the cross hair pointer around the circle to select the desired hue 3 Click in the inner square and drag horizontally to select the desired saturation Click in the inner square and drag vertically to select the desired value To Click Incorporate the change OK Ignore the change Cancel Revert to the default colors Reset 6 Close the Edit Dye Display Information dialog box 1 See the Essential Guide to User Interface Design W O Galitz 1996 John Wiley amp Sons 5 14 Software Section Controlling the Instrument Using Manual Control In This Section The following topics are covered in this section Topic See Page Manual Control Commands 5 16 Using Manual Control Commands 5 17 Software 5 15 Manual Control Commands Table of Commands The following table displays the manual control options as they are organ
116. bases on different computers is illustrated below Run Module File Data copied to create a file Stored in hard drive directory of your choice Data copied to re create original run module in another database Import nstrument Database Database About Exporting a A run module cannot be transferred directly The data in a run module must first be Module copied into a file that is created and stored on a hard drive This is known as exporting the module because you are exporting it from the database The file created has the file name format filename modexp The hard drive to which the run module file is saved could be the local drive of the donor or acceptor computer or it could be a server that is accessible to both computers Software 5 23 5 24 Exporting a Run To export a run module Software Module Step Action 1 Click the Module Editor button on the toolbar to open the Module Editor dialog box EA i Module Editor x Modules Module Parameters Sequencing GeneScan Calibration Hame none selected Template none selected _ Parameter Name Value Range IHTGS36_POP4a3DefauttModule Comments New _ seve Save As E Import m Make sure that the module you want to export is selected in the Modules group box In the Modules group box click Export This opens the Export browser dialog box Lookin ani El E E
117. bbles and then proceed eter f with instrument preparation sufficient in quantity to complete prep your runs Note To remove any air bubbles see page 8 28 greater than 1 week old or Fill the syringes and the upper polymer block with polymer by following the Change Polymer wizard For instructions see page 8 10 E 13100 Data Collection Software Fie View Instrument EMES Service Help HE E i Plate Editor Module Editor Change Polymer Wizard insufficient in quantity to complete your runs Plate View Run view Instrument Condition Install Capillary Array Wizard Autosampler Calibration Vizard Perform Spatial Calibration Display Spatial Calibration Display Spectral Calibration CHEMICAL HAZARD POP polymer may cause eye skin and respiratory tract irritation Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Use for research and development purposes only a A run uses 50 80 pL of polymer This is equivalent to 60 100 runs from one 5 mL syringe A minimum of 1 mL of polymer is required for the instrument to operate IMPORTANT Always replace polymer that is older than 1 week IMPORTANT Ensure there are no air bubbles in the upper polymer block before proceeding To remove any air bubbles see page 8 28 Performing a Run 3 21 Preparing Buffer and Filling the Reservoirs Required Materials
118. becomes the database plate ID and must be unique among all existing plates Having the name field in addition to the plate ID field allows you to delete a plate record from the plate import table and then re import it with the same name but a different Plate ID Working with Plate Records 6 21 The name must be the same as the plate name given to the header in the BLOB equivalent of the tab delimited text file It can be up to 32 characters and must not contain any restricted characters IMPORTANT Use only the following characters which are a subset of the characters allowed by the Windows NT operating system letters numbers and _ Status There are three status options Status Assigned when Set by New the data is ready for transfer LIMS Old a plate table has been successfully imported 3100 Data Collection software Bad the transfer was unsuccessful 3100 Data Collection software The status of any data set stored in the plate import table can be checked at any time through the LIMS software Plate BLOB The plate BLOB is an array of binary data that is equivalent except in language to a Definition tab delimited text file used for data import The plate BLOB is written from a table in the LIMS database that contains data and formatting equivalent to a tab delimited text file or spreadsheet used for data import The plate ID in the header of the binary BLOB must exactly match the plate name i
119. c Analyzer is currently supported by Applied Biosystems for use with Chemistries DNA sequencing samples that are fluorescently labeled with ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit ABI PRismM BigDye Primer Cycle Sequencing Ready Reaction Kit ABI PRism dRhodamine Terminator Cycle Sequencing Ready Reaction Kit Note These chemistries use the dyes in Dye Set E Fragment analysis samples that are labeled with the fluorescent primers supplied with the ABI PRism Linkage Mapping Set LD20 MD10 or HD5 Note These chemistries use the dyes in Dye Set D System Overview 2 19 Polymers Overview Supported Polymers 2 20 Chemical Hazard Storage and Expiration Proper Disposal System Overview The ABI PRISM 3100 Performance Optimized Polymer POP is used as a replaceable sieving medium that separates the DNA fragments by size during electrophoresis POP is shipped ready to use Two polymers are used with the 3100 Genetic Analyzer as follows Polymer Name Use for Part Number ABI PRISM 3100 POP 4 polymer Fragment analysis 4316355 ABI Prism 3100 POP 6 polymer DNA sequencing 4316357 7 NOVIO CHEMICAL HAZARD POP polymer may cause eye skin and respiratory tract irritation Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Use for research and development purposes only
120. ce The Diskspace Utility Function File Name and Directory When to Perform Checking the Space The Diskspace utility lists the amount of space that the database uses the amount that is free for use and the percent filled The Diskspace utility is named diskspace bat and is located in the following directory D AppliedBio abi 3100 Bin Run the Diskspace utility at least once per week to ensure you have adequate space for your sample files To check the space using the utility Step Action 1 Ensure OrbixWeb Daemon is running 2 Quit the ABI PRism 3100 Data Collection software 3 Using the Microsoft Windows NT Explorer navigate to the following directory D AppliedBio abi 3100 Bin Locate and double click diskspace cmd Diskspace Utility p rkin e lnersabi J180 Bindsvrngr3 Bdickspace cgl Oracle Server Mannger Release 3 86 5 6 8 Troduction lt c Copyright 1 7 Oracle Corporation All Rights Reserved OracleaX He lease NX H h H H Prnodurtinn PL SQL Release 3 8 5 8 6 Pruduvt iun Connected Statement processed us MEGS 1 row selected Statement prucessed Servcr Manager complctc The Diskspace ulility pruyvides the anuvuit uf database space used Please run cleanupdh bat once the amount of USED_MEGS exceeds 8608 negabytes Press any key to continuc If USED_MEGS is greater than 8000 MB run the Cleanup Database utility See page 7 8 I
121. ch explains the hazard and any user action required A safety alert symbol which indicates a potential personal safety hazard See the ABI Prism 3100 Genetic Analyzer Site Preparation and Safety Guide for an explanation of all the safety alert symbols provided in several languages As the generator of potentially hazardous waste it is your responsibility to perform the actions listed below Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure the health and safety of all personnel in your laboratory Ensure that the instrument waste is stored transferred transported and disposed of according to all local state provincial or national regulations Note Radioactive or biohazardous materials may require special handling and disposal limitations may apply Ensure that everyone involved with the operation of the instrument has Received instruction in general safety practices for laboratories Received instruction in specific safety practices for the instrument Read and understood all related MSDSs 7 eT NERA Le Avoid using this instrument in a manner not specified by Applied Biosystems Although the instrument has been designed to protect the user this protection can be impaired if the instrument is used improperly Operating the computer correctly prevents stress producing effects such as fatigue pain and strain To minimize
122. ct1 DT3100P0 6 BD Ww2 mob 1X DOAN SAMPLING 3100_Project1 DT31 faasala mob 1X DOAN SAMPLING 3100_Project1 RE RUN CF LRS DILUTION EXPERIMENT E Note It may take a while for the new plate record to be saved to the database and added to the Pending Plate Records table as shown below 3100 Data Collection Software Yersion 1 0 LRS_DILUTION_2 pending irs_dlution2 C 3 40 Performing a Run Linking and Unlinking a Plate Introduction The procedure below describes how to link a plate on the autosampler to the plate record you have created This must be done before a plate can be run IMPORTANT A plate can be linked even if there are no run modules selected for its samples In this case there is no error message and runs for samples in the plate will not be scheduled Linking a Plate toa To link a plate to a plate record Plate Record Step Action 1 Click the Plate View tab on the 3100 Data Collection software window to go to the Plate View page Plate View tab Plate View l Run View Status View Array View Capillary View On the Plate View page a In the Pending Plate Records table click the plate record for the plate you are linking b Click the plate position indicator that corresponds to the plate you are linking Pending Plate Records Plate Name Application Wells Status 96 pe
123. ction From the Instrument menu select Manual Control From the Command Category drop down list select Capillary From the Command Name drop down list select Fill From the Value drop down list select the appropriate array length and polymer Array with Polymer Using Manual Step Control 1 2 3 4 5 Click Send Command Checking Capillary To check capillary alignment using the capillary ruler Action Place the ruler beside the capillaries and detach a side of the ruler to the bottom of the holder Check the capillaries to match the lines of the ruler Check both sides of the capillaries Place the capillary array holder on the flat surface and stand the ruler up at the end of capillaries Alignment Using the Capillary Ruler _St P 1 3 5 Check the cross points of line on the ruler to match the end of capillaries If some of capillaries are bent adjust each capillary carefully 8 16 Maintenance Storing a Capillary Array on the Instrument When to Store on the Instrument Storing the Array on the Instrument Store the capillary array on the instrument when the capillary array will be unused for less than 1 week To store the capillary array on the instrument follow the instructions to perform a short term shutdown on page 8 8 Storing a Capillary Array off the Instrument When to Store off the Instrument Storing the Capillary Array of
124. ctrophoresis is on Oven Oven is off Oven is on Front Doors Doors are closed Doors are open Oven Door Door is closed Door is open Autosampler Autosampler is homed Autosampler is forward The Events box lists the Instrument s recent actions Status of each capillary as passed or failed at the end of a spectral calibration Calibration data at the end of a spatial calibration Some of the events listed in the Events box provide information for service engineers Performing a Run 3 51 Errors Box The Errors box lists errors that have occurred during the current run Some of the error messages provide information for service engineers A fatal error usually requires that you restart the Data Collection software Status Bar The Status bar indicates the instrument s current state or operation 3 52 Performing a Run Array View Page Function Features Use this scroll box to view data block by block Click the Array View tab during or after a run to examine the quality of your data which is displayed as individual electropherograms and as color data for the entire capillary array IMPORTANT Always exit from the Array View and the Capillary View windows During a run do not leave these pages open for extended periods This may cause unrecoverable screen update problems Leave the Status View window open This is an example of the Array View page Raw multicom
125. d The Microsoft Windows NT operating system includes a simple text only word processor called Notepad located in the Accessories menu Notepad will open any text only file even if the file was created by a program using the Macintosh operating system In this case though the end of line characters may need to be re entered 6 10 Working with Plate Records About Creating Tab Delimited Text Files Introduction Although it is possible to create tab delimited text files by typing the required information directly into a word processing program it is easier to first enter the data into a spreadsheet and then save the spreadsheet as a tab delimited text file Spreadsheets are easier to work with because they organize data clearly in columns and because repetitive typing can be reduced by using their fill down function Overview of the The typical method for importing information to create a plate record is outlined below Method Create a new empty spreadsheet or open the template Enter the plate and sample data into the spreadsheet following the examples in this section Save the file as a tab delimited text file filename plt In the Plate View page of the 3100 Data Collection software select Import Select the file in the Open File dialog box Omitting You do not need to include all of the information required for a run before importing a Information tab delimited text file into the instrument database Information can be
126. d as a plate record As a result there is little need to keep the imported data in the plate import table once the success of the import has been verified We recommend that you periodically delete data from the plate import table It is best to do this when the 3100 Data Collection software is not running To delete data from the plate import table consult your Oracle database administrator If an error occurs while importing data from the plate import table the error is registered in the following locations Errors pane on the Status View page Run log table on the Run Log page Plate import table status will be set to Bad A LIMS entry into the plate import table must contain the following five fields Field Format Plate ID Up to 32 characters Name Up to 32 characters Status Up to 32 characters Plate BLOB BLOB Plate BLOB version Integer The plate ID is a unique identifier or primary key for the plate This ID should not be the same as the plate name The plate ID must be unique The instrument database will not allow entry of a plate ID if that value is already used by another row in the plate import table The name is the name of the plate This name should not be the same as the plate ID The name is not a unique identifier for the plate in the plate import table and can be used more than once within the plate import table However once the data is used to create a plate record the name
127. d format is shown below OK Canzel Click OK The preferences will be applied to your highlighted plate Continue your setup and run your samples as usual When the run has completed the sample files will be extracted to the BioLIMS database automatically However you must view the debug log file to see if the extraction completed successfully Continue with After Extracting to the BioLIMS Database on page 5 55 After Extracting to the BioLIMS Database Introduction After your samples have run you must view the run s log file debug log to ensure the extraction to the BioLIMS database was successful IMPORTANT You will not receive any error messages if the extraction was not completed successfully e g if the database connection was not established if the BioLIMS project information was entered incorrectly etc The only way to check the status of the extraction is to view the debug log file Viewing a Run s Log To view a run s log file File Step Action 1 Open the directory that contains the 3100 Data Collection software and navigate to the ExtractedRuns folder In most cases the path will be D AppliedBio abi 3100 DataExtractor ExtractedRuns 2 Open the ExtractedRuns folder A directory is created in this folder for each run you ve performed on the 3100 Genetic Analyzer 3 Find the run for which you want to check the status and open its directory All the data co
128. dard Files SZS x Cancel 14 In the File name text box type a file name for the size standard file 15 Click Save The browser dialog box closes and the file is saved to the correct directory location for Auto Extractor to read In the newly created Filename szs dialog box click the Close button Creating a GeneScan To create a GeneScan analysis module Analysis Module Step Action 1 From the File menu select New This opens the Create New box ull x Create New Software 5 43 5 44 Software To create a GeneScan analysis module continued Step Action 2 Click the Analysis Parameters icon This opens the untitled 3 dialog box untitled 3 TEA fioo eceee Fill out the untitled dialog box according to the directions given in the ABI PRISM GeneScan Analysis Software v 3 5 NT User s Manual In the AutoAnalysis Only group box select the size standard file that you just created from the Size Standard drop down list To create a GeneScan analysis module continued Step Action 4 From the File menu select Save This opens a browser dialog box Navigate to the Params folder in the following directory D AppliedBio abi Shared Analysis Sizecaller Params Save this document as ix Save in ja Params z c z Resource frk a GS3504nalysis gsp js GS4004nalysis gsp a GS400CubicAnalysis as
129. ds the entry for the plate record appears in the Pending Plate Records table of the Plate Setup page 4 22 Spatial and Spectral Calibrations Linking the Plate To link the plate record to the plate Step Action 1 In the Pending Plate Records table select the plate record that you just created 2 Click the plate graphic that corresponds to the plate on the autosampler 3100 Data Collection Software x Fie View Instrument Tools Service Help cal gt m cle Plate View Run View Status View Array View Capillary View Pending Plate Records Plate Name Application Wells Status Place a plate into plate position B Linked Plate Records Plate Name SpectralCalibrati Application Spectral 96 Wells Status pending r Processed Plate Records Plate Name Application Wells Status Edt Delete Import Note When a plate is linked the Plate graphic changes from yellow to green Plate record moves from the Pending Plate Records table to the Linked Plate Records table This may take up to 30 sec The Run Instrument button on the toolbar is enabled meaning that the instrument is ready to run Starting the To start the calibration Calibration Step Action 1 If you want to review the run schedule before beginning the run click the Run View tab Click the Run Instrument button on
130. e or expense directly or indirectly arising from the purchase or use of the Instrument Applied Biosystems makes no warranty whatsoever with regard to products or parts furnished by third parties This warranty is limited to the initial purchaser and is not transferable THIS WARRANTY IS THE SOLE AND EXCLUSIVE WARRANTY AS TO THE INSTRUMENT AND IS IN LIEU OF ANY OTHER EXPRESS OR IMPLIED WARRANTIES INCLUDING WITHOUT LIMITATION ANY IMPLIED WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE AND IS IN LIEU OF ANY OTHER OBLIGATION ON THE PART OF APPLIED BIOSYSTEMS D 2 Limited Warranty Statement Index Numerics 3100 instrument preparation 3 19 3100 software CDs 5 4 3100 system components 2 4 A ab1 files See ABIF sample files ABI Sample File Toolkit 5 7 ABIF sample files access through developer s toolkit 5 7 discussed 7 4 Adobe Acrobat Reader 5 8 air bubbles clearing 8 28 alignment capillary 8 16 analysis module provided modules 6 7 selecting for GeneScan 3 34 selecting for sequencing 3 39 analysis parameter files See sequencing analysis modules analyzing GeneScan data 3 62 3 63 Array View page discussed 3 53 array See capillary array array fill syringe See also syringe volume and function 8 20 auto extractor 5 7 autoextraction failure 3 60 autosampler 3 23 calibrating 8 29 controlling See manual control commands placing plates 3 25 won t move forward 9 2 B basecaller settings file creating 5 30 Big
131. e Cleanup Database utility Step Action 1 Ensure OrbixWeb Daemon is running 2 Quit the 3100 Data Collection software 3 Using Windows NT Explorer navigate to the following directory D AppliedBio abi 3100 Bin 4 Locate and double click CleanUpDB bat This runs the Cleanup Database utility which takes a few seconds to complete 5 Shut down and then relaunch OrbixWeb Daemon 7 eT Neale If you do not perform this step any new run data will not be saved to the database Note There is no need to re import the spatial spectral and run calibration methods or the calibration data obtained from the last calibration runs 7 8 System Management and Networking Another Method to Deleting the plate record for a plate of samples is another way to delete processed Delete Processed frame data stored in the instrument database Frame Data Directions for deleting individual plate records start on page 6 39 System Management and Networking 7 9 Importing a New Spatial or Spectral Calibration Method The New Method Import Utility About Method Files Spatial and spectral method files contain the parameters that define the calibration run conditions along with the SCPI commands that direct the operation of the instrument Note Spatial and spectral calibration method files are not the same as the spatial and spectral calibration files which contain results of calibration runs Function New methods prov
132. e Run View page see page 3 50 Note Although individual runs can be deleted the order in which the runs are scheduled cannot be altered For more information on run scheduling see page 3 46 3100 Data Collection Software Version 1 0 ee Unlinking a Plate To unlink a plate record Record Step Action 1 In the Linked Plate Records table of the Plate View page select the plate record that you want to unlink 2 Click Unlink If the plate record is Then the plate record will completed Go to the Processed Plate Records not completed Return to the Pending Plate Record table and the plate position indicator will return to yellow Performinga Run 3 43 3 44 Performing a Run Section Running the Instrument In This Section The following topics are covered in this section Topic See Page About Run Scheduling 3 46 Controlling the Run 3 47 Run Times 3 48 Performing a Run 3 45 About Run Scheduling Introduction Plate Run Order Sample Run Order 3 46 Performing a Run To view the run schedule click the Run View tab The order in which the runs are scheduled cannot be altered Run scheduling depends on the factors listed below If two plates are being run the order in which they are run is based on the following factors in the order listed If one plate is a spectral calibration run it w
133. e available from our web site in the future Mobility files for dye sets other than the ABI PRISM BigDye sets must be provided by the manufacturer Run Modules A module is a collection of routines that perform a task Run modules define the run conditions for a sample For a list of conditions you can set for running a sample see Modifiable Run Module Parameters on page 5 22 Run Modules Provided The following run modules are provided with the 3100 software Run Type Run Module GeneScan GeneScan36_POP4DefaultModule Standard DNA sequencing StdSeq50_POP6DefaultModule Rapid DNA sequencing RapidSeq36_POP6DefaultModule 6 6 Working with Plate Records Analysis Modules A module is a collection of routines that perform a task Analysis modules tell the AutoAnalyzer which parameters to use for data analysis You can use the analysis modules provided and or create your own to define different analysis parameters Analysis Modules Provided The following analysis modules are provided with the 3100 software You can examine the settings for each of the files using the analysis software Note The meanings of the settings are described in the ABI PRism DNA Sequencing Analysis Software v 3 6 NT User s Manual and the ABI PRISM GeneScan Analysis Software v 3 5 NT User s Manual Analysis Module Run Type BC 3100_SeqOffFtOff saz Standard DNA sequencing BC 3100RR_SeqOffFtOff saz Rapid DNA
134. e block The syringe should be finger tight in the block Place the array fill syringe tip in the right port on the top of the upper polymer block and screw the syringe tip clockwise into the polymer block IMPORTANT Always hold the syringe by the metal sleeve not the glass when screwing the syringe into the block The syringe should be finger tight in the block Push the polymer block all the way against the instrument Replace the syringe guard Removing Syringes To remove the syringes from the instrument Step Action 1 Remove the syringe guard 2 Grasp the polymer reserve syringe just above the fitting or at the base not the glass barrel and rotate the syringe counterclockwise Do not loosen this fitting while removing the syringe GR1876 IMPORTANT Be careful not to remove the fitting There are several rings and check valves that could come out if this fitting is removed Grasp the array fill syringe and rotate the syringe counterclockwise Properly dispose of any remaining polymer Proceed to Cleaning and Inspecting Syringes on page 8 21 Maintenance 8 23 8 24 Maintenance Section Polymer Blocks In This Section The following topics are covered in this section Topic See Page Removing the Polymer Blocks 8 26 Cleaning the Polymer Blocks 8 27 Removing Air Bubbles from the Upper Polymer Block 8 28 Mainte
135. e imported despite a warning When this happens the purpose of the warning is to prompt you to examine and correct the data in the plate editor Working with Plate Records 6 35 To import one or more tab delimited text files to create plate records continued Step Action 4 Review the plate records in the plate editor 5 Link the plate record to the plate 6 36 Working with Plate Records Section Deleting Plate Records and Run Data In This Section The following topics are covered in this section Topic See Page Introduction 6 38 Cleanup Database Utility 6 38 Deleting Individual Plate Records 6 39 Working with Plate Records 6 37 Introduction When to Delete Plate Records and Run Data Two Procedures Recommended Procedure Delete the plate records and run data when the used space on drive E is more than 8 GB See Checking Database Space The Diskspace Utility on page 7 5 There are two ways to delete the processed frame data that is associated with plate records You can Use the Cleanup Database utility CleanUpDB bat Delete individual plate records The Cleanup Database utility is the recommended way to delete plate records because Itis much faster to delete the processed frame data than to delete individual plate records It prevents problems that result from incomplete deletion of data Cleanup Database Utility Reference to the Cl
136. e in polymer block Pause the run check the polymer path for bubbles and remove them if present A slow leak may be present in the system Check polymer blocks and syringes for leaks Tighten all fittings Incorrect buffer concentration Prepare 3100 1X running buffer Add 3 mL 3100 10X buffer with EDTA to 27 mL deionized water Not enough buffer in anode reservoir Add buffer up to the fill line Clogged capillary Refill capillary array and check for clog Arcing Check for moisture in and around the septa the reservoirs the oven and the autosampler Poor performance of capillary array used for fewer than 100 runs Poor quality samples possible cleanup problems Desalt samples using a recommended purification protocol Poor quality formamide Prepare fresh HiDi formamide and re prep samples Incorrect buffer Use 3100 10X running buffer with EDTA to prepare 3100 1X running buffer Migration time becomes progressively slower Leak in system Tighten all ferrules screws and check valves Replace any faulty parts Improper filling of polymer block Check polymer pump force If the force needs to be adjusted call a service representative Expired polymer Check expiration of polymer If necessary change the lot Migration time becomes progressively faster Water in syringe resulting in diluted polymer Clean the syringe and dry it wit
137. e installation Instrument and chemical safety ABI PRISM 3100 Genetic Analyzer User procedures 4315834 Gere Manuel Instrument maintenance Troubleshooting ABI PRISM 3100 Genetic Analyzer Abbreviated procedures for performing a 4315832 Quick Start Guide for Fragment fragment analysis run Analysis ABI PRISM 3100 Genetic Analyzer Abbreviated procedures for performing a 4315833 Quick Start Guide for Sequencing sequencing run Software ABI PRISM DNA Sequencing Detailed procedures for analyzing 4308924 Analysis Software v 3 6 NT Users sequencing data Manual ABI Prism DNA Sequencing Analysis Software Release Notes ABI PRISM GeneScan Analysis Detailed procedures for analyzing 4308923 Software v 3 5 NT User s Manual fragment analysis data ABI PRISM GeneScan Analysis Software Release Notes Chemistry ABI PRISM 3100 Genetic Analyzer Detailed chemistry procedures 4315831 Sequencing Chemistry Guide specific for the 3100 Genetic Analyzer Chemistry troubleshooting for the 3100 Genetic Analyzer ABI PRISM Automated DNA A description of DNA sequencing 4305080 Sequencing Chemistry Guide instruments chemistries and software Detailed procedures for preparing DNA templates performing cycle sequencing and preparing extension products About User Bulletins User bulletins are the mechanism we use to inform our customers of technical information product improvements and related new products and laboratory 1 4 Introducti
138. e syringes Syringes 4 Whenever they are removed from the instrument or at least once per week Each time the polymer is replaced including when switching to a new type or lot of polymer Cleaning Syringes IMPORTANT Wear gloves while performing the following procedure and any other time you handle the capillary array glass syringes septa or buffer reservoirs To clean a syringe Step Action 1 Remove the syringe guard 2 Remove the syringes as described on page 8 23 3 Clean the syringe thoroughly by rinsing the inside and outside of the syringe barrel and the syringe tip with warm water IMPORTANT Be sure there is no dried polymer left in the syringes Rinse the syringe barrel and tip with deionized water Blow dry with compressed air Reassemble the syringe and then inspect it as described below Inspecting a Syringe IMPORTANT After cleaning a syringe always inspect it for missing O rings to avoid leaks during your run To inspect the syringe Step Action 1 Inspect the syringe for two O rings P N 221102 one behind the ferrule and one around the ferrule O rings 0 N i 0 05 0 1 0 15 0 2 0 25 GRO0413 2 Verify that the ferrule is firmly seated in the end of the syringe Maintenance 8 21 Priming and Filling Syringes Priming and Filling the Polymer Reserve Syringe Priming and Filling the Array Fill Syringe 8 22 Maintenance Follow this
139. eScan Analysis Software v 3 6 NT User s Manual Note For more information on module files see Chapter 5 Software If you want to run the same sample again select a second run module and a second analysis module You can run a sample in a linked plate up to five times Run Module 2 Analysis Module 2 Samples will be automatically grouped so that all samples with the same run module are run sequentially To enter sample information and save the plate record continued Step Action Make sure the plate record is correct and then click OK Plate Editor GeneScan_samplet 3100_Project1 HTGS36_PO GS400HDAnalysis gsp GeneScan_sample2 3100_Project1 HTGS36_PO GS400HDAnalysis gsp GeneScan_sample3 HTGS36_PO GS400HDAnalysis gsp GeneScan_sample4 3100_Projectt HTGS36_PO GS400HDAnalysis gsp GeneScan_samples 3100_Project1 HTGS36_PO GS400HDAnalysis gsp Note added t Tisienecancepeerecee E It may take a while for the new plate record to be saved to the database and o the Pending Plate Records table as shown below 3100 Data Collection Software my_plate_record GS Performing a Run 3 35 Creating a Plate Record for DNA Sequencing Analysis Entering Plate Note You cannot create a plate record while a run is in progress Record Infor
140. each sample is positioned at the bottom of its tube or well To check the plate of samples Step Action 1 Hold the plate up to a light source Your samples should Look like this Not look like this Not look like this g aA p A amp U U The sample is positioned The sample lies on the An air bubble lies at the correctly in the bottom of side wall because the bottom of the well the well plate was not because the plate was centrifuged not Centrifuged with enough force or Centrifuged for enough time 2 If any of the samples are not positioned at the bottom of their tube or well recentrifuge the plate 3 8 Performing a Run Working with Plate Assemblies Plate Assembly The plate assembly components are assembled as follows Components Plate retainer Septa Sample plate Plate base Preparing a Plate To prepare a plate assembly Assembly Step Action 1 Secure a clean and dry septa strip on the sample plate IMPORTANT Never use warped plates IMPORTANT Ensure the septa strip lies flat on the plate 2 Place the sample plate into the plate base Snap the plate retainer onto the plate and plate base Performing a Run 3 9 3 10 Performing a Run To prepare a plate assembly continued Step Action 4 Ensure the plate retainer holes are aligned with the holes in the septa strip IMPORTA
141. eanup Database Utility NANON The Cleanup Database utility deletes all run data and plate records from the database Before running the utility be sure that all runs have been extracted from the atabase To delete plate records and run data from the instrument database using the Cleanup Database utility see Deleting Processed Frame Data The Cleanup Database Utility on page 7 8 6 38 Working with Plate Records Deleting Individual Plate Records Introduction When a plate record is deleted the run data associated with samples in the plate is also deleted from the instrument database Note A new run cannot be started while a plate record is being deleted IMPORTANT You cannot delete a linked plate record but plate records for unlinked partially processed plates can be deleted If the processed runs from unlinked partially processed plates have not yet been extracted the run information will be deleted from the database The pending plate record table is where unlinked partially processed plates are listed Make sure that processed runs have been extracted by looking in the ExtractorEventLog txt file When to Delete Use this method if you want to delete only Individual Plate Plate records that have no associated run data Records Certain plate records Deleting Individual To delete individual plate records Plate Records Step Action 1 Click the Plate View tab in the 3100 Data Collection software This op
142. een the loading bar cathode and the electrode located on the lower polymer block anode The voltage drives the movement of negatively charged DNA fragments through the polymer in the capillaries towards the anode From the anode the current flows back in electrical wires through the power supply to the cathode to complete the circuit 7 AARNE ELECTRICAL SHOCK HAZARD To reduce the chance of electrical shock do not remove covers that require tool access No user serviceable parts are inside Refer servicing to Applied Biosystems qualified service personnel 2 26 System Overview Section Fluorescence Detection Overview In This Section The following topics are covered in this section Topic See Page Introduction 2 28 Laser 2 29 Transmission Grating 2 29 CCD Camera 2 29 System Overview 2 27 Introduction Detection Overview Detection Components 2 28 System Overview The dye labeled DNA fragments are separated by electrophoresis within the capillary array Once the fragments enter the detection cell they pass through a laser beam The light excites the attached dye labels causing them to fluoresce The detection components work together to collect the fluorescence and convert the information into electronic form The electronic information is then processed and displayed by the 3100 Data Collection software The main components of the detection system and their function are listed in the follo
143. eets to Create Tab Delimited Text Files 0 00 0 6 12 Spreadsheet or Tab Delimited Text File Information 0 0 0 0 6 14 Running the Same Sample with Different Conditions 000 6 18 Section Creating a Plate Record by Importing LIMS Data 000005 6 19 Data Transfer ces mocni eke hho ee ten Se E eas CA Ae Es ren 6 20 Plate Import Table s speia nianma ta ihe uaa A A EEA asta Saag ilk 6 21 Section Creating Plate Files 0 ccc ccc ccc cence ene en ee ee eenees 6 23 Creating a Plate File Using a Provided Template 00 0 00000005 6 24 Creating a Plate File from a New Spreadsheet 0 00 0 cece ee eee ee 6 28 Creating a Plate File from a Custom Spreadsheet Template 00 6 29 Creating a Plate File from an Edited Plate Record 0 0 00 0000 00 00 0c eee 6 30 Section Importing Plate Files and Linking Plate Records 00000 6 31 About Importing Tab Delimited Text Files and Linking Plate Records 6 32 Simultaneously Importing and Linking a Plate Record 00 02 0005 6 33 Sequentially Importing and Linking a Plate Record 0 00 0 00 0000 0 6 35 Section Deleting Plate Records and Run Data 00 e cece cece eee 6 37 IntroductiOn ps 34 tata kee eke tik dashes eagle ea abe eats 6 38 Cleanup Database Utility 0 0 cee eee eee 6 38 Deleting Ind
144. egroom for adequate movement ING ie ELECTRICAL SHOCK HAZARD To reduce the chance of electrical shock do not remove covers that require tool access No user serviceable parts are inside Refer servicing to Applied Biosystems qualified service personnel PHYSICAL INJURY HAZARD Do not attempt to lift the instrument or any other heavy objects unless you have received related training Incorrect lifting can cause painful and sometimes permanent back injury Use proper lifting techniques when lifting or moving the instrument Two or three people are required to lift the instrument depending upon instrument weight System Overview Overview In This Chapter The following topics are covered in this chapter Topic See Page Section 3100 Instrument Overview 2 3 ABI PRISM 3100 System Components 2 4 What the Instrument Does 2 5 How the Instrument Works 2 6 Section Instrument Hardware Overview 2 9 Front View 2 10 Front View with Doors Open 2 11 Back View 2 12 Section Computer and Software Overview 2 13 Computer Workstation 2 14 Software 2 15 Section Chemistry Overview 2 17 Supported Dye Sets 2 18 Labeling Chemistries 2 19 Polymers 2 20 Injection Solution 2 21 Section Electrophoresis Overview 2 23 Capillary Array 2 24 Electrophoresis 2 25 Electrophoresis Circuit 2 26 Section Fluorescence Detection Overview 2 27 Introduction 2 28 Laser 2 29 Transmission G
145. elect the mobility file that you want to use for processing each sample For the names of the mobility files provided with the ABI Prism 3100 Genetic Analyzer Software and for which file to use when see page 6 6 Mobility files have the general format filename mob They must never be moved from the Mobility folder located in the following directory D AppliedBio Abi Shared Analysis Basecaller Mobility Technical Support Technical Support Contacting Technical Support To Contact Technical Support by E Mail Hours for Telephone 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 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 Peptide and DNA Synthesis corelab appliedbiosystems com Biochromatography PerSeptive DNA PNA and Peptide Synthesis systems CytoFluor FMAT Voyager and Mari
146. elect the spectral source file mcl to override the unsatisfactory profile and then click OK El Select file x fo SpectralCal 3100_RabbitRun_3100_Rabbit_2000 06 08_1 al ia CapO1 me a Cap02 me ia CapO3 me a CapO4 me ba CapOS me a Cap06 me a CapO7 me a CapO8 me a CapO9 me a Cap11 me A OK Spectral Calibration Data mcl x Cancel Look in File name Files of type Spatial and Spectral Calibrations 4 31 To override a spectral calibration profile with previously collected data continued Step Action 6 In the Use the matrix from file dialog box confirm that the correct file appears next to Data Source From file and then click the appropriate button ook ae Sana MGS Raa Goa r a oc on f a WY a Te oo interi oy Bd Aire im Ti nh Ghevdiy miini Me wini terddian Humbe 83 whee Coed fois Seuece Frome Cae ree Dasti ap i gym Gm pi aiir Click OK 4 32 Spatial and Spectral Calibrations Section Advanced Features of Spectral Calibration In This Section The following topics are covered in this section Topic See Page Fine Tuning a MatrixStandard Calibration 4 34 Spectral Calibration Matrices 4 36 Spectral Calibration Log Files 4 37 Spectral Calibration Parameter Files 4 38 Spectral Calibration Parameters 4 40 dataType Parameter 4 41 m
147. emplate File Names D AppliedBio Abi Support Files Data Collection Support Files Plate Import Files 4 Exploring D nerkin elmer abi Support Files Data Collection Support Files Plale Impor Fal x File Edit View Tools Hels L Plate Inport Files M l 1 Alale AllFolders Contents of D p rkin elmer abi S upport Files Data Colecti abi A Name mal 3100 3 FullPlate_GSWell_384 alt G GeneScan 3 FullPlate_GSell_96 pt _ SegAnal 2 FulPlate_Secwell_384 gt plt D Gj Shared 3 FullPlate_SecWell_Yb plt _ Suppor Files C Data Collection Suppott Files _ Calibration Data __ Method Files an Plate Import Files _ Service Modules Poa E gt 4 opject s 826KB Disk free space 4 23GB This table lists the file names and types of the templates Template File name Type of Template FullPlate_GSWell_384 plt 384 well for fragment analysis FullPlate_GSWell_96 plt 96 well for fragment analysis FullPlate_SeqWell_384 plt 384 well for sequencing analysis FullPlate_SeqWell_96 plt 96 well for sequencing analysis Creating a Plate To create a plate record using a template Record Using a Template Step Action 1 Start Microsoft Excel 6 24 Working with Plate Records To create a plate record using a template continued Step Action 2 From the File menu select Open This opens the Open dialog box Cem ot
148. ending on the earlier of Limited Warranty one year from the completion of installation or fifteen 15 months from the date of Statement Shipment to the customer the Warranty Period the ABI PRISM 3100 Genetic Analyzer purchased by the customer the Instrument will be free from defects in material and workmanship and will perform in accordance with the published performance specifications contained in the 3100 Genetic Analyzer Specification Sheet the Specifications publication number 106SP02 01 During the Warranty Period if the Instrument s hardware becomes damaged or contaminated or if the Instrument otherwise fails to meet the Specifications Applied Biosystems will repair or replace the Instrument so that it meets the Specifications at Applied Biosystems expense However if the 3100 Genetic Analyzer becomes damaged or contaminated or if the chemical performance of the Instrument otherwise deteriorates due to solvents and or reagents other than those supplied or expressly recommended by Applied Biosystems Applied Biosystems will return the Instrument to Specification at the customer s request and at the customer s expense After this service is performed coverage of the parts repaired or replaced will be restored thereafter for the remainder of the original Warranty Period This Warranty does not extend to any Instrument or part which has been a the subject of an accident misuse or neglect including but not limited
149. ens the Plate Setup page 2 In either the Pending or Processed Plate Record table select the row that names the plate record you want to delete Note You can select more than one row at a time by pressing CTRL while selecting additional rows 3 Click Delete Working with Plate Records 6 39 System Management and Networking Overview In This Chapter The following topics are covered in this chapter Topic See Page Section Managing Hard Drive and Instrument Database Space 7 3 How Run Data Is Stored 7 4 Checking Database Space The Diskspace Utility 7 5 Re Extracting Processed Frame Data The Re Extraction Utility 7 6 Deleting Processed Frame Data The Cleanup Database Utility 7 8 Importing a New Spatial or Spectral Calibration Method The New Method 7 10 Import Utility Removing Run Modules from the Instrument Database The Remove Run 7 11 Modules Utility Reinitializing the Instrument Database The Initialize Database Utility 7 12 Section Networking 7 13 Networking Options 7 14 Networking the Computer Workstation 7 16 Requirements for a Networked Computer 7 18 System Management and Networking 7 1 7 2 System Management and Networking Section Managing Hard Drive and Instrument Database Space In This Section The following topics are covered in this section Topic See Page How Run Data Is Stored 7 4 Checking Database Space T
150. ent analysis or rapid DNA sequencing 50 cm array for DNA sequencing Type of plate Either a 4 96 well plate 384 well plate Method of creating plate records There are six different ways to create plate records Which analysis module to use Either Select one of the supplied analysis modules Create your own analysis module in the downstream application Which run module to use Either Select one of the supplied run modules Edit one of the supplied run modules to change the conditions used for a run How many times to run your samples To run your samples only once use only one run module column and one analysis module column when creating the plate record To run each sample up to five times use identical run module columns and identical analysis module columns Whether to run the same sample again under different run conditions Prepare two run module columns when creating the plate record filling in the second with a different run module Whether to perform a single run or a batch run Either A single run that electrophoreses up to 16 samples A batch run that performs several sequential runs without needing operator attention Performing a Run 3 5 Decision Table continued Decision Comments Whether to save data If you do not enable BioLIMS in the Setting Preferences dialog to a BioLIMS box the sample files are stored in the fol
151. erent modules for different samples the samples will be automatically grouped so that all samples with the same run module are run at the same time Runs are scheduled alphanumerically by run module name not by the order indicated in the plate record nor by sample name Performing a Run 3 33 3 34 Performing a Run To enter sample information and save the plate record continued Step Action 6 For each sample select the appropriate Analysis Module from the drop down list IMPORTANT The AutoAnalysis On preference must be selected if analysis is to take place automatically after the run see page 3 29 Analysis Module 1 sno selection lt no selection GS3504nalysis gsp GS400CubicAnalysis gsp GS400HDAnalysis qsp GS4000rd24nalysis gsp GSs004nalysis gsp The following table shows which analysis module to select based on the number of fragments in your size standard If using size standard Select this analysis module GS400HD GS400HDAnalysis gsp GS350 GS350Analysis gsp GS500 GS500Analysis gsp GS500 see footnote GS400CubicAnalysis gsp GS4000rd2Analysis gsp a These modules are for advanced users with specific sizing needs See the AB PRISM GeneScan Analysis Software v 3 5 NT User s Manual Note You can examine the settings for each of these files using GeneScan Analysis Software The meanings of the settings are described in the ABI PRISM Gen
152. et as a tab delimited file Using the spreadsheet examples and the information about each token starting on page 6 14 type your information into the file From the File menu select Save As In most spreadsheet programs the Save As dialog box will open Type in a name for the tab delimited file that you are about to create IMPORTANT Use only the following characters which are a subset of the characters allowed by the Windows NT operating system letters numbers and _ Do not use spaces Save the file with the following file name format filename plt In the File Type text box or equivalent select the text file tab delimited file type or equivalent Note If you close Microsoft Excel before performing this step the Office Assistant opens Click Yes and then Save Follow the directions starting on page 6 32 for importing a tab delimited text file to create a plate record 6 28 Working with Plate Records Creating a Plate File from a Custom Spreadsheet Template Introduction This method can be used to create a read only spreadsheet template which you can save as a different name and then modify to suit your needs Creating the Template Modifying the Template If you are using similar samples and run conditions this method allows you to type less each time you want to create a new plate record There are two parts to the procedure Creating the template Modifying the
153. f the Instrument Store the capillary array off of the instrument when the capillary array will be unused for longer than 1 week Before storing the capillary array for long periods we recommend filling the capillaries with fresh polymer IMPORTANT If you intend to capillary reuse the array do not let the capillaries dry out Store the capillary array with both ends in fresh 1X running buffer IMPORTANT Wear gloves while performing the following procedure and any other time you handle the capillary array glass syringes septa or buffer reservoirs To store the capillary array off the instrument Step Action 1 Fill the capillary array with fresh polymer using the Change Polymer wizard or manual control commands Remove the syringe guard Remove both syringes from the upper polymer block and properly dispose of any remaining polymer Wash the syringes Remove the capillary array from the instrument using the Install Replace Capillary Array wizard For instructions see Installing and Removing the Capillary Array on page 8 15 Replace the cover over the detection cell Fill a buffer reservoir with fresh 1X running buffer and cover with a septa strip Insert the capillary tips into the buffer 8 Fill a 1 5 mL conical tube with deionized water and insert the detection end of the capillary array 9 Wrap the tube with laboratory film such as Parafilm to prevent evaporation 10 S
154. faultModule madexp File narn2 OK Files ofipe Run Module Export File mode cancel 4 Navigate to the folder in which you saved the run module file Select the file Note Due to software limitations you cannot select a folder on the desktop 5 Click OK to import the file The transferred run module has the same name except for a unique appended number El Module Editor Mikl Ei r Modules r Module Parameters Sequencing cenesoan Others Name Sequencing _1DefaultModule Template Sequencing _1 EJ Parameter Name Value Range Sequencing1_1DefaultModule Sequencing1_1DefaultModule932657 4 Flat field corrected yes boolean yes no a 2 integration size 1 int 0 5 pixels 3 Run Temp 60 int 25 70 Deg C 4 Cuvette Temperature Jao int 25 50 Deg C 6 Cap Pressure 1200 int 50 1500 0 1 psi inal Comments Import complete a L Indicates that the file data was successfully transferred Software 5 25 5 26 Software Section Working with Sequencing Analysis Modules In This Section The following topics are covered in this section Topic See Page Viewing and Editing Analysis Modules for DNA Sequencing 5 28 Creating a Sequencing Analysis Module 5 30 Introduction Sequencing analysis modules created with DNA Sequencing Analysis software provide the Auto Extractor with the parameters needed to analyze sequencing da
155. ferring data from a LIMS database is completely automatic The data transfer process is automatic it does not need to be initiated by a manual import command in the Data Collection software When the software is configured to import LIMS data it Periodically polls the plate import table described below for new data transferred into it by the LIMS database Automatically Creates plate records from the transferred data Enters an event describing the import in the Events log Registers the plate record in the Pending Plate Record table of the Plate View page To use the automatic LIMS data transfer feature the 3100 Data Collection software must be configured to automatically poll the instrument database for plate import table entries BioLIMS is a type of LIMS database For information about BioLIMS see page 5 47 6 20 Working with Plate Records Plate Import Table Introduction Plate Import Table Capacity Plate Import Errors Required Fields Plate ID Name The instrument database contains a plate import table It is the only part of the database that can be safely accessed by outside programs as there are no links to other tables in the database The number of sets of plate data that can be accommodated in the plate import table is dependent on the amount of available space in the instrument database Once the data in the table has been successfully imported into the main database the data is store
156. for computing the Q values for matrixStandard and known dye sets Dye Sets D and E sequenceStandard dataType parameters are slightly different For this reason the minQ values for the unknown dye sets sequenceStandard dataType should be set lower than the values for the known matrixStandard or sequencingStandard dataType 4 42 Spatial and Spectral Calibrations High Q Values_ In rare cases a high Q value can be computed for a poor matrix This can happen if the matrix standard is contaminated leading to the creation of one or more extra peaks The extra peak s causes the true dye peak to be missed by the algorithm By chance this can lead to a higher Q value than would be computed with the correct peak The best way to intercept this error is to visually inspect the spectral calibration profile for each capillary see Displaying a Spectral Calibration Profile on page 4 25 Spatial and Spectral Calibrations 4 43 conditionBounds Parameter Definition Condition Number C Value Using the Correct Values How the conditionBounds Value Is Used by the Software The conditionBounds parameter value comprises two numbers that represent the lower and upper bounds of the matrix condition number also called the c value The conditionBounds value format is lowest allowable c value highest allowable c value The condition number is a single number that indicates the amount of overlap between the dye peaks in the fluorescence emissio
157. for long periods may affect data quality Complete the current run a Stop and E Stop the other scheduled runs b After run in the Question dialog box E Question xi 2 Stop now or after current run i Cancel Stop the current run and Stop the other scheduled runs a Stop E b Now in the Question dialog box Question x 2 Stop now or after current run Cancel When you click Now the run files extract automatically The files will be automatically analyzed if the AutoAnalysis preference is enabled To recover data from a stopped run see Recovering Data if Autoextraction Fails on page 3 60 Stop the current run and Continue the other scheduled runs Skip to Next Run To recover data from a stopped run see Recovering Data if Autoextraction Fails on page 3 60 Performing a Run 3 47 Run Times DNA Sequencing Run Times GeneScan Run Times 3 48 Performing a Run The following table lists the approximate run times of common DNA sequencing analysis runs Type of Analysis Run Module Run Time Standard DNA sequencing StdSeq50_POP6DefaultModule 2 hr 30 min Rapid DNA sequencing RapidSeq36_POP6DefaultModule 1 hr The following table lists the approximate run times of common GeneScan analysis runs Type of Analysis Run Module Run Time GeneScan GeneScan36_POP4DefaultModule 45
158. formation xi P oject Name ji Project Information The instrument handles sequ build 5 seq_ _ project info Add Procct Delete Project 2 If you want to Then add a new project a Click Add Project A blank row appears b Continue with step 3 delete an existing project a Highlight the project you want to delete b Click Delete Project c Skip to step 4 3 Enter the appropriate information in the text fields Text Field Description Constraints Project Name Type a descriptive name of your choice Note The Project Name will be the Collection Name in the BioLIMS database Project Owner Type in your name Note The Project Owner will be the Creator in the BioLIMS database Project Information Type in any comments if desired Note The Project Information will be the Comment in the BioLIMS database 5 48 Software To set up the BioLIMS project information continued Step Action 4 Click OK to save your changes The new project s will be listed in the drop down list under the BioLIMS Project column in the Plate Editor window 5 Continue with Preparing a Plate for Extracting to BioLIMS on page 5 50 Software 5 49 Preparing a Plate for Extracting to BioLIMS Introduction After you have set up the BioLIMS project information you must prepare a plate record in the 3100 Data Collection software for extraction to the Bio
159. g at Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 The ABI PRISM 3100 Genetic Analyzer includes patented technology licensed from Hitachi Ltd as part of a strategic partnership between Applied Biosystems and Hitachi Ltd as well as patented technology of Applied Biosystems ABI PRISM and its design Applied Biosystems BioLIMS GeneScan Genotyper and MicroAmp are registered trademarks of Applied Biosystems or its subsidiaries in the U S and certain other countries ABI BigDye Factura Hi Di POP POP 4 and POP 6 are trademarks of Applied Biosystems or its subsidiaries in the U S and certain other countries AmpliTaq is a registered trademark of Roche Molecular Systems Inc Microsoft Windows and Windows NT are registered trademarks of the Microsoft Corporation in the United States and other countries Oracle is a registered trademark of the Oracle Corporation pGEM is a registered trademark of Promega Corporation All other trademarks are the sole property of their respective owners 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 Record information about your software below Software CD Serial Number Version Number Registration Code 3100 Software Or
160. gions in the data to pass the calibration A typical maximum number of scans analyzed value is 6000 frames startptRange Parameter Introduction Purpose Examples If you use the sequenceStandard dataType parameter you can select the starting point range startptRange parameter The startptRange not shown in the summary diagram defines the minimum and maximum frame numbers that bound the start of data analysis The startptRange parameter gives you fine control over the first frame number that the algorithm uses for analysis Although you can select the starting point offset the Autostart point is selected by the algorithm based on when the first dye is detected By using just the startptOffset parameter you do not control the actual point at which the algorithm starts its analysis However by specifying the starting point range you can have this control The startotRange parameter forces the analysis starting frame to lie within the selected range It is applied after the Autostart computation and startptOffset are applied To set an exact start point set the lower and upper bounds to be the same value for example 8000 3000 The following example shows the effect on the starting frame number using a startptRange value of 8000 4000 Analysis Starting Frame Analysis Starting Frame Number Without Setting the Number When startptRange Is startptRange Set Comments 3300 3300 Frame number lies within range so
161. grouped so that all samples with the same run module are run sequentially Performing a Run 3 39 To enter sample information and save the plate record continued Step Action 8 Make sure the plate record is correct and then click OK Plate Editar DT3100PO gt 6 BD Ww2 mob STD SEQ INJECTION 3100_Project1 DT3100PO0 6 BD w2 mob STD SEQ INJECTION 3100 Proiect1 DT3100P0 6 BD jvz mob STD SEQ INJECTION 3100_Preiect1 DT3100PO gt 6 BD Ww2 mob STD SEQ INJECTION 3100_Project1 OT3100P0 6 BD jvz mob STD SEG INJECTION 3100_PrgectI DT3100P0 6 BD jvz mob STD SEQ INJECTION 3100_Proect1 DT3100P076 BD jvz mob STD SEQ INJECTION 3100_Preiect1 DT3100P0 6 BD jz mob STD SEQ INJECTION 3100_Proect1 DT3100P0 6 BD jvz mob STD SEQ INJECTION 3100_Proect1 DT3100P0 6 BD jv2Z niub STD SEQ INJECTION 3100_Prujeult DT3100P0 6 BD jz mob STD SEQ INJECTION 3100_Project1 DT3100P0 6 BD jvz mob STD SEQ INJECTION 3100_Preiect1 DT3100P0 6 BD jz mob STD SEQ INJECTION 3100_Proect1 DT3100P0 6 BD jz mob STD SEQ INJECTION 3100_Project1 DT31 OOP O G D0D jwz inob STD SCQ INJECTION 3100_Project1 DT3100P0 6 BD Ww2 mob STD SEQ INJECTION 3100_Preiect1 DT3100P0 6 BD jz mob 1X DOWN SAMPLING 3100_Project1 DT3100PO0 6 BD Ww2 mob 1X DOAN SAMPLING 3100_Project1 DT3100PO 6 BD jvz mob 1X DOAN SAMPLING 3100_Proiect1 DT31 00PO G DD jvz mob 1X DOAN SAMPLING 3100_Project1 DT3100P0 6 BD jvz mob 1X DOAN SAMPLING 3100_Proie
162. h compressed air Extra peaks in the electropherogram Data off scale Dilute the sample and re inject the sample Possible contaminant in sample Re amplify the DNA Sample renaturation Heat denature the sample in good quality formamide and immediately place on ice Peaks exhibit a shoulder effect in GeneScan applications Sample renaturation Heat denature the sample in good quality formamide and immediately place on ice Purging of polymer from the polymer reserve syringe Arcing in the anode gel block Replace the lower polymer block Bubbles in syringes Remove bubbles Leaking polymer at the top of either syringe Insufficient seal around the Teflon tip of the plunger Make sure to wet the Teflon before filling the syringe with polymer If the leaking persists replace the syringe Note Do not mix and match barrels and plungers Troubleshooting 9 7 Observation Possible Cause Recommended Action Leaking polymer at the bottom of the polymer reserve syringe Improper tightening of the array ferrule knob to the syringe or and to the polymer block Ensure the array ferrule knob is tightened Error message Leak detected appears The run aborts Air bubbles in the polymer path Check for bubbles and remove if present Then look for leaks Buffer jar fills very quickly with polymer Air bubbles in the polymer path
163. he following punctuation only _ Do not use spaces 4 When done click Finish The Plate Editor spreadsheet opens File Edit Plate Name EEO wel Sample Neme Dyes Color Into Clor Comnent BIOLIMS Project Dye Set Runt Al B1 Performing a Run 3 31 3 32 Entering Sample To enter sample information and save the plate record Information Performing a Run Step Action 1 In the Plate Editor spreadsheet type the names of all the samples in the Sample Name column Note Inthe default naming convention the sample name you type is incorporated into the sample file name For example MySample A010 nea Capillary position i Well position Sample name you type The sample file naming convention used can be changed in the Preferences dialog box See page 3 28 for details IMPORTANT When naming the samples you can use letters numbers and the following punctuation only _ Do not use spaces IMPORTANT Be sure that sample file names are not longer than 55 characters An underscore separates each preference selected so be sure to count the underscore in the number of characters There is no automatic error checking for sample names that exceed this limit Sample files with long names cannot be opened by the analysis software Optional For each sample enter Color Info and Color Comment text Enter a BioLIM
164. he Diskspace Utility 7 5 Re Extracting Processed Frame Data The Re Extraction Utility 7 6 Deleting Processed Frame Data The Cleanup Database Utility 7 8 Importing a New Spatial or Spectral Calibration Method The New Method 7 10 Import Utility Removing Run Modules from the Instrument Database The Remove Run 7 11 Modules Utility Reinitializing the Instrument Database The Initialize Database Utility 7 12 System Management and Networking 7 3 How Run Data Is Stored Types of Run Data Run data is stored in different forms depending on the configurations selected in the Storage Preferences and Auto Extractor dialog boxes Data Storage Type Where Stored Approx Data Storage Space Processed frame data In the instrument database of the local computer 100 MB for a 2 5 hr run workstation ABIF sample file On the local or networked hard drive at a directory 250 KB per sample file for a location specified in the Extraction Directory dialog box ABI PRISM 3100 POP 4 of Auto Extractor sequencing analysis run The default setting is to store ABIF sample files in the 210 KB per sample file for a following directory ABI PRISM 3100 POP 6 D AppliedBio abi 3100 Data Extractor ExtractedRuns fragment analysis run 300 KB per sample file for a ABI PRISM 3100 POP 4 fragment analysis run BioLIMS data BioLIMS database on another networked computer 7 4 System Management and Networking Checking Database Spa
165. he following topics are covered in this section Topic See Page Supported Dye Sets 2 18 Labeling Chemistries 2 19 Polymers 2 20 Injection Solution 2 21 Overview This section provides an overview of the chemistry For more detailed information see the ABI PRISM 3100 Genetic Analyzer Sequencing Chemistry Guide System Overview 2 17 Supported Dye Sets Overview DNA fragments are detected and identified by the fluorescent dyes with which they are chemically labeled Dyes are purchased and used as dye sets which are optimized for particular applications Table of Supported Two dye sets are currently supported by Applied Biosystems for use with the Dye Sets 3100 Genetic Analyzer Note Other dye sets can also be used for sequencing with the 3100 Genetic Analyzer see dataType Parameter on page 4 41 Dye Set Comprises Use for D 6FAM HEX NED ROX Fragment analysis dRhodamine and ABI PRISM BigDye versions of dROX dTAMRA dR6G dR110 DNA sequencing Ea SNP Detection Snapshot a The ABI PRism BigDye dye set has a similar spectral profile as Dye Set E Customers have successfully used Dye Set E matrix standards for BigDye dyes For best performance however we recommend that you create the matrix from Long Read standards 2 18 System Overview Labeling Chemistries Supported Labeling The 3100 Geneti
166. he screen at one time If you select more than four a scroll bar appears so that you can access the others An electropherogram is a graph of relative dye concentration against time plotted for each dye The data displayed is multicomponented The relative dye concentration is determined by applying chemometric algorithms to the collected fluorescence data There are two plots for each dye The plots represent the upper and lower confidence limits associated with the measured fluorescence intensity Display Order The capillaries are displayed in the order in which the boxes are checked For example to display capillary 1 under capillary 15 a Clear all check boxes b Select check box 15 c Select check box 1 Instrument Status Monitor Function The Instrument Status Monitor displays the current run conditions Viewing the To view the Instrument Status Monitor Instrument Status Monitor Step Action 1 From the View menu select Instrument Status Monitor or double click the Instrument Status Monitor button on the toolbar Running Run_B6_1999 12 11_3 Remaining this run 00 10 Run Time 01 17 I Current 0 0 I voltage 0 0 I Temp 28 0 C Note The Instrument Status Monitor can remain open while viewing other pages Performing a Run 3 57 3 58 Performing a Run Section Working with Data In This Section The following topics are covered in this section Topic See Page
167. he upper and lower polymer blocks Weekly 8 27 Replace the polymer in the syringes upper polymer block Weekly or as 8 10 and capillary array needed Check the storage conditions of the used arrays Weekly 8 4 Maintenance As Needed Tasks Perform these tasks as needed Maintenance Task Frequency See Page Clean the drip trays As needed Change the array As needed 8 15 Remove any dried polymer from the capillary tips Use a As needed lint free wipe moistened with deionized water Calibrate the autosampler Very rarely 8 29 Routine Cleaning General Cleaning To clean the instrument Step Action 1 Press the Tray button on the front of the instrument to move the autosampler to the forward position Wipe off any liquid on or around the autosampler using a lint free tissue 3 Clean out the drip trays with deionized water and lint free tissue Clean off any polymer build up crystals on the instrument including the capillary tips and the stripper plate with deionized water and lint free tissue IMPORTANT Never use organic solvents to clean the instrument Maintenance 8 5 Moving and Leveling the Instrument Before Moving the PHYSICAL INJURY HAZARD Do not attempt to lift the instrument or any Instrument ther heavy objects unless you have received related training Incorrect lifting
168. her These differences may cause the condition numbers to be systematically higher on one side of the array than on the other IMPORTANT Do not manually override matrices that have slightly higher c values on one side of the array because these matrices are still the most accurate for the capillaries they describe and will result in the smallest amount of pull up pull down To determine a suitable condition number range for a dye set Step Action 1 Perform a spectral calibration without setting a conditionBounds value such as with the SeqStd AnyDyeSet par file or the MtxStd AnyDyeSet par file Examine the spectral calibration profiles for each capillary 3 Open the spectral calibration log file in the following directory D AppliedBio abi 3100 DataCollection SpectralCalLogs Record the computed condition numbers for each capillary Plot a frequency distribution histogram of the condition numbers Do this by grouping the values into ranges and plotting the number of capillaries that fall within each range The histogram will probably be a skewed normal distribution curve 6 Use your judgment to determine minimum and maximum condition numbers You are aiming to set numbers that are Close enough to the mean to eliminate outliers Not so close to the mean that unnecessary failures on subsequent calibration runs are caused by Normal variation across the capillary array Instrument to instrument vari
169. here are two ways to reset the ABI PRISM 3100 Genetic Analyzer Press the Reset button on the front of the instrument to dump and reload the firmware and to reset the electronics Try this method first Shut down and restart the computer and the instrument Resetting Using the To reset the instrument Reset Button Step Action 1 Close the instrument doors 2 Using a long narrow implement such as a straightened paper clip press the Reset button on the front of the instrument Reset button Resetting by To reset the instrument Powering Down Step Action 1 Close the instrument doors 2 Turn off the instrument by pressing the On Off button on the front of the instrument 3 Restart the computer a From the Start menu select Shutdown b In the Shutdown Windows dialog box select Restart and click OK IMPORTANT Wait until the computer has completely restarted before proceeding 4 Turn on the instrument Note When the instrument is shut down the firmware is not saved Upon restart the instrument will reload a copy of the firmware and the calibration file from the computer 5 Open the Data Collection software Maintenance 8 7 Shutting Down the Instrument When to Perform Perform the appropriate shutdown procedure as follows Each Shutdown Procedure If the instrument will be unattended for Perform this shutdown procedure no more tha
170. herogram An electropherogram plots relative dye concentration y axis against time x axis for each of the dyes used to label the DNA fragments Each peak in the electropherogram represents a single fragment 800 400 11 6 117 11 8 11 9 12 12 1 Intensity vs Run Tit Automatic Data Extraction and Data Analysis The processed data is automatically extracted from the instrument database and analyzed The positions and shapes of the electropherogram peaks are used to determine either the base sequence or fragment profile depending on the type of run selected The analyzed data is stored as sample files on the hard drive of the computer Auto Extractor e Extraction e Analysis Sample Files Viewing the Results The analyzed data is viewed with either DNA Sequencing Analysis software for sequencing or GeneScan Analysis software for fragment analysis If necessary the data is reanalyzed using different analysis parameters CDIOSCEH MIAVSS SOftWaAE IES System Overview 2 7 2 8 System Overview Section Instrument Hardware Overview In This Section The following topics are covered in this section Topic See Page Front View 2 10 Front View with Doors Open 2 11 Back View 2 12 System Overview 2 9 Front View Diagram The following diagram shows the front of the instrume
171. icient filling of array Check for broken capillaries and refill the capillary array Expired matrix standards Check the expiration date and storage conditions of the matrix standards If necessary replace with a fresh lot Spikes in the data Expired polymer Replace the polymer with a fresh lot using the Change Polymer Wizard Air bubbles especially in the polymer block tubing assembly Refill the capillaries using manual control Possible contaminant or crystal deposits in the polymer Properly bring the polymer to room temperature do not heat to thaw rapidly Swirl to dissolve any solids Replace the polymer if it has expired 9 4 Troubleshooting Run Performance Observation Possible Cause Recommended Action No data in all capillaries Bubbles in the system No sample injection Visually inspect the polymer block and the syringes for bubbles Remove any bubbles using the Change Polymer Wizard Or follow the procedure on page 8 28 for manual bubble removal If bubbles still persist perform the following a Remove the capillary array b Clean out the polymer block and syringes c Replace polymer with fresh polymer Make sure to draw the polymer into the syringe very slowly No signal Autosampler calibration is not optimal Check the injection with 20 uL samples If the injection is OK recalibrate the autosampler using the Autosa
172. icular dyes so that it does not appear in the displays Open the Set Color dialog box to change the colors shown See Using the Set Step Action 1 From the Instrument menu point to Data Acquisition and select Set Color This opens the Edit Dye Display Information dialog box as shown below The operations of the Edit Dye Display Information dialog box are summarized in the diagram below Dialog Box Click in the Name text box to change the name of the dye Click to open the Set Color dialog box Click to store any changes you make in the Set Color dialog box and close the Edit Dye Display Information dialog box Click to test the effect of any changes you make E Edit Dye Display Information Name Color 2 color 2 a Iv 3 color 3 co Iv Visible Of x Intensity Factor afos i 5 oors E Vv _ x without storing the changes 5 12 Software Cancel Slide to increase decrease the color intensity Clear to hide the data for this dye in the displays Click to undo test changes Using the Set Color Dialog Box Why Change the Display Colors Two Ways to Change the Display Colors Changing the Display Colors Using the RGB System It is a good idea to change the colors used in the electropherogram and capillary displays if you find it hard to distinguish the default colors There are two ways to change the color used to represent the conce
173. ided by Applied Biosystems must be imported into the instrument database before they can be used The New Method Import utility imports these methods File Name and The New Method Import utility is named NewMethodImportUtility bat and is located in Directory the following directory D AppliedBio abi 3100 Bin Importing a New To import a method into the instrument database Method Step Action 1 Ensure OrbixWeb is running 2 Quit the 3100 Data Collection software 3 Copy the new method file into the Method Files folder in the following directory D AppliedBio abi Support Files Data Collection Support Files Method Files 4 Using Windows NT Explorer navigate to the Bin folder in the following directory D AppliedBio abi 3100 Bin 5 Right click NewMethodImportUtility bat 6 From the pop up list select Edit This opens the utility s batch file in Notepad 7 Look on the third line of the text for a string representing a file path to the method file The end of that string states INSERTNAME mtd 8 Replace the INSERTNAME text with the actual name of the new method file e g SeqXR101 Note Do not replace or delete the mtd file extension 9 From the File menu select Save This opens the Save As dialog box 10 In the Save As dialog box type a name for the method and click Save 11 From the File menu select Exit This closes Notepad 12 Double click the file that you just created to run the
174. ill be run first For two plates of either fragment analysis or DNA sequencing the plates will be run in the order in which the plates were linked The order in which the samples are run is based on the following factors in the order listed First samples will be sorted alphabetically by run module name Samples with module names beginning with capital letters come before those that begin with lower case letters e g Z before a If samples have the same run module name the samples on the plate that was linked first will be run first Secondly samples within a plate will be run in the order of their well designation i e A1 B1 C1 etc Lastly samples with more than one run module specified will be run in the order that the run modules appear Note The analysis module of a sample plays no part in the order in which it will be run Controlling the Run Controlling the Run Using the Instrument Menu File View Tools Servi HES Run SKIPENE Pause HOP You can use the Instrument menu to start skip pause or stop a run E 3100 Data Collection Software Controlling the Run You can also use the toolbar at the top of the 3100 Data Collection software window to Using the Toolbar control the run To Click Comment Start the run Run Instrument This begins all scheduled runs The run starts only when set temperature is reached Pause the run Pause Pausing
175. in a word processing program is a text file Using tab stops to separate sections of text and end of line characters to separate lines of text makes a file a tab delimited text file Examples A tab delimited text file created in Microsoft Word is shown below The symbols do not appear when the file is printed First line token one token two tokenthree token four gt token fivef Secondline tokenmone token two tokenthree token four token fiveff Third line token one tokentwo token three token four token siff With the nonprinting symbols turned off the file looks like this First line token one token two token three token four token five Second line token one token two token three token four token five Third line token one token two token three token four token six Word Wrapped _ As in word processed documents tab delimited text files with long lines wrap around Example to the next line First line token one token two token three token four which is a long token thatwraps around to the next line token five Second line token one token two which is anotherlong token token three token four token five Third line token one token two token three which is a furtherlong token that wraps around to the nextline and makes the file difficult to read token four token six Word wrapping does not affect the performance of a file but it does make the information more difficult to comprehend Notepa
176. inQ Parameter 4 42 conditionBounds Parameter 4 44 numDyes and writeDummyDyes Parameters 4 46 numSpectralBins Parameter 4 46 Parameters Specific to sequenceStandard dataType 4 47 startptOffset Parameter 4 48 maxScansAnalyzed Parameter 4 49 startptRange Parameter 4 49 minRankQ Parameter 4 50 Spatial and Spectral Calibrations 4 33 Fine Tuning a MatrixStandard Calibration Introduction Use the procedure below to fine tune the parameter for a calibration run for DNA sequencing with Dye Set E Fragment analysis with Dye Set D Fine Tuning a To create a spectral calibration parameter file for matrixStandard dataType Calibration Run Step Action 1 Navigate to the Spectral Calibration folder in the following directory D AppliedBio abi Support Files Data Collection Support Files Calibration Data Spectral Calibration 2 Double click the ParamFiles folder This opens the folder displaying the stored parameter files amp D perkin elmer abi S upport Files D ata Collection Support Files Calibration Data Spectral Calibration ParamFiles File Edit View Help E ParamFiles z a ee amp lale aaaea fal MtaStdiAnyDyeS et par 1K3 PAR ile 4 12 00 1 11 PM A a MtxStd GeneScarSetD car 1K3 PAR ile 5 30 00 2 30 PM A a MtxStd Sequencing SetE par 1K3 PAR ile 4 12 00 1 11 PM A a SegStd dnyDyeSet par 1K3 PAR ile 4 12 00 1 11 PM ja SeqStd Sequencng SetE par 1K3 PAR ile 4
177. indows During a run do not leave these pages open for extended periods This may cause unrecoverable screen update problems Leave the Status View window open 5 Alternatively to view electropherogram data from several capillaries at once click the Capillary View tab to display the Capillary View page Note For information on the Capillary View page see page 3 56 IMPORTANT Always exit from the Array View and the Capillary View windows During a run do not leave these pages open for extended periods This may cause unrecoverable screen update problems Leave the Status View window open Performing a Run 3 61 Viewing Analyzed GeneScan Data Introduction Locating Sample Files Run Folder Default 3 62 Name Performing a Run After a run has been extracted to sample files you can use the GeneScan Analysis software to view the electropherogram data both raw and analyzed Refer to the ABI PRISM GeneScan Analysis Software v 3 5 NT User s Manual P N 4308923 for details on viewing and analyzing GeneScan data When a run is finished the analyzed sample files are extracted into a run folder along with a run log in the directory D AppliedBio abi 3100 DataExtractor ExtractedRuns An example of the run folder and its contents is shown below O x D perkin elmer abii31004D ataE xtractor ExtractedRuns Run_demo_3100_2000 07 05_21 File Edit View Help E Run demo 310c 2000 07 05 gt fei
178. ion 8 Click the Close button in the upper right corner of the window Either a standard Windows NT message box or an equivalent Office Assistant message box is displayed i lt 9 Microsoft Excel SamplePlate2 plt is not in Microsoft Excel 97 format Do you want to save your changes To save your changes in Microsoft Excel 97 format click Yes and then click Microsoft Excel Workbook in the Save As Type box e To save your changes in the existing format and replace the original file click Yes and then click Save Some types of changes may be lost To close the file in its existing format without saving changes click No No Cancel Click Yes This opens the Save As dialog box 10 In the File name drop down list of the Save As dialog box delete the name of the file that you selected and type a new name for the edited file Make sure that you add the plt extension Click Save This saves the edited file as a new file 11 Follow the directions starting on page 6 32 for importing a tab delimited text file to create a plate record Working with Plate Records 6 27 Creating a Plate File from a New Spreadsheet Creating a Plate File To create a plate file plt file from a new spreadsheet from a New Spreadsheet Step Action 1 On acomputer using a Windows NT operating system open a new spreadsheet file in a program that allows you to save a spreadshe
179. ion Parameters 4 40 dataType Parameter 4 41 minQ Parameter 4 42 conditionBounds Parameter 4 44 numDyes and writeDummyDyes Parameters 4 46 numSpectralBins Parameter 4 46 Parameters Specific to sequenceStandard dataType 4 47 startptOffset Parameter 4 48 maxScansAnalyzed Parameter 4 49 startptRange Parameter 4 49 minRankQ Parameter 4 50 Spatial and Spectral Calibrations 4 1 4 2 Spatial and Spectral Calibrations Section Spatial Calibration In This Section The following topics describe how to perform a spatial calibration Topic See Page About Spatial Calibrations 4 4 About Spatial Calibration Data 4 5 Performing a Spatial Calibration 4 6 Displaying a Spatial Calibration Profile 4 10 Evaluating a Spatial Calibration Profile 4 11 Overriding the Current Spatial Calibration Map 4 12 Spatial and Spectral Calibrations 4 3 About Spatial Calibrations When to Calibrate A spatial calibration must be performed after each time you Install or replace a capillary array Temporarily remove the capillary array from the detection block What a Spatial A spatial calibration provides information about the position of the fluorescence from Calibration Tells each capillary on the CCD It does not provide information about the performance of You the capillaries 4 4 Spatial and Spectral Calibrations About Spatial Calibration Data Spatial Maps Spatial Calibration Files Spatial Calibration
180. ions 2 0 cc eee 4 4 About Spatial Calibration Data 0 cence 4 5 Performing a Spatial Calibration 2 0 eee eee 4 6 Displaying a Spatial Calibration Profile 0 0 0 eee eee ee 4 10 Evaluating a Spatial Calibration Profile 0 0 0 eee eee eee 4 11 Overriding the Current Spatial Calibration Map 0 00 00 c eee ee eee 4 12 Section Spectral Calibration 6 0 0 ccc ccc ccc nee ene n ee ee eens 4 15 About Spectral Calibrations 0 0 0 cee eee ene 4 16 Performing a Spectral Calibration Using Default Processing Parameters 4 18 Displaying a Spectral Calibration Profile 0 0 0 e cece cee eee 4 25 Overriding a Spectral Calibration Profile 02 0 0 eee eee eee 4 28 Section Advanced Features of Spectral Calibration 000 cc wce eens 4 33 Fine Tuning a MatrixStandard Calibration 0 00 0 cece eee eee 4 34 Spectral Calibration Matrices 2 0 ccc cee eee 4 36 Spectral Calibration Log Files 0 0 0 0 0 eee cee eee ee 4 37 Spectral Calibration Parameter Files 0 0 0 eee cc cee eee 4 38 Spectral Calibration Parameters 1 2 0 0 cee ce cece eens 4 40 data Type Parameter su oroarea eaa ss fol eth co Seas Sega eae Lae hance and lac ERNER Lea 4 41 minQ Parameter a se s2s eer eels Sear Ra BON ae aed ge ad ad noe ba lars Gane 4 42 conditionBounds Parameter 0 0 0 0 ccc eee ee eens 4 44 numDyes and writeDummyDyes Parameters
181. ions of the instrument such as opening valves The firmware is largely controlled by the commands sent from the computer workstation It acts as the link between the software commands and hardware operations About the 3100 Firmware Unlike the previous ABI PRism instruments for DNA analysis the 3100 firmware resides on the computer workstation The 3100 firmware is downloaded when the instrument is started Therefore the instrument and the computer workstation must be running to perform any functions Function The 3100 Data Collection software performs the following functions Works in conjunction with the 3100 firmware to control the mechanical operation of the instrument such as moving the autosampler and switching on the oven Collects and stores plate record data and preference settings in the instrument database Automatically schedules samples to particular runs Monitors and displays the status of the instrument and saves it to the instrument database as EPT data Collects and processes fluorescence emission data from the CCD camera during runs Stores the processed data in tables in the database and in temporary files on the hard drive Displays electropherograms for the current run or any previous run still stored in the instrument database Provides wizards which guide you through routine maintenance procedures Provides utilities which when launched automatically perform database maintenance Software
182. it Search Help 96 Well Sample Name Dye Set Mobility File Comment BioLIMS Project Sample Tracking Id Run Module std E DT3166P0P6 lt BD u2 mob 3166_Project1 StdSeq56_POP6b1DefaultModule std E DT3166P0P6 BD u2 mob 3166_Project1 StdSeq56_POP6b1DefaultModule std E DT3166P0P6 BD v2 mob 3166_Project1 StdSeq56_POP6b1DefaultModule Example of An example of a Microsoft Excel spreadsheet for samples intended for fragment Fragment Analysis analysis is shown below Spreadsheet Version number Plate header Column header 1 6596 Full GS 96 Well Well Sample NeColor Num Standard CDye Set Color Info Color Com BioLIMS P Sample Tr Run Modul Analysis Module Al std 1 4D 4 3100_Proje GeneScan GS400HD GeneScan GE 1 Sample data Al std 2 4D 2 4 3100_Proje GeneScan GS400HD GeneScan GE L_ Al std 3 4D 3 4 3100_Proje GeneScan GS400HDy GeneScan GE Al std 4 4D 4 4 3100_Proje GeneScan GS400HDy GeneScan GE id 41 gt Pi FullPlate_GSWell_96 ih SE ey 6 12 Working with Plate Records Example of When the above spreadsheet is converted to a tab delimited text file and opened with Fragment Analysis the Notepad program it looks like the example below Tab Delimited Text File For an explanation of the labels see page 6 14 1 68 FullPlate_GSWell_96 Notepad iof x File Edit Search Help 1 6 GS96_FullPlate GS 96 Well Sample Name Color Number Standard Dye Dye Set Color Info Color Comment BioLIMS Proj std 1 3166 _
183. iteria Height Similar heights for all peaks Red crosses One red cross marking the top of every peak No misplaced crosses To move a cross a Change the value in a Capillary Position box b Click outside of that box c Click OK to accept the new value Shape Single sharp peak for each capillary Small shoulders are acceptable Spacing Position values are 13 16 higher than the previous one for every capillary Theoretical spacing between capillaries is 15 Example of Passing Profile 400007 300007 200007 i 100007 0 10000 4 o 20 40 60 80 100 120 140 160 180 200 220 240 260 Intensity vs Pixel Number Capillary Number 1 10 114 13 14 Capillary Position a E E a E e r E T canes Example of Failed Profile 50000 40000 30000 20000 10000 0 10000 it 20 40 60 80 100 120 140 160 180 200 220 240 260 Intensity vs Pixel Number Capillary Number 1 10 11 does E bs fobs bo fis Fre feo fsa hoe fre fee for fe Bae a Cancel Spatial and Spectral Calibrations 4 11 Overriding the Current Spatial Calibration Map Introduction Once the spatial calibration run has completed and you have accepted it the new spatial calibration map is stored in the instrument database and sent to the instrument This new map will be used to process sample run data If the current run did not provide good data you can override the new data wi
184. ividual Plate Records 0 eee cee cee eee 6 39 7 System Management and Networking OVEIVIEW or ea dS cae ine Cn LES ea pk ae dus ab ees 7 1 Section Managing Hard Drive and Instrument Database Space 7 3 How Run Data Is Stored 2 0 cece eects 7 4 Checking Database Space The Diskspace Utility 0 0000 00 00 0008 7 5 Re Extracting Processed Frame Data The Re Extraction Utility 0 0 0 7 6 Deleting Processed Frame Data The Cleanup Database Utility 0 0 7 8 Importing a New Spatial or Spectral Calibration Method The New Method Import Utility 0 0 0 2 ee eee 7 10 Removing Run Modules from the Instrument Database The Remove Run Modules Utility 0 0 eee eee 7 11 Reinitializing the Instrument Database The Initialize Database Utility 7 12 Section Networking 6 0 0 0 ccc cece cece eee eee eee ee eee ee eens 7 13 Networking Options os css acere ieas SAN aa hae bh pak L beg ea ele eo eae 7 14 Networking the Computer Workstation 0 0 0 0 cece eee ee eee 7 16 Requirements for a Networked Computer 0 0 00 cee e eee eee eee 7 18 8 Maintenance OVELVIEW 2c Bice OS eee we a eA eA Sat Ra De abe 8 1 Section Instrument Maintenance 00 ccc ec eee teen eens 8 3 Maintenance Task Lists i aene deka eee ba eae thb E oe GM Gea Gena hee Ry ee 8 4 Routine Cleaning o perne eai he SNR Da AER Rend Ge eae ees 8
185. ized in the Data Collection software Command Category Command Name Value Electrophoresis Set Power Supply On Off Set Voltage A number between 0 and 20 kV Laser Set State Idle On Off Set Power A number between 0 and 25 mW Open Close Shutter Open Closed Oven Set State On Off Set Temperature A number between 18 and 65 C Autosampler Move Forward N A Return N A Move Up down A number between 500 and 500 steps Move to Site Site 1 left front for 1X running buffer Site 2 left rear for deionized water Site 3 right front for deionized water Site 4 right rear for deionized water Array fill syringe Move Home N A Move Up A number between 1 and 1200 steps Move Down A number between 1 and 1200 steps Polymer reserve Move Home N A syringe Move Up A number between 1 and 1200 steps Move Down A number between 1 and 1200 steps Pin valve Set Position Open Closed Capillary Fill 50 cm POP6 36 cm POP4 36 cm POP6 Using Manual Control Commands Sending a Manual IMPORTANT The oven and instrument doors must be closed for manual control commands to Control Command amp ecute Note You cannot send a manual control command during a run To send a manual control command Step Action 1 From the Instrument menu select Manual Control The Manual Control dialog box appears Manual Control
186. k DNA quality Troubleshooting 9 5 Observation Possible Cause Recommended Action Elevated baseline Possible contaminant in the polymer path Wash the polymer block with hot water Pay particular attention to the upper polymer block the ferrule the ferrule screw and the peek tubing Dry the parts with compressed air before replacing them onto the instrument See Routine Cleaning on page 8 5 IMPORTANT Do notwash syringes in hot water because the Teflon plungers will get damaged Possible contaminant or crystal deposits in the polymer Bring the polymer to room temperature swirl to dissolve any deposits Replace the polymer if it has expired Poor spectral calibration Perform new spectral calibration Detection cell is dirty Place a drop of methanol onto the detection window and dry with compressed air Use only light air force Loss of resolution Too much sample injected Dilute the sample and re inject Poor quality water Use high quality ultra pure water Poor quality or dilute running buffer Prepare fresh running buffer from 10X 3100 buffer with EDTA Poor quality or breakdown of polymer Use a fresh lot of polymer Capillary array used for more than 100 injections Replace with new capillary array Degraded formamide Use fresh HiDi and ensure correct storage conditions High salt concentration in samples Use a
187. l The following table describes the preferences that can be set within this page Preference Description AutoAnalysis On Select AutoAnalysis On to have the samples automatically analyzed by the analysis software after the run Note Selecting this option will not prevent you from reanalyzing your sample data BioLIMS Use these settings to have data extracted to a BioLIMS database instead of to sample files on the hard drive Sample File Name Prefix Format Specify the format for the sample file names by using the drop down lists to reorder the identifiers Identifier Origin Run ID Generated by the Data Collection software Sample Name Taken from the Plate Editor spreadsheet entry Well Position Taken from the sample s position on the plate column letter and row number e g C3 Plate Name Taken from the Plate Editor dialog box entry Instrument ID Taken from the Data Collection page preferences entry Array ID Taken from the Install Capillary Array wizard entry Note In addition to the four identifiers you set with the drop down lists all names are automatically appended with the capillary number and a file extension Therefore in the Data Analysis page example shown above the sample name will be Sample Name_Well Position_Capillary Number sa or abl for sequencing Performing a Run 3 29 About Plate Records Introduction When to Create a
188. lary Array Introduction Before you reinstall a capillary array it is recommended that you Clean the front of the detection cell Check that the cathode bar is dry Cleaning the This procedure is unnecessary for new arrays unless you have accidently touched the Detection Cell detection cell To clean the detection cell Step Action 1 Put one drop of methanol on the front surface of the detection cell TREE Front surface of detection cell CHEMICAL HAZARD Methanol is a flammable liquid and vapor Exposure may cause eye skin and respiratory tract irritation and central nervous system depression and blindness Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Blow dry the cell using clean pressurized air Checking the When putting a used array back on the instrument be sure that the cathode bar is dry Cathode Bar A wet bar could lead to arcing 7 AER NINE ELECTRICAL SHOCK FIRE HAZARD Do not leave liquid in the cathode bar This can lead to electric shock or even fire if not properly maintained 8 14 Maintenance Ensure the cathode bar is dry especially in the center Installing and Removing the Capillary Array When to Change a Capillary Array Installing or Removing the Capillary Array Using the Wizard A capillary array should last approximately 100 runs The following
189. llected and extracted for this run is stored in this directory along with a log file for the run debug log 4 Open the run s debug log file If the extraction was Then completed successfully The following message appears after each sample file listed Successfully uploaded to BioLIMS not completed A message appears after each sample file listed successfully explaining why the extraction was not successfully completed For example Failed to open connection with database using specified credentials or database was not alive You must manually upload the extracted data using Sample2DB See the Sample2DB User s Manual for instructions Software 5 55 Working with Plate Records Overview In This Chapter The following topics are covered in this chapter Topic See Page Section Introduction 6 3 About Creating Plate Records 6 4 About the Plate Record Fields 6 5 Section Tab Delimited Text Files 6 9 Introduction to Tab Delimited Text Files 6 10 About Creating Tab Delimited Text Files 6 11 Using Spreadsheets to Create Tab Delimited Text Files 6 12 Spreadsheet or Tab Delimited Text File Information 6 14 Running the Same Sample with Different Conditions 6 18 Section Creating a Plate Record by Importing LIMS Data 6 19 Data Transfer 6 20 Plate Import Table 6 21 Section Creating Plate Files 6 23 Creating a
190. lowing directory database optional or ae aes to ABIF sample files D AppliedBio abi 3100 Data Extractor Extracted Runs Whether to perform a A spatial calibration must be performed after each time you spatial calibration Install or replace a capillary array Temporarily remove the capillary array from the detection block Whether to perform a A spectral calibration must be performed Spectral calibration Whenever you use a new dye set on the instrument Whenever you change the type of polymer used After the laser or CCD camera has been realigned by a service engineer If you begin to see a decrease in spectral separation pull up and or pull down peaks 3 6 Performing a Run Section Working with Samples and Plate Assemblies In This Section The following topics are covered in this section Topic See Page Sample Preparation 3 8 Working with Plate Assemblies 3 9 Performing a Run 3 7 Sample Preparation References for For information on required materials sample preparation and plate centrifugation Sample Preparation refer to the appropriate guide as follows For Refer to the DNA sequencing samples ABI Prism Automated DNA Sequencing Chemistry Guide P N 4305080 Fragment analysis samples ABI PRISM 3100 Genetic Analyzer Quick Start Guide for Fragment Analysis P N 4315832 Checking the Plate After centrifuging the plate of samples ensure
191. mation To enter plate record information Step Action 1 Click the Plate View tab on the 3100 Data Collection software window to go to the Plate View page Plate View tab 2 In the Plate View page click New Or double click the Plate Editor button on the toolbar The Plate Editor dialog box appears Plate Editor S6 Well This is an example plate record 3 Use the Plate Editor dialog box to name your plate and to specify the application and plate type Entering comments is optional IMPORTANT When naming the plate you can use letters numbers and the following punctuation only _ Do not use spaces 4 When done click Finish The Plate Editor spreadsheet opens Plate Editar 3 36 Performing a Run Entering Sample To enter sample information and save the plate record Information Step Action 1 In the Plate Editor spreadsheet type the names of all the samples in the Sample Name column Note Inthe default naming convention the sample name you type is incorporated into the sample file name For example Mysample AII p ap Capillary position L Well position Sample name you type The sample file naming convention used can be changed in the Preferences dialog box See page 3 28 for details IMPORTANT When naming the samples you can use letters numbers and the following punctuation only _ Do not use
192. min Section Monitoring a Run In This Section The following topics are covered in this section Topic See Page Run View Page 3 50 Status View Page 3 51 Array View Page 3 53 Capillary View Page 3 56 Instrument Status Monitor 3 57 Introduction This section describes the functions and features of The 3100 Data Collection software pages that are used to monitor a run and The Instrument Status Monitor which provides a summary of the current run conditions Performing a Run 3 49 Run View Page Function Features Scheduled runs in order Click the Run View tab to monitor the status of the scheduled runs This is an example of the Run View page 23100 Data Collection Software x Fie View Instrument Tools Service Help Hila gt e Plate View Run View status View Array View Capilary View Run Schedule Capillary Use Indicator Plate Image Indicators Delete Button 3 50 Performing a Run Run Run Type Module Status C a p i l a ry 1 310 Sample _POPSa3 Pending 2 Run_demo_310 Sample StdSeq50_POP6a3 Pending use 3 Run_demo_310 Sample StdSeq50_POP6a3 Pending 16 2 4 Run_demo_310 Sample StdSeq50_POP6a3 Pending indicator Run_demo_310 Sample StdSeq50_POP6a3 Pending A my_plete_record B Li 6 Run_demo_310 Sample _ StdSeq50_POPEa3 Pending a Plate Images
193. mpler Calibration Wizard Pay particular attention to the Z axis If the injection is not OK perform the procedures below Dead space at bottom of sample tube Centrifuge the sample tubes Bent capillary array Replace the capillary array and recalibrate the autosampler using the Calibrate Autosampler Wizard Failed reaction Repeat reaction Cracked or broken capillary Visually inspect the capillary array including the detector window area for signs of breakage Signal too high Sample concentration is too high Dilute the sample Decrease the injection time Too much DNA added to the reaction resulting in uneven signal distribution Optimize chemistry Low signal strength Poor quality formamide Use a fresh lot of HiDi Formamide Pipetting error not enough sample Increase the amount of DNA added Recalibrate the pipets Sample has high salt concentration Dilute in high quality water Desalt using a column purification method Insufficient mixing Vortex the sample thoroughly and then centrifuge the tube to condense the sample to the bottom of the tube Autosampler out of calibration Check the injection with 20 uL samples If the injection is OK recalibrate the autosampler using the Autosampler Calibration Wizard Pay particular attention to the Z axis Weak amplification of DNA Re amplify the DNA Chec
194. mporting method files 7 10 plate files 6 31 plate records froma LIMS 6 19 to 6 22 run modules from file 5 25 incident fluorescence on CCD camera A 4 Initialize Database utility 7 12 Injection Time run module parameter 5 22 Injection Voltage run module parameter 5 22 instrument cleaning routine 8 5 components 2 4 documents 1 4 hardware 2 9 to 2 12 moving and leveling 8 6 operation 3 45 overview 2 3 to 2 7 resetting 8 7 safety 1 5 setup 3 19 shutdown 8 8 startup 3 13 status lights 2 10 waste handling 8 12 instrument database 5 8 configuring data flowin A 7 deleting from 7 8 plate import table 6 21 See also processed frame data Instrument Status Monitor discussed 3 57 Internet address Documents on Demand B 4 IP address for networking to LAN 7 16 J Java Runtime Environment 5 8 L labeling chemistry 2 19 LAN See networking 7 14 laser controlling See manual control commands discussed 2 29 hazard warning 2 29 Leak detected 9 8 limited warranty D 1 LIMS database importing plate records from 6 19 to 6 22 option 7 15 Link to dialog box 6 33 6 35 linking a plate 3 41 loading standards 4 20 log spatial calibration log files 4 5 log Spectral calibration log files 4 37 log file viewing forarun 5 55 logging on checking username 7 16 loss of resolution 9 6 low signal strength 9 5 M Macintosh computer using to view data 7 15 maintenance task lists 8 4 manual control comma
195. n on page 2 21 Appendix C Part Numbers What the Instrument Does Types of Analysis The 3100 Genetic Analyzer performs two kinds of analysis DNA Analysis Purpose Sequencing analysis Separates a mixture of DNA fragments according to their lengths Provides a profile of the separation Determines the order of the four deoxyribonucleotide bases Fragment analysis Separates a mixture of DNA fragments according to their lengths Provides a profile of the separation Determines the length of each fragment in basepairs Estimates the relative concentration of each fragment in the sample System Overview 2 5 How the Instrument Works Description of a The following table describes a typical run on the 3100 instrument Typical Run Stage Description Diagram 1 Sample Preparation During sample preparation the DNA fragments in a sample are chemically labeled with fluorescent dyes The dyes facilitate the detection and identification of the DNA Typically each DNA molecule is labeled with one dye molecule but up to five dyes can be used to label the DNA sample Both the type of fluorescent labeling and the sample composition vary with the sample preparation method used Samples are prepared in 96 or 384 well plates Fluorescent LAS label nrc 2 Software Setup The operator creates a plate record and specifies the sample type and run module in
196. n the plate import table Converting the data into a plate BLOB format requires a knowledge of SQL and is a topic beyond the scope of this manual Plate BLOB Version The plate BLOB takes its version number from the header of the table used to create Number the plate BLOB This number is 1 0 for the current release of the software which is identical to the version in the tab delimited text files prepared for import into the instrument database 6 22 Working with Plate Records Section Creating Plate Files In This Section The following topics are covered in this section Topic See Page Creating a Plate File Using a Provided Template 6 24 Creating a Plate File from a New Spreadsheet 6 28 Creating a Plate File from a Custom Spreadsheet Template 6 29 Creating a Plate File from an Edited Plate Record 6 30 Definition A plate file is a tab delimited text file saved with the file name extension plt Working with Plate Records 6 23 Creating a Plate File Using a Provided Template Introduction This method uses a tab delimited text file template and Microsoft Excel to create a plate file Templates are provided with the 3100 software and are listed below In Microsoft Excel you are able to view a tab delimited text file template in a spreadsheet format without actually saving it as a spreadsheet Locating the The templates provided with the 3100 software are located in the following directory Templates T
197. n 1 week with a full buffer reservoir Short term IMPORTANT The key to a successful short term shutdown is keeping the capillary array in 1X running buffer This prevents the polymer from drying in the capillaries for more than 1 week Long term Performing a To perform a short term shutdown Short Term Shutdown Step Action 1 Fill the capillaries with fresh polymer For instructions see page 8 16 2 Push the Tray button to move the autosampler forward 3 Fill the buffer reservoir with 1X running buffer to just below the top of the reservoir 4 Fill other reservoirs with fresh deionized water 5 Secure a septa onto the reservoir and place the reservoir in position 1 on the autosampler 6 Close the instrument doors The autosampler will move to position 1 leaving the capillary tips in the buffer reservoir 7 Shut down the computer and turn off the instrument Performing a To perform a long term shutdown Long Term Shutdown 8 8 Maintenance Step Action 1 Follow the procedure on page 8 17 to remove and store the capillary array off of the instrument 2 Remove from the instrument Syringes from the upper polymer block For instructions see page 8 23 Upper polymer block For instructions see page 8 26 Lower polymer block For instructions see page 8 26 3 Remove from the autosampler Plate assemblies Reservoirs 4 Wipe the autosampler
198. n Ea sea ea ed ee aie E E A E Waele aa E 5 22 Transferring Run Modules Between Computers 0 0 00 00 0c eee eee eee 5 23 Section Working with Sequencing Analysis Modules 000 c ce aceaes 5 27 Viewing and Editing Analysis Modules for DNA Sequencing 5 28 Creating a Sequencing Analysis Module 00 0 0 eee eee eee eee 5 30 Section Working with GeneScan Analysis Modules 00 0cccceceees 5 37 Viewing and Editing Analysis Modules for GeneScan 0 00002 eee eee 5 38 Creating a GeneScan Analysis Module 0 0 0 0 cee eee 5 40 Section Working with BioLIMS 000 ccc ccc ween ene e eee enees 5 47 Setting Up BioLIMS Project Information 0 0 0 eee eee eee 5 48 Preparing a Plate for Extracting to BioLIMS 0 00 5 50 After Extracting to the BioLIMS Database 00 0 0 ee eee eee 5 55 Working with Plate Records OVELVICW i sda tee hte dette tbe deeds Geet Hoe eo bid oaa o wets dead 6 1 S ction Introductions sssssi sirane nni case se dece 10s ae eRe eee eel ea 6 3 About Creating Plate Records 0 0 ee ccc eens 6 4 About the Plate Record Fields 0 0 0 0 ccc een ee 6 5 Section Tab Delimited Text Files 00 60 c ccc cece ee cece eee eee eee eeecs 6 9 Introduction to Tab Delimited Text Files 00 0 0 0 eee eee eee eee 6 10 About Creating Tab Delimited Text Files 0 0 00 0 0 eee eee 6 11 Using Spreadsh
199. n run the software analyzes the matrices and assigns a analysis capillary status value to each capillary The matrix passes if it Exhibits four distinct fluorescence emission maxima Meets the criteria specified in the selected spectral calibration parameter text file A passed matrix must be assigned to every capillary before a sample run can be performed The software automatically replaces matrices for failed capillaries with matrices created from capillaries that passed The replacements are made from the next nearest capillary with the left side taking priority over the right side Even though the algorithm has passed a calibration matrix from a capillary it does not mean that the calibration data should necessarily be used for sample data analysis We recommend that you examine all 16 calibration matrices before electing to save and use them for sample data processing Ideally each capillary has its own passed matrix If you see a matrix that you do not want to use you can use the Override Spectral Calibration command to replace the matrix with one from a neighboring capillary Spatial and Spectral Calibrations 4 17 Performing a Spectral Calibration Using Default Processing Parameters Introduction Preparing the Equipment DNA Sequencing Sample Types for Spectral Calibration DNA Sequencing Preparing the Matrix Standard for Dye Set E Matrices 4 18 Spatial and Spectral Calibrations Use the proced
200. n spectra of the dyes in the dye set If there were no overlap in a dye set the c value would be 1 0 the lowest possible value The condition number increases with increasing peak overlap Because of slight variations in optics between instruments a range is used to define the conditionBounds As the expected range of condition numbers is different for different dye sets the conditionBounds values are different for calibration runs that use different dye sets Use the following table to select the correct conditionBound values If you are Then creating a parameter file and are intending you do not need to change the to perform a spectral calibration for either conditionBounds values The conditionBounds dye set values are listed below D with Matrix Standard Set DS 30 condition E with Matrix Standard Set DS 01 or Bounds Parameter File Value sequencing reaction MtxStd Sequencing SetE par 3 0 5 0 SeqSid Sequencing SetE par 8 0 5 0 MtxStd GeneScan SetD par 4 0 7 0 calibrating for a different dye set using the determine the appropriate conditionBounds sequenceStandard dataType value range for that dye set you do not want to set the use the supplied parameter file conditionBounds value SeqStd AnyDyeSet par or recommended the first time you use a MtxStd AnyDyeSet par new dye set other than D and E After a spectral calibration run the softwa
201. n to add the computer workstation to a LAN you should be aware of the following The person logged in as 3100User must have system administration rights on the computer workstation The computer workstation has two network interface cards Administrator For installation and upgrades to the 3100 software the person logged in as 3100User Privileges must be a member of the Administrators group Network Interface The computer workstation has two network interface cards These are Cards The card on the motherboard which is connected to the instrument The card installed in an expansion slot in the system unit which can be used to connect to the network This card requires that drivers be installed IMPORTANT Use only the network interface card in the expansion slot to connect to the LAN The network interface card on the motherboard is reserved for the Ethernet connection to the instrument IP Address Your network s system administrator must provide you with an IP address for networking to the LAN This is not the same as the Internet Protocol IP address already being used to connect the computer workstation to the instrument IMPORTANT Do not modify the given IP address Windows NT User IMPORTANT Do not change the default Windows NT logon user name from 3100User This Name will break the connection with the 3100 Data Collection software and make the software inoperable To see the Windows NT logon user name S
202. nance 8 25 Removing the Polymer Blocks Removing the Upper To remove the upper polymer block Polymer Block Step Action 1 Remove the syringe guard 2 Remove the syringes as described on page 8 21 3 Disconnect the capillary array from the polymer block a Press the Tray button b Open the instrument oven and detection block doors c Loosen the capillary array nut d Pull out the polymer block part way e Remove the detection cell from the detection block f Remove the capillary array sleeve from the polymer block g If the capillary array is to be reused store it as described on page 8 17 4 Disconnect the lower polymer block by unscrewing the polymer block tube fitting on the upper polymer block s under right side 5 Grasp the upper polymer block with two hands and pull it straight out 6 The upper polymer block rides on two steel shafts and slides out easily after a spring moves past a check point Removing the Lower To remove the lower polymer block Polymer Block 8 26 Maintenance Step Action 1 Remove the anode reservoir and properly dispose of the buffer 2 Grasp the lower polymer block and pull it straight out 3 Disconnect the polymer block tube fitting Cleaning the Polymer Blocks When to Clean the Clean the upper and lower polymer blocks Polymer Blocks Before replacing the polymer on the instrument When the polymer has been on the instrumen
203. ncing or fragment analysis Do not change this value writeDummyDyes Integers from 0 to 9 0 Allows for the use of an 4 46 additional dye Do not change this value numSpectralBins Integer from 2 to 50 20 Indicates the number of 4 46 pixel bins on the CCD Do not change this value startptOffset Integer from 0 to 5000 200 Use only with the 4 48 sequenceStandard dataType maxScansAnalyzed Integers greater than 6000 for Use only with the 4 49 or equal to 100 SeqStd AnyDyeSet par sequenceStandard dataType startptRange Integer for both the matrixStandard none Use only with the 4 49 minimum and sagienesiandard sequenceStandard i mini i taType maximum minimum 800 500 dataType maximum minRankQ Any number from 0 5 Indicates spectral data 4 50 0 0 to 1 0 purity threshold Use for matrixStandard only 4 40 Spatial and Spectral Calibrations dataType Parameter Types of Algorithm Selecting the Type Pass Fail Stringency Parameters There are two types of spectral calibration that correspond to two types of algorithm They are matrixStandard assumes one peak detected per dye sequenceStandard assumes multiple peaks detected per dye By selecting the type of algorithm you are deciding whether to perform a calibration run using one of the matrix standard sets or using other dye sets about which the 3100 Data Collection software has no prior information The following table describes when to use each type of calibra
204. nd Linking Plate Records In This Section The following topics are covered in this section Topic See Page About Importing Tab Delimited Text Files and Linking Plate Records 6 32 Simultaneously Importing and Linking a Plate Record 6 33 Sequentially Importing and Linking a Plate Record 6 35 Introduction This section describes how to import tab delimited text files to create plate records and how to link the plate records to their plates Working with Plate Records 6 31 About Importing Tab Delimited Text Files and Linking Plate Records Introduction To create and link a plate record by importing a plate file into the instrument database you must Import the data Place the plates Link the plates Options In general the options for importing placing and linking are as follows Simultaneously import the file and link the plate record to its plate This is the faster option and is highly recommended for importing data for 384 well plates but you can only import and link one plate record at a time Directions for this procedure start on page 6 33 Sequentially import one or more files and then link the plate record s to their plates Directions for these two procedures start on page 6 35 The options are summarized in the diagram below Create plt files Simultaneously import and link Sequentially import and link Place plates now or later Place plates
205. nding my_plate_record GS Place a plate into plate position B Plate Name Application Processed Plate Records Plate Name Application Wells Status Click anywhere on the plate position indicator Click the plate record Performing a Run 3 41 3 42 Performing a Run To link a plate to a plate record continued Step Action 3 Verify that the plate has been linked Once the plate has been linked the Run Instrument button on the toolbar is enabled meaning that the instrument is ready to run Plate position indicator for the linked plate becomes green Plate record moves from the Pending Plate Records table to the Linked Plate Records table Run Instrument Plate position button is enabled indicator is green E 3100 Data Collection Software File View Instrument Tools Service Help ele gt ll ulm ale Plate View Run View Status view Array View Capillary View Pending Plate Records Place a plate into plate position B Linked Plate Records Plate Name Application Wells Status 96 Ajmy_plate_record GS pending B Plate record is in the Linked Plate Records table Repeat steps 1 3 to link a second plate if applicable To link a plate to a plate record continued Step Action 5 Click the Run View tab to view the run schedule For more information about th
206. nds 5 16 manual set 1 4 C 3 mapping capillary to CCD pixel A 4 matrices for spectral calibration 4 36 matrix standards preparing for fragment analysis 4 19 preparing for sequencing 4 18 maxScansAnalyzed parameter 4 40 4 49 mcl spectral calibration files 4 36 method files importing 7 10 Microsoft Excel creating plate records 6 12 6 24 to 6 27 middleware See Orbix Desktop migration time too fast or too slow 9 7 minQ parameter 4 40 to 4 43 minRankQ parameter 4 50 mob files See mobility files mobility files A 8 directory path A 8 provided 6 6 selecting 3 38 modexp run module files 5 23 5 25 moving the instrument 8 6 MSDS 1 6 multicomponenting A 6 N networking 7 14 to 7 17 New Method Import utility 7 10 New Method Import utility overview 5 7 No candidate spectral files found 9 4 numDyes parameter 4 46 numspectralbins parameter 4 46 O Oracle database See also BioLIMS database See instrument database Orbix Desktop 5 8 OrbixWeb Daemon creating a shortcut 3 14 OrbixWeb software 5 7 oven controlling See manual control commands overriding spatial calibration file 4 12 spectral calibration profiles 4 28 Index 3 P parameters spectral calibration 4 40 partitions computer hard drive 2 14 parts list C 1 to C 3 password 3 12 pausing arun 3 47 pdf portable document format files 5 8 peaks troubleshooting 9 7 Persistence Object Layer 5 8 pixel grouping in CCD camera A 3 plate file creating 6 23 to 6 30
207. ner Mass Spectrometers tsupport appliedbiosystems com Applied Biosystems MDS Sciex api3 support sciex com Chemiluminescence Tropix tropix appliedbiosystems com 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 Technical Support B 1 To Contact Technical In North America Support by To contact Applied Biosystems Technical Support use the telephone or fax numbers Telephone or Fax given below To open a service call for other support needs or in case of an B 2 Technical Support 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
208. nformation on filling out a spreadsheet see page 3 32 for GeneScan analysis and page 3 37 for DNA sequencing Click OK You will receive a Please wait message before the software returns to the 3100 Data Collection software window Plate Editor Eg Please Wait Software 5 51 To specify a BioLIMS project in the plate s sample sheet continued Step Action 6 If it is not already selected select the Plate View tab 43100 Data Collection Software Version 1 0 SEQ_SPEC_BS 7 Make sure your plate is listed under Pending Plate Records 8 Highlight your plate and continue with Setting BioLIMS Preferences on page 5 52 Setting BioLIMS To set BioLIMS preferences for the plate Preferences Step Action 1 From the View menu select Preferences The Setting Preferences window appears Setting Preferences demo_3100 5 52 Software To set BioLIMS preferences for the plate continued Step Action 2 Select the Data Analysis tab Setting Preferences x Data Cilectinn Data Analysis p AutoAnalysis 7 AutoAnalysis On DioLIME V Enable BioLIMS User Name pimig2c Password freee Database Name Pimig2c Serve Name fon Sample Fie Name Prefix Format Samate Name z Rur ID x fenone gt z xi Cansel 3 If you want to send Then raw d
209. nsion and 14 pixels in the spectral dimension are combined to form a bin This process is called binning Binning increases the signal without adding noise For each capillary 20 adjoining spectral bins are read creating a full spectrum profile for the dye The 20 bin data can be viewed in the spectral calibration profiles see page 4 25 and the spectral calibration matrix files see page 4 36 The 20 bin data is converted using the spectral matrix into intensity values for the four or five dyes This process is called multicomponenting Using full spectrum data reduces the amount of noise in the multicomponented data 123 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Bins 14 pixels G R2002 Data Flow A 3 Incident Fluorescence Fluorescence Pattern Capillary Mapping A 4 Data Flow The fluorescence from the dye labeled DNA molecules passing through the detection window of all 16 capillaries simultaneously falls onto the CCD in the pattern illustrated below 200nm ng pay ou Aseyjidea OOOO sxe jeneds sjexid OSZ CCD GR1998 512 pixels Spectral axis The collected emission spectrum ranges from 500 nm blue to 700 nm red Note Dyes that emit green fluorescence which is towards the shorter wavelength end of this spectrum are referred to as blue dyes because they emit on the blue side of the red dyes which emit true red fluorescence The position of the fluorescence o
210. nt Doors Light switch Tray button Reset button Status lights On Off button Description Part Function Light switch Switches on and off the interior lights On Off button Switches on and off the instrument Reset button Resets all of the electronics on the instrument including the firmware and the calibration file IMPORTANT Use this button only as a last resort when the instrument is not responding See page 8 7 for procedure Tray button Brings the autosampler to the forward position Note This button works only when the instrument and oven doors are closed Status lights Indicates the status of the instrument as follows Light Appearance Instrument Status All off Power off Yellow solid Loading firmware Yellow blinking Loading calibration file Initializing subsystems Green solid Ready for use Green blinking Running Red blinking Error 2 10 System Overview Front View with Doors Open Diagram The following diagram shows inside the instrument s doors Description Upper polymer Polymer reserve syringe Array fill syringe Oven Detection cell Capillary array Buffer and water reservoirs Autosampler L Lower polymer block Anode buffer reservoir Part Function Anode buffer reservoir Contains 9 mL of 1X running buffer Buffer and water reservoirs four Co
211. ntains 16 mL of 1X running buffer or water Autosampler Holds the sample plates and reservoirs and moves to align the samples water or buffer with the capillaries Capillary array Enable the separation of the fluorescently labeled DNA fragments by electrophoresis It is a replaceable unit composed of 16 silica capillaries Detection cell Holds the capillaries in place for laser detection Lower polymer block Contains the anode electrode The anode buffer reservoir connects to this block Oven Maintains uniform capillary array temperature Polymer reserve syringe Contains and dispenses the polymer that fills the polymer blocks and the array fill syringe A 5 mL syringe Array fill syringe Contains and dispenses the polymer under high pressure to fill the capillaries A 250 pL syringe Upper polymer block Connects the two syringes and the detection end of the capillary array System Overview 2 11 Back View Diagram The following diagram shows the back of the instrument Chassis fan Laser fan Ethernet outlet Power cord Air filter holder Air inlet vents Description part Function Air filter holder Holds the filter that cleans the air entering the instrument Air inlet vents Allows air into instrument IMPORTANT To ensure adequate air flow do not place paper under the instrument Ethernet outlet Pro
212. ntinue on to Viewing Successful Results and Saving the Data below failed an error message box appears providing some information about the reason for the failure E Perform Spatial Calibration x Spatial calibration progress Spatial calibration failed Bad spacing I Fill capillaries Details _ ok _ Cancel a Click Details to view the Spatial Calibration Profile window b Do one of the following Click Cancel and then click Start to repeat the calibration Take corrective action as outlined on page 4 9 Spatial and Spectral Calibrations 4 7 Viewing Successful To view the spatial calibration results and save the data Results and Saving the Data Step Action 1 Evaluate the spatial calibration profile Note For information about the profile see Evaluating a Spatial Calibration Profile on page 4 11 Spatial Calibration Profile 20 40 60 80 100 120 140 160 180 200 220 240 260 Intensity vs Pixel Number Capillary Number 1 2 3 4 5 6 gL ey fa eee Ae i f 7 8 g 2 ATAN Capillary Position fiz 27 2 68 73 488 fos 119 134 fias 165 180 figs 210 225 241 cae When you are finished click OK to close the Spatial Calibration Profile box If the spatial calibration profile is Then satisfactory Continue on to step 3 unsatisfactory a Click Cancel to close the Details box and then click Start to repeat the calibration or b
213. ntly For instructions see Spectral Calibration on page 4 15 Placing the Plate onto the Autosampler Placing the Plate To place the plate onto the autosampler onto the Autosampler Step Action 1 Place the plate assembly on the autosampler as shown below Note There is only one orientation for the plate with the notched end of the plate base away from you IMPORTANT Ensure the plate assembly fits flat in the autosampler Failure to do so may allow the capillary tips to lift the plate assembly off of the autosampler 2 When the plate is correctly positioned the plate position indicator on the Plate View page changes from gray to yellow Check to ensure this has happened IDF ss Plate placed in position A r plate position B 4 E No plate in position B T at m EE 3 Close the instrument doors Note Closing the doors returns the autosampler to the home position placing the tips of the capillaries in buffer Performing a Run 3 25 3 26 Performing a Run Section Setting Up the Software In This Section The following topics are covered in this section Topic See Page Setting Software Preferences 3 28 About Plate Records 3 30 Creating a Plate Record for GeneScan Analysis 3 31 Creating a Plate Record for DNA Sequencing Analysis 3 36 Linking and Unlinking a Plate 3 41 Performing a Run 3 27 Setting Software Introduction Viewing the Setting P
214. nto the CCD is not fixed The fluorescence will fall in a slightly different place in the spectral axis if you move the detection end header of the capillary array This means that you must run a new spatial calibration each time you re install or replace the capillary array Note A new spectral calibration is not required when you change the capillary array because the spectral position is a function of the laser position which does not change when the capillary array is replaced For the fluorescence data collected from DNA samples to be meaningful each bin must be mapped to the fluorescence pattern falling onto the CCD This mapping is achieved by performing a spatial calibration see page 4 3 followed by a spectral calibration see page 4 15 The calibrations generate a spatial map and a spectral matrix which are used in the processing of sample data Frame Data About Frame Data How It Works Raw Frame Data Sets Processed Frame Data Sets Re Extracting Processed Frame Data Deleting Processed Frame Data Frame data is a two dimensional spectral dimension vs spatial dimension matrix that represents a single frame of data captured by the CCD camera When light falls on the CCD camera pixels that are struck by the light become electrically charged in proportion to the intensity of the light The information carried by the fluorescence is therefore converted into electronic information The 3100 Data Collection softw
215. ntration of dye in the 3100 Data Collection software user interface Using the red green blue RGB color system Using the hue saturation value HSV color system The RGB system uses the three primary colors red green and blue in various proportions to create the other colors To change the displayed dye color using the RGB system Step Action 1 From the Instrument menu point to Data Acquisition and select Set Color 2 Within the Edit Dye Display Information dialog box click the Color box of the color you want to change The Set Color dialog box is displayed Click the RGB tab if it is not already selected ey Set Color x Red AELE AUMIA A SLAS 0 85 170 255 Green PST ISS ES E ISS SSL A C SELES SIEN VEL DALIN 0 85 170 255 eE SER A S a AANI S LSL G 0 85 170 255 OK Cancel Reset Move the sliders to mix the three colors until you produce the display color that you want To Click Incorporate the change OK Ignore the change Cancel Revert to the default colors Reset Close the Edit Dye Display Information dialog box Software 5 13 Changing the The Hue Saturation Value HSV system describes colors in terms of three Display Colors Using properties the HSV System Property Description Hue The wavelength composition of the color e g blue S
216. o not override original spectral calibration parameter files Spatial and Spectral Calibrations 4 39 Spectral Calibration Parameters About Spectral Calibration Spectral calibration algorithms use variables known as spectral calibration parameters Each parameter has one or more options that you can set Parameters How Parameters Are Stored Parameters List Spectral calibration parameters are stored in text files called spectral calibration parameters files These text files have a par extension and they are editable The spectral calibration parameters that have values you can define are listed below Parameter Name Allowed Values Default Value Comments See Page dataType matrixStandard Selects the type of 4 41 algorithm to use Use the t segupncseiandard matrixStandard algorithm except when using a sequencing sample to generate a matrix minQ Any number from 0 95 for all parameter Use with both dataType 4 42 0 0 to 1 0 files except parameters SeqStd AnyDyeSet par which is 0 92 conditionBounds Any number from 1 0 3 0 5 0 for Use with both dataType 4 44 for both minimum sequencing analysis parameters No allowable c value 4 0 7 0 for fragment conditionBound numbers maximum allowable are used for the Any Dye analysis c value see default as Set par files an example numDyes Integers from 2 to 7 4 Indicates number of dyes 4 46 used in seque
217. odule used for the sample IMPORTANT This cell or token must always be next to last from left to right IMPORTANT The name of the run module must be typed correctly If the name is typed incorrectly the plate will be imported but the run module will not be entered in the plate record 6 16 Working with Plate Records Cell or Token Function Analysis Module Specifies the analysis module used to run the sample GeneScan analysis modules have the file format file name gsp IMPORTANT This cell or token must always be last from left to right IMPORTANT You must always select an analysis module if you want the data to be extracted and analyzed IMPORTANT The name of the analysis module must be typed correctly If the name is typed incorrectly the plate will be imported but the analysis module will not be entered in the plate record Note The Dye Set Comment and Project Name tokens can be presented in any order Sample Data The sample data begins on row 4 of a spreadsheet A 96 well plate for sequencing analysis contains up to 96 rows of sample data one row for each sample and therefore each well A 96 well plate for fragment analysis contains a multiple of 96 rows since one well can contain several dye channels each labeled with a differently colored dye Working with Plate Records 6 17 Running the Same Sample with Different Conditions Sample Run Options How to Set Up Multiple Runs You can
218. oint to Applied Biosystems and select OrbixWeb Daemon Note To create a shortcut a Navigate to orbixd exe in the following directory D dbtools iona orbixweb3 2 bin b Right click the file c Click Create Shortcut This creates a shortcut named Shortcut to orbixd exe d Drag the shortcut to the desktop IMPORTANT OrbixWeb Daemon must be started before the 3100 Data Collection software can run Starting the Data To start the Data Collection software Collection Software 3 14 Performing a Run Step Action 1 From the Start menu point to Applied Biosystems and select 3100 Data Collection Note To create a shortcut a Navigate to 3100Collection bat in the following directory D AppliedBio abi 3100 Bin b Right click the file c Click Create Shortcut This creates a shortcut named Shortcut to 3100 Collection Software d Drag the shortcut to the desktop The 3100 Data Collection software opens and the following window is displayed File View Instrument Tools Service Help Hele Mle cle Plate View Run View Status View Array View Capillary View Pending Plate Records Plate Name Application Wells Status A B Place a plate into Place a plate into plate position 4 plate position B Linked Plate Records Plate Name Application Wells Status A B Processed Plate Records Plate Name Application Wells Status
219. oltage connection to the capillary electrodes Available Lengths Length cm Use for 36 Fragment analysis Rapid DNA sequencing 50 Standard DNA sequencing For More The following table lists capillary array topics covered elsewhere in this manual Information Topic See Page Changing a capillary array 8 15 Storing a capillary array 8 17 Part numbers for capillary arrays C 1 2 24 System Overview Electrophoresis Overview Samples are electrophoretically separated as they travel through the polymer in the Temperature capillary array Electrophoresis temperature is controlled by housing the capillary array in a sealed oven The following table lists the normal electrophoresis temperature for each type of run Temperature Type of Run C Standard DNA sequencing 50 Rapid DNA sequencing 55 Standard fragment analysis 60 System Overview 2 25 Electrophoresis Circuit Overview A high voltage electrical circuit facilitates the electrophoresis of DNA fragments The electrical charge is conducted through the circuit by DNA and buffer ions in the polymer Buffer ions in the buffer Electrons in the electrical wires and electrodes Diagram The electrophoresis circuit is shown below Capillaries containing polymer Loading bar cathode Electrode anode Description During electrophoresis a high voltage is applied betw
220. olymer Blocks oes coiii eaii cece eee 8 27 Removing Air Bubbles from the Upper Polymer Block 00000000 8 28 Section Autosampler Calibration 00 c ccc ccc cece cent n eee eeees 8 29 Troubleshooting OVERVIEW resene ran Aires Latins hbk ead Meee eG ete 9 1 Instrument Startup e s 4 Ses te bee ai da aioe an dae die adie 9 2 Spatial Calibrations coat eae ae eis Stas Sac eed as eh oes 9 3 Spectral Calibrations vs 2c s ds hea ea iy n eae Ae Ree dws Sega 9 4 Run Performante sessies i ealiete diese E ENEE EE E hatha dee eeelaa akan EE 9 5 Data Flow OVERVICW 3 ote ete A oa a eg ie eo oad eo Se ee as A 1 About Data FLOW risie eeii eai ee R RA E EE eet te ETE A 2 Organization of the CCD iee niii eens be eae aS wares E wae se als wR Sty A 3 Incident Fluorescence sicreti eia e e a A a a SEa A E een e a ees A 4 Prame Data sais poser neice tie oh E OE E E klk eld E EEE ee wine EP LES A 5 Multicomponenting 0 inr a ence nena A 6 Configuring Data FLOW ici ccd ie cen a Re Pees Ge eee pe Dee S A 7 Mobility Shift Correction for DNA Sequencing 0 0 eee eee A 8 Technical Support Technical Support oss coped ede ede pte 4 Oded Gee Oe ee ede ie B 1 Part Numbers Applied Biosystems Part Numbers 0 0 cece ce eee ees C 1 Limited Warranty Statement Introduction and Safety Overview In This Chapter The following topics are covered in this chapter Topic See Page ABI PRISM 31
221. on Plates 4305505 Reservoir septa 4315932 Description Part Number 96 well plate retainer 4317241 96 well plate base AB 4317237 384 well plate retainer 4317240 384 well plate base 4317236 Reservoirs for buffer water and waste 628 0163 Glass syringe 5 0 mL polymer reserve 628 3731 Glass syringe 250 pL array fill 4304470 Syringe O rings 221102 Syringe ferrule 005401 Anode buffer reservoir jar 005402 Upper polymer block drip tray 628 3720 Lower polymer block drip tray 628 3088 Autosampler drip tray 628 3059 Polymer block tubing assembly 628 3732 Array calibration ruler 628 3214 Array comb holders 628 3403 Array ferrule sleeves 628 0165 Array ferrule knob 628 3730 Reference Materials Description Part Number ABI PRISM 3100 Genetic Analyzer User s Manual 4315834 ABI PRISM 3100 Genetic Analyzer Sequencing Chemistry Guide v 3 6 4315831 ABI PRISM GeneScan Analysis v 3 6 NT User s Manual 4308923 ABI PRISM 3100 Genetic Analyzer Quick Start Guide for Fragment Analysis 4315832 ABI Prism Sequencing Analysis Software v 3 6 NT User s Manual with 4308924 v 3 6 1 update ABI PRISM 3100 Genetic Analyzer Quick Start Guide for Sequencing 4315833 ABI PRISM 3100 Genetic Analyzer Operator Training CD 432559 Part Numbers C 3 Limited Warranty Statement Applied Biosystems Applied Biosystems warrants to the customer that for a period
222. on and Safety techniques User bulletins related to the use of this instrument will be mailed to you We recommend storing the bulletins in this manual A tab labeled User Bulletins has been included for this purpose Safety Documentation User Attention Words Chemical Hazard Warning Chemical Waste Hazard Warning Site Preparation and Safety Guide Five user attention words appear in the text of all Applied Biosystems user documentation Each word implies a particular level of observation or action as described below Note Calls attention to useful information IMPORTANT Indicates information that is necessary for proper instrument operation 7 Neo NU gale Indicates a potentially hazardous situation which if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices PNG Mel Indicates a potentially hazardous situation which if not avoided could result in death or serious injury WN Wella Indicates an imminently hazardous situation which if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations 7 Eafe CHEMICAL HAZARD Some of the chemicals used with Applied Biosystems instruments and protocols are potentially hazardous and can cause injury illness or death Read and understand the material safety data sheets MSDSs provided by the chemical manufacturer before you store handle or work wi
223. ows normal body and head posture This height depends upon the physical proportions of the user Adjust vision factors to optimize comfort and efficiency by Adjusting screen variables such as brightness contrast and color to suit personal preferences and ambient lighting Positioning the screen to minimize reflections from ambient light sources Positioning the screen at a distance that takes into account user variables such as nearsightedness farsightedness astigmatism and the effects of corrective lenses When considering the user s distance from the screen the following are useful guidelines The distance from the user s eyes to the viewing screen should be approximately the same as the distance from the user s eyes to the keyboard For most people the reading distance that is the most comfortable is approximately 20 inches The workstation surface should have a minimum depth of 36 inches to accommodate distance adjustment Adjust the screen angle to minimize reflection and glare and avoid highly reflective surfaces for the workstation Use a well designed copy holder adjustable horizontally and vertically that allows referenced hard copy material to be placed at the same viewing distance as the screen and keyboard Keep wires and cables out of the way of users and passersby Choose a workstation that has a surface large enough for other tasks and that provides sufficient l
224. p a GS400HDAnalysis asp GS4000rd2Analysis asp a GS5004nalysis gsp Fle name sve Save as type analysis Params File GSP z Cancel In the File name text box type a file name for the analysis parameter file 6 Click Save The browser dialog box closes and the file is saved to the correct directory location for the Auto Extractor to read In the newly created Filename szs dialog box click the Close button Software 5 45 5 46 Software Section Working with BioLIMS In This Section The following topics are covered in this section Topic See Page Setting Up BioLIMS Project Information 5 48 Preparing a Plate for Extracting to BioLIMS 5 50 After Extracting to the BioLIMS Database 5 55 Software 5 47 Setting Up BioLIMS Project Information Introduction In order to extract sample files into the BioLIMS database you must first set up information for your BioLIMS project s in the 3100 Data Collection software BioLIMS projects are the 3100 Data Collection software equivalent of collections in BioLIMS When samples are extracted into the BioLIMS database they are added to the specified BioLIMS project Setting Up BioLIMS To set up the BioLIMS project information Project Information Step Action 1 From the View menu select BioLIMS Project Info The Setting Up BioLIMS Project Information window appears Setting Up BioLIMS Project In
225. partitioned to Partitions create the following logical drives Physical Hard Size Disk Drive GB Function 1 C 2 System operating files Note You may also install your own programs on this drive D 7 Reserved for the 3100 software and the analysis software 2 E 9 Reserved for the instrument database 2 14 System Overview Software Overview The software installed on your computer workstation consists of Data Collection software that controls monitors and collects data from the instrument An analysis application that either analyzes raw sequencing data or sizes and quantifies DNA fragments Software that automatically extracts and analyzes the data A database Utilities that enable you to manage the files in the database 5 gt gt gt A toolkit that enables you to develop customized applications For a complete list of the ABI PRISM 3100 software installed on your computer see page 5 5 Note Other programs are available from Applied Biosystems to align sequences identify previously unsequenced regions archive data identify patterns of heredity and perform other kinds of data manipulation See your Applied Biosystems representative Note To avoid software conflicts it is recommended that you do not install third party software onto the computer attached to the 3100 instrument System Overview 2 15 2 16 System Overview Section Chemistry Overview In This Section T
226. patial Calibration Data scl x Cancel 2 Select the spatial calibration file that you want to use 4 12 Spatial and Spectral Calibrations To override the current spatial calibration map continued Step Action 3 Click OK This opens the Spatial Calibration Profile from box with the data for the selected file displayed 4 Click OK This data is now the current spatial calibration map and the Spatial Calibration Profile from box closes Spatial and Spectral Calibrations 4 13 4 14 Spatial and Spectral Calibrations Section Spectral Calibration In This Section The following topics are covered in this section Topic See Page About Spectral Calibrations 4 16 Performing a Spectral Calibration Using Default Processing Parameters 4 18 Displaying a Spectral Calibration Profile 4 25 Overriding a Spectral Calibration Profile 4 28 Spatial and Spectral Calibrations 4 15 About Spectral Calibrations Introduction A spectral calibration creates a matrix that corrects for the overlapping of fluorescence emission spectra of the dyes When to Calibrate You must perform a spectral calibration Whenever you use a new dye set on the instrument After the laser or CCD camera has been realigned by a service engineer If you begin to see a decrease in spectral separation pull up and or pull down peaks Procedure Overview The procedures for
227. pens the Select Dye and Analysis Parameters dialog box f Select Dye and Analysis Parameters GS3504nalysis asp CaaS Coa From the Dye drop down list select the dye that was used to label the size standard DNA fragments Software 5 41 5 42 Software To create a size standard file continued Step Action 9 From the Analysis Parameters drop down list select lt Analysis Parameters gt This references the current analysis parameter settings rather than an analysis parameter file i Select Dye and Analysis Parameters Select the Dye and Analysis Parameters to use in creating the New Size Standard Definition 10 Click OK This opens the untitled dialog box untitled x ei 1400 2800 4200 5600 7000 8400 9800 11200 Template File Info File Eb10d2 3 fsa Run Date Tue Feb 02 1999 Run Time 10 41 53 AM Dye R Parameters lt Analysis Parameters 11 12 From the File menu select Save This opens the Save this document as browser dialog box To create a size standard file continued Step Action 13 Navigate to the SizeStandards folder located in the following directory D AppliedBio abi Shared Analysis SizeCaller SizeStandards The folder contains a number of size standard szs files Save this document as RARS Save in Si SizeStandards x El c File name MEE Save Save as type Size Stan
228. performing a spectral calibration for fragment analysis or DNA sequencing are basically the same Performing a spectral calibration is similar to performing a sample run except that matrix calibration standards are run in place of samples and a spectral calibration method file is used in place of a run module Parts of the Spectral Calibration Procedure Part Description Software setup You will begin the procedure by preparing the instrument and calibration standards Next you will set up the run using the Plate View page of the 3100 Data Collection software During the software setup you will be prompted to select a specific Spectral run module determines the run conditions for each array type Dye set configures the software for the dye set you are using Spectral parameter file selects the type of algorithm you want to use to process the data matrixStandard or sequenceStandard Standards calibration During the calibration dye labeled DNA standards are electrophoresed and the fluorescence data is collected and stored as temporary files The matrix making software analyzes this data and creates a spectral calibration matrix which is used for sample data Application of this matrix to the raw data is called multicomponenting see page A 6 4 16 Spatial and Spectral Calibrations Parts of the Spectral Calibration Procedure continued Part Description Data After the calibratio
229. pillaries by 4 dyes or 5 dyes Note For more information on multicomponenting see Multicomponenting on page A 6 The processed frame data sets are stored in the instrument database If a sample file created from the processed frame data is deleted or damaged the processed frame data stored in the instrument database can be re extracted to make a replacement sample file For more information see Re Extracting Processed Frame Data The Re Extraction Utility on page 7 6 For information on deleting processed frame data see Deleting Processed Frame Data The Cleanup Database Utility on page 7 8 Data Flow A 5 Multicomponenting Background Definition Function Chemometric Method Confidence Limits A 6 Data Flow Each dye in a dye set has a unique fluorescence emission but the emission spectra are sufficiently broad for there to be overlap between them This spectral overlap is corrected for during the chemometric processing described below Multicomponenting is the process of using a spectral calibration matrix to correct for the overlapping fluorescence emission spectra of the dyes in a dye set It is carried out by the 3100 Data Collection software Multicomponenting reduces the data to four or five data points one per dye in the spectral dimension During a spectral calibration all dyes in a dye set are run in each capillary and their fluorescence emission profiles are collected A mathematical de
230. ponented Capillary color data electropherogram display for display selected capillary PED 00 Data Collection Software Version 1 0 x Fie View Instryma nt Toos Service Helg A le loe olel Plate View Run iew Status View Array View cepitary View E j i 69 3 696 70 l Intensity vs Run Time mins 540 3780 pes 2980 180 SSS 1980 1080 180 Jig p 2 4 6 8 10 12 14 16 18 20 4 6 8 10 12 14 16 Capillary Number 14 Intensity vs Spectral Bin Intensity vs Ca gt Number Collecting cata Selected capillary to be Fluorescence emission spectrum Total intensity of collected displayed in the center plot for the selected capillary signal for each capillary Performing a Run 3 53 Capillary Color Data Display Capillary Display Fluorescence Emission Spectrum Electropherogram Display 3 54 Performing a Run Each cell in the capillary color data display represents one capillary The status of that capillary is indicated by the color of the cell as described in the following table Cell Color Status of the Capillary Comment Green Operational Yellow Questionable This capillary did not pass the spectral calibration and has been assigned the spectral profile of its nearest passing neighbor There may be a problem with data collected from this capillary Red Nonoperational All capillaries will have a red cell until a spatial calibration is
231. positions are A H and 1 12 For 384 well plates the well positions are A P and 1 24 IMPORTANT This cell or token must always be first from left to right Sample Name Identifies the sample The sample name you assign must not exceed 63 characters IMPORTANT When naming the sample you can use letters numbers and the following punctuation only _ Do not use spaces IMPORTANT You must limit the sample name to 63 characters 59 character filename and 4 character extension If you do the name may be truncated when exported from the 3100 Data Collection software IMPORTANT This cell or token must always be second from left to right Dye Set Specifies the dye set used to label the DNA This name must match the names stored in the instrument database Note If you select the wrong dye set you will have to re run your samples You cannot correct this problem after the run Mobility File Specifies the dye mobility file used for processing the fluorescence data Note This is identical to the dye set primer file used with previous ABI Prism genetic analyzers Comment Allows you to enter comments about the sample Project Name Designates the BioLIMS Genetic Information Management System Collection name into which this sample will be added Run Module Specifies the run module used for the sample IMPORTANT This cell or token must always be next to last from left to right IMP
232. pta are firmly seated and flat Before each run Ensure the plate assemblies were put together properly Before each run 3 9 IMPORTANT The holes in the plate retainer must align with the holes in the septa or the capillary tips will be damaged Ensure the plate assemblies are positioned on the plate Before each run deck properly Plates should sit snugly on the deck IMPORTANT Never use warped plates Replenish the water and 1X running buffer reservoirs on Daily or before 3 22 the instrument each run Check for bubbles in the polymer block and polymer block Daily or before 8 28 channels and remove each run Check the loading end header to ensure the capillary tips Daily or before are not crushed or damaged each run Check the level of polymer in the polymer reserve syringe Daily or before to ensure there is at least 1 mL each run Check the polymer block to ensure it fits securely on the Daily _ instrument Clean the instrument surfaces Daily _ Check for dried polymer around the polymer block and Daily clean as necessary Check for leaks around the syringes and screw nut Daily Check data base space Delete plate records from the Daily 7 5 instrument database and archive sample files Weekly Tasks Perform these tasks at least once per week Maintenance Task Frequency See Page Clean the syringes Weekly or as 8 21 needed Clean the water and buffer reservoirs with warm water Weekly Clean t
233. ra 3100_Proj 3100_Proj 3100_Pra 3100_Proj 3100_Proj 3100_Pra 3100_Proj 3100_Pra 3100_Pra 3100_Proj 3100_Proj mann A StdSeqS0_BC 3100_ StdSeq50_E StdSeqS0_BC 3100_ StdSeqS0_E StdSeqS0_BC 3100_ StdSeqS0_E StdSeqS0_BC 3100_ StdSeqS0_E StdSeqS0_BC 3100_ StdSeqS0_E StdSeqS0_BC 3100_ StdSeqS0_E StdSeqS0_BC 3100_ StdSeqS0_E StdSeqS0_ BC 3100_ StdSeqS0_t StdSeq50_ BC 3100_ StdSeqS0_E StdSeq50_ BC 3100_ StdSeqS0_E StdSeq50_BC 3100_ StdSeqS0_E StdSeqS0_BC 3100_ StdSeqS0_E StdSeq50_BC 3100_ StdSeqS0_E StdSeq50_BC 3100_ StdSeq50_E StdSeqS0_BC 3100_ StdSeq50_E StdSeq50_BC 3100_ StdSeq50_E StdSeq50_BC 3100_ StdSeq50_E StdSeq50_BC 3100_ StdSeq50_E mam ra MA nann fem rA r Modify any data in the cells by clicking them and retyping To save time use the Fill Down command Select the cell containing the information that you want to copy From the Edit menu select Copy Drag the fill down handle in the bottom right corner of the cell to copy the information into adjacent cells Al B1 C1 D1 E1 F1 G1 H1 Seq96_FulSQ Well Sample Name std std std std std Dye Set E E E E E E E E E E E E E E E E E E E E E E E E E E E E Mobili DT31C DT31C DT31C DT3I1C T31C 9999999999999 aa T31 z P FuliPiate_seqwen 4 e 7 Working with Plate Records To create a plate record using a template continued Step Act
234. ral calibration run a spectral calibration log file is automatically created This file provides a list of the capillaries that passed and failed the calibration run Locating a Spectral Spectral calibration log files are stored in the following directory Calibration Log Files D AppliedBio abi 3100 DataCollection Spectral Cal Logs File Naming Spectral calibration log files have the following file name convention Convenes SpectralCal instrument name Run_instrument name_year_date_run no for day log Example File An example of a file opened in Notepad is shown below Opened in Notepad SpectralCal demo_3100 Run_demo_3100_2000 06 08_4 log Notepad File Edit Search Help Spectral calibration result for run SpectralCal demo_3166 Run_demo_3166 2666 66 68 4 for dye set E Capillary Capillary Capillary Capillary Capillary Capillary Capillary Matrix produced Matrix produced Matrix produced Matrix produced Matrix produced Matrix produced Matrix produced Capillary Matrix produced Please verify Capillary Matrix produced c 3 58 Please verify Capillary 16 Failed q 6 936 lt Low quality Used matrix from capillar Capillary 11 Matrix produced Capillary 12 Matrix produced Capillary 13 Matrix produced Please verify Please verify Please verify Please verify Please verify Please verify Please verify ananaaan punt tot ut tt Go to Go GO GO GO OO o I ow pal wi 1 2 3 4 5
235. rating 2 29 CCD Camera 2 29 System Overview 2 1 2 2 System Overview Section 3100 Instrument Overview In This Section The following topics are covered in this section Topic See Page ABI PRISM 3100 System Components 2 4 What the Instrument Does 2 5 How the Instrument Works 2 6 System Overview 2 3 ABI PRISM 3100 System Components Table of The ABI PRism 3100 Genetic Analyzer system includes the following components Components 2 4 System Overview Component For detailed information see ABI PRISM 3100 Genetic What the Instrument Does on page 2 5 Analyzer How the Instrument Works on page 2 6 Instrument Hardware Overview on page 2 9 Computer workstation with Computer Workstation on page 2 14 Microso Windows NT Managing Hard Drive and Instrument Database operating system Space on page 7 3 ABI PRISM 3100 Genetic Analyzer software Software on page 2 15 Chapter 5 Software ABI PRISM DNA Sequencing Analysis or GeneScan Analysis software ABI Prism DNA Sequencing Analysis Software v 3 6 NT User s Manual P N 4308924 ABI PRISM GeneScan Analysis Software v 3 5 NT User s Manual P N 4308923 Capillary Array Capillary Array on page 2 24 Appendix C Part Numbers Reagent consumables gt gt oe o Polymers on page 2 20 Injection Solutio
236. re then enter the part number or keyword s in the field on this page If you have The MSDS document number or the Document on Demand index number The product part number Keyword s c You can open and download a PDF using Adobe Acrobat Reader of the document by selecting it or you can choose to have the document sent to you by fax or email By automated telephone service Use To Obtain Documents on Demand under Technical Support By telephone in the United States Dial 1 800 327 3002 then press 1 By telephone from Canada To order in Dial 1 800 668 6913 and English Press 1 then 2 then 1 again French Press 2 then 2 then 1 By telephone from any other country See the specific region under To Contact Technical Support by Telephone or Fax under Technical Support For chemicals not manufactured or distributed by Applied Biosystems call the chemical manufacturer Instrument Safety Labels About Waste Disposal Before Operating the Instrument Safe and Efficient Computer Use Safety labels are located on the instrument Each safety label has three parts A signal word panel which implies a particular level of observation or action e g CAUTION or WARNING If a safety label encompasses multiple hazards the signal word corresponding to the greatest hazard is used A message panel whi
237. re computes the condition number of the spectral calibration matrix obtained for each capillary If the condition number falls outside the conditionBounds range set in the selected parameter file the capillary will fail and the matrix from a neighboring capillary will be used in its place In other words the conditionBounds parameter allows you to discard spectral matrices that do not conform to the overlap pattern expected for the dye set used IMPORTANT If you select a parameter file that is prepared for a particular dye set and then use matrix standards for a different dye set the capillaries will not pass the calibration In addition if you have a contaminant in your dyes that affects the spectra the calibrations are likely to fail 4 44 Spatial and Spectral Calibrations What the Condition Number Allows You to Compare What the Condition Number Does Not Allow You to Compare Determining Suitable conditionBounds Values for a Dye Set An ideal dye set has no spectral overlap The condition number allows you to compare how close different dye sets are to the ideal which is one The lower the condition number the smaller will be the overlap from neighboring dyes The condition number does not allow you to compare the quality of a matrix from one capillary with a matrix from another capillary collected during the same calibration run This is because of slight differences in the optics from one side of a capillary array to the ot
238. reating a Run Module 5 21 Run Module Parameters 5 22 Transferring Run Modules Between Computers 5 23 Section Working with Sequencing Analysis Modules 5 27 Viewing and Editing Analysis Modules for DNA Sequencing 5 28 Creating a Sequencing Analysis Module 5 30 Section Working with GeneScan Analysis Modules 5 37 Viewing and Editing Analysis Modules for GeneScan 5 38 Creating a GeneScan Analysis Module 5 40 Section Working with BioLIMS 5 47 Setting Up BioLIMS Project Information 5 48 Preparing a Plate for Extracting to BioLIMS 5 50 After Extracting to the BioLIMS Database 5 55 Software 5 1 5 2 Software Section About the 3100 Software In This Section The following topics are covered in this section Topic See Page ABI PRISM 3100 Genetic Analyzer Software CD ROMs 5 4 3100 Genetic Analyzer Software Suite 5 5 Types and Locations of Files 5 9 Software 5 3 ABI PRISM 3100 Genetic Analyzer Software CD ROMs Introduction The ABI PRiSM 3100 Genetic Analyzer software was installed on your computer by an Applied Biosystems service engineer This software is provided on a set of six compact discs Contents of the CDs The software CD ROMs and their contents are listed below CD Title Contents 3100 Software ABI PRISM 3100 Firmware ABI PRism 3100 Data Collection software Auto Extractor Re extraction utility Clean up database utility NewMethodImport utility Remove Run Modules utili
239. recommended protocol for salt removal Dilute salts with water Poor resolution in some capillaries Insufficient filling of array Refill array and look for cracked or broken capillaries If problem persists contact Technical Support Re inject the same samples Poor quality samples Check the sample preparation No current Poor quality water Use only high quality ultra pure water Water placed in buffer reservoir position 1 Replace with fresh 3100 1X running buffer Not enough buffer in anode reservoir Add buffer up to the fill line Buffer too dilute Prepare 3100 1X running buffer Add 3 mL 3100 10X buffer with EDTA to 27 mL deionized water Bubble s present in the polymer block and or the capillary and or PEEK tubing Pause run and inspect for the instrument for bubbles They may be hidden in the PEEK tubing Remove any bubbles according to the remove bubble procedure in the Replace Polymer Wizard 9 6 Troubleshooting Observation Possible Cause Recommended Action Elevated current Decomposed polymer Open fresh lot of polymer and store at 4 C Incorrect buffer dilution Prepare 3100 1X running buffer Add 3 mL 3100 10X buffer with EDTA to 27 mL deionized water Arcing in the gel block Check for moisture in and around the septa the reservoirs the oven and the autosampler Fluctuating current Bubbl
240. red in the spectral calibration folder as mcl files Overriding with Note To ensure the highest quality data we recommend that you do not override capillary Data from Another P ofiles Capillary To override a spectral calibration profile with data from another capillary Step Action 1 From the File menu select Override Spectral Calibration The Select the dye set to display dialog box appears E Select the dye set to display x B x OK Cancel 4 28 Spatial and Spectral Calibrations To override a spectral calibration profile with data from another capillary continued Step Action 2 From the drop down list select the appropriate Dye Set and then click OK The current spectral profile is displayed E Spectral Calibration Profile for E DA 2 3 4 5 6 8 9 10 41 12 13 14 15 16 az 18 19 20 Intensity vs Bin Number 3000 4000 5000 Intensity vs Scan Number Capillary Number 1 al Condition Number 3 39 Q Value 0 992 Data Source Override matrix from another source From capillary From data file oe a 3 Use the slider bar to select the capillary to be overridden Click From capillary Spatial and Spectral Calibrations 4 29 To override a spectral calibration profile with data from another capillary continued 5 Step Action Use the slider bar to select the source capillary to override the unsatisfactory profile
241. references Dialog Box Data Collection Page 3 28 Performing a Run Preferences The Data Collection software preferences are set during instrument installation however you can view or change these preferences in the Setting Preferences dialog box To view the Setting Preferences dialog box Step Action 1 From the View menu select Preferences or click the Preferences button on the toolbar 3100 Data Collection Software File 4GA Instrument Tools Service Help wi Toolbar v Status Bar Plate View Ctri 1 Run View Ctrl 2 Status View Ctri 3 Array View Ctrl g Capillary View Ctr 5 Instrument Status Monitor Ctr S Preferences BioLIMS Project Info The dialog box has two pages as described below Setting Preferences x Data Collection Data Analysis Instrument Instrument Name fiemo_31 00 OK Cancel The following table describes the preferences that can be set within this page Preference Description Instrument Name This field automatically populates with demo_3100 You can change it to any name e g the instrument s serial number Data Analysis Page Setting Preferences x Data Collection Data Analysis AutoAnalysis IV AutoAnalysis On BioLIMS Enable BioLIMS User Name J Password Database Name Server Name Sample File Name Prefix Format Sample Name z ven Position x fenone x OK Cance
242. rent 9 7 high signal 9 5 length of time 3 48 loss of resolution 9 6 low signal 9 5 monitoring discussed 3 49 to 3 57 no current 9 6 no signal 9 5 options 6 18 planning 3 5 scheduling 3 46 settings 3 51 setup for multiple runs 6 18 slow migration time 9 7 starting stopping skipping pausing 3 47 status 3 57 summary 3 4 viewing run schedule 3 50 run modules 5 19 to 5 25 editing or creating 5 21 exporting to file 5 24 importing and exporting 5 23 importing from file 5 25 parameters described 5 22 provided 6 6 removing from the database 7 11 selecting for GeneScan 3 33 selecting for sequencing 3 39 transferring between computers 5 23 to 5 25 viewing 5 20 Run Time run module parameter 5 22 Run View page discussed 3 50 Run Voltage run module parameter 5 22 running buffer making and storing 3 22 S safety 1 5 sample files maximum length 3 32 3 37 sample preparation 3 8 saz sequencing analysis module files creating 5 33 to 5 35 SCSI device installing 3 64 Select the run to display dialog box 3 61 seq Sequence text files option to write 5 33 sequenceStandard dataType parameters 4 47 sequencing analysis modules creating 5 30 to 5 35 analysis modules discussed 5 27 to 5 35 analysis modules selecting 3 39 dye set 3 37 matrix standards 4 18 mobility files 3 38 polymer 2 20 preparing Big Dye sample 4 19 run modules 3 39 runtimes 3 48 viewing analyzed data 3 63 Saz Sequencing analysis module file viewing
243. ring samples for sequencing and fragment analysis is given in other Applied Biosystems manuals see the table below Networking which is needed if you want to integrate the 3100 Genetic Analyzer into your existing laboratory data flow system The following table lists the sources of specific information to help you get started quickly For instruction on how to Refer to prepare DNA templates perform cycle sequencing prepare extension products ABI Prism 3100 Genetic Analyzer Sequencing Chemistry Guide P N 4315831 prepare samples for sequencing analysis perform a sequencing analysis run and view run data ABI Prism 3100 Genetic Analyzer Quick Start Guide for Sequencing P N 4315833 prepare samples for fragment analysis perform a fragment analysis run and view run data ABI Prism 3100 Genetic Analyzer Quick Start Guide for Fragment Analysis P N 4315832 calibrate this instrument Chapter 4 Spatial and Spectral Calibrations operate this instrument detailed Chapter 3 Performing a Run Introduction and Safety 1 3 Additional Documentation List of User The following table lists the complete ABI PRISM 3100 Genetic Analyzer document set Documents for users Title Contents P N Instrument ABI PRISM 3100 Genetic Analyzer Laboratory requirements for 4315835 Site Preparation and Safety Guid
244. rrection has not been applied There are two formats for viewing the raw data within the 3100 Data Collection software Inthe Array View page in much the same way that you might view the gel file output from an ABI PRism slab gel instrument Inthe Capillary View page capillary by capillary Note Only current run data can be viewed during a run you cannot view data from previous runs while the instrument is running IMPORTANT Always exit from the Array View and the Capillary View windows During a run do not leave these pages open for extended periods This may cause unrecoverable screen update problems Leave the Status View window open Viewing Raw Data To view raw data from a completed run Step Action 1 In the 3100 Data Collection software click the Array View tab to display the Array View page 2 From the Instrument menu point to Data Acquisition and choose Display Run Data This opens the Select the run to display dialog box Select the run to display x Run_demo_3100_2000 04 06_23 X OK Cancel 3 From the drop down list select the run that you want to display and click OK Note You can view any completed run that remains in the instrument database Note It may take a few moments to retrieve the data 4 Use the scroll features on the Array View page to view the data Note For information on the Array View page see page 3 53 IMPORTANT Always exit from the Array View and the Capillary View w
245. rt the other ferrule into the lower polymer block and rotate clockwise until finger tight IMPORTANT Do not overtighten 5 Install clean drip trays if they are not already on the instrument Preparing and IMPORTANT Wear gloves while performing the following procedure and any other time you Installing the handle the capillary array glass syringes septa or buffer reservoirs Syringes To prepare and install the syringes Step Action 1 Clean and inspect the syringes as instructed on page 8 21 2 Prime and fill the syringes as instructed on page 8 22 3 Install the syringes as instructed on page 8 23 3 20 Performing a Run Installing or If necessary install a capillary array using the Install Capillary Array wizard For Replacing the instructions see Installing and Removing the Capillary Array on page 8 15 Capillary Array File View Instrument WEWES Service Help Hela E i Plate Editor Module Editor Plate View l Run View Change Polymer Wizard Instrument Condition Install Capillary Array Wizard Autosampler Calibration Wizard Perform Spatial Calibration Display Spatial Calibration Display Spectral Calibration Adding or Changing Determine whether to add or change the polymer on the instrument before proceeding Polymer With instrument preparation If polymer on the instrument is Then less than 1 week old and Ensure there are no air bu
246. run module the polymer used during the previous run may not be completely replaced This could lead to an accumulation of residual large DNA fragments in the capillaries over time causing an increase in background signal Prerun Voltage The voltage applied across the capillaries during the prerun period of electrophoresis A prerun is performed to equilibrate the ionic strength across the capillary array before electrokinetic injection Prerun Time The duration of the prerun period of electrophoresis Injection The voltage applied across each capillary during electrokinetic injection Voltage The injection voltage is directly proportional to the amount of DNA injected This works in conjunction with the Injection Time to control the amount of DNA injected Injection Time The duration of electrokinetic injection This works in conjunction with the Injection Voltage to control the amount of DNA injected Run Voltage The voltage applied across each capillary during a run Data Delay The period of electrophoresis between the completion of electrokinetic Time injection and the time at which the software starts to collect data Run Time The duration of electrophoresis including the Data Delay Time The maximum run time for DNA sequencing and fragment analysis runs is 16 000 seconds Transferring Run Modules Between Computers Overview The process of transferring run modules between two instrument data
247. ry Array Wizard Autosampler Calibration Aizard Perform Spatial Calibration Display Spatial Calibration Display Spectral Calibration This opens the Module Editor dialog box In the Modules group box click either the Sequencing or GeneScan tab as appropriate 5 Module Editor x Modules Module Parameters Sequencing GeneScan Calibration Hame G2neScan36_POP4DefauttModule Template GeneSzan36_POP4 kGeneScan36_POP4CefauttModule Parameter Name Value Range 1 Run emperature 60 int 25 35 Deg C 2 Cap Fill Volume 184 int 1 830 steps 3 Pre Run Voltace 15 float 0 15 kYolte 4 Pre Run Tine 160 inl 1 1300 seu 5 Injection voltage 3 float 1 15 k olts 6 Injection Time 10 int 1 630 sec 7 Run Yoltage 15 float 15 15 kVots 8 DataDelay Time 1 int 1 3500 sec 9 Run ime 1500 Int 300 14000 sec Comments NOTE dick on the rows to see comnents for he parameters new Tee Tea Double clic lt in the value column to edit values Se e e Note The Calibration tab lists the spatial and spectral calibration modules To view the parameters for a particular module select the name of the module from the list All the parameters for the run module are displayed Note The Run Voltage value is fixed It cannot be edited 5 20 Software Editing or Creating a Run Module Editing or Creating To edit an existing run module or to create a new run module a Run Module Step
248. s Type Run Module Standard DNA sequencing StdSeq50_POP6DefaultModule Rapid DNA sequencing RapidSeq36_POP6DefaultModule Note If you select different modules for different samples the samples will be automatically grouped so that all samples with the same run module are run at the same time Runs are scheduled alphanumerically by run module name not by the order indicated in the plate record nor by sample name To see the scheduled order of the runs select the Run View tab For each sample select the appropriate Analysis Module from the drop down list IMPORTANT The AutoAnalysis preference must be selected if analysis is to take place automatically after the run see page 3 29 Analysis Module 1 BC 3100RR_SeqOttFtoft saz BC 3100_SeqOtfFtOtf saz The following table shows the analysis module to select based on your run type Run Type Analysis Module Standard DNA sequencing BC 3100_SeqOffFtOff saz Rapid DNA sequencing BC 3100RR_SeqOffFtOff saz Note You can examine the settings for each of these files using DNA Sequencing Analysis software The meanings of the settings are described in the ABI PRISM DNA Sequencing Analysis Software v 3 6 NT User s Manual If you want to run the same sample again select a second run module and a second analysis module You can run a sample in a linked plate up to five times Run Module 2 Analysis Module 2 Samples will be automatically
249. s range Runs where the red data differs significantly i e extra or missing peaks To create a size standard file Step Action 1 Review the size standard data and optimize the analysis parameters 2 Quit the 3100 Data Collection software if it is running 3 Start GeneScan Analysis software You may have a program icon for the GeneScan Analysis software on the Start menu or a shortcut icon on your desktop If not you can find the application GeneScan exe in the following directory D AppliedBio abi GeneScan Bin 5 iii Genescan exe 4 From the File menu select New This opens the Create New box ill Lx Create New ay Bi AA Project Foalysis Size Parameters Standard To create a size standard file continued Step Action 5 Click the Size Standard icon This opens a browser dialog box Select Sample File a iame ritr ane Navigate to the Extracted Runs folder in the following directory D AppliedBio abi 3100 DataExtractor ExtractedRuns Note This folder is created when Data Extractor is first used Select the GeneScan sample file with the extension fsa that you want to use as a template Select Sample File EE feo Eb10d2 2 fsa j pO Eb10d2 3 fsa l l J Eb10d2 4 fsa 0jEb110171 fsa BO Eb1148 1 tsa Eb11c6 1 fsa Eb10d2 3 fsa 3 a meee ce Click Open This o
250. safety guide is a separate document sent to all customers who have purchased an Applied Biosystems instrument Refer to the guide written for your Introduction and Safety 1 5 About MSDSs Ordering MSDSs 1 6 Introduction and Safety instrument for information on site preparation instrument safety chemical safety and waste profiles Some of the chemicals used with this instrument may be listed as hazardous by their manufacturer When hazards exist warnings are prominently displayed on the labels of all chemicals Chemical manufacturers supply a current MSDS before or with shipments of hazardous chemicals to new customers and with the first shipment of a hazardous chemical after an MSDS update MSDSs provide you with the safety information you need to store handle transport and dispose of the chemicals safely We strongly recommend that you replace the appropriate MSDS in your files each time you receive a new MSDS packaged with a hazardous chemical NVON CHEMICAL HAZARD Be sure to familiarize yourself with the MSDSs before using reagents or solvents You can order free additional copies of MSDSs for chemicals manufactured or distributed by Applied Biosystems using the contact information below To order MSDSs Then Over the Internet a Go to our Web site at www appliedbiosystems com techsupp b Click MSDSs Then Enter one of these numbers in the appropriate field on this page Select Click He
251. scription of the spectral overlap for each capillary is generated and stored as a matrix This data is used to correct the sample run fluorescence data during the chemometric processing The mathematical method used to perform multicomponenting on the 3100 Genetic Analyzer is called the chemometric method As the data is collected for each capillary a comparison is made between the dye set matrix collected during the spectral calibration and the sample data being collected Using this comparison at each spectral bin the portion of the fluorescence emitted from a neighboring dye in the dye set is subtracted from the data thus compensating for the spectral overlap The confidence limits associated with the measured fluorescence intensity are also calculated during multicomponenting The confidence limits do not indicate systematic errors that may be introduced during multicomponenting such as pull up error but rather indicate a random error associated with noise in the detection system This error is displayed as confidence bands in the real time electropherogram views of the Array View Two traces are displayed for each dye Each pair represent the upper and lower limits of detection for that dye The greater the separation between two plots of color the less confidence we have in the detection of the dye Separation of confidence bands will be normally higher at the end of a run and at the peak baseline Configuring Data Flow Data Flow
252. sequencing GS400HDAnalysis gsp GeneScan using size standard 400HD GS350Analysis gsp GeneScan using size standard GS350 GS500Analysis gsp GeneScan using size standard GS500 GS400CubicAnalysis gsp GS4000rd2Analysis gsp a These modules are for advanced users with specific sizing needs See the ABI PRISM GeneScan Analysis Software v 3 5 NT User s Manual Working with Plate Records 6 7 6 8 Working with Plate Records Section Tab Delimited Text Files In This Section The following topics are covered in this section Topic See Page Introduction to Tab Delimited Text Files 6 10 About Creating Tab Delimited Text Files 6 11 Using Spreadsheets to Create Tab Delimited Text Files 6 12 Spreadsheet or Tab Delimited Text File Information 6 14 Running the Same Sample with Different Conditions 6 18 Introduction The topics in this section explain tab delimited text files provide an overview of how to create them and give examples of tab delimited text files for DNA sequencing and fragment analysis Working with Plate Records 6 9 Introduction to Tab Delimited Text Files Introduction Tab delimited text files can be imported directly into the instrument database to create plate records What They Are Tab delimited text files are text only files that contain groups of information called tokens separated by tabs or end of line characters Any text only file containing no graphics or tables created
253. spaces IMPORTANT Be sure that sample file names are not longer than 55 characters An underscore separates each preference selected so be sure to count the underscore in the number of characters There is no automatic error checking for sample names that exceed this limit Sample files with long names cannot be opened by the analysis software 2 For each sample select the appropriate Dye Set from the drop down list For DNA Sequencing analysis select Dye Set E IMPORTANT Be sure to select the correct dye set for your run s Data collected with the incorrect dye set selected cannot be saved and the runs will have to be repeated because multicomponenting is applied during collection Performing a Run 3 37 3 38 Performing a Run To enter sample information and save the plate record continued Step Action 3 For each sample select the appropriate Mobility File from the drop down list MOEIIty File DP3100P0 6 BD 21M13 v1 mob DP3100P0 6 BD M 3Rev v1 mob DT3100PO 6 BD vz mob DT3100PO 6 dRhod w1 mob Note You may need to resize the column to see the whole file name To do this place the cursor between the column headers it will become a double headed arrow and drag right Mobility File gt Comment The following table shows which mobility file to select based on your sequencing chemistry DNA Sequencing Chemistry Mobility File ABI PRISM BigDye Primer
254. stems Technical Support Web site at Internet for fax or http www appliedbiosystems com techsupp e mail delivery 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 Technical Support B 5 Part Numbers Applied Biosystems Part Numbers Introduction Instrument Hardware Plate Assembly Kits Software Kits DNA Sequencing Reagents and Consumables Part numbers for many consumables are given in this appendix Refer to these part numbers when ordering from Applied Biosystems More information about Applied Biosystems kits and consumables is available from your sales representative or on the web at http www appliedbiosystems com Description Part Number ABI Prism 3100 Genetic Analyzer with Dell Workstation 3100 01 Printers sold only with ABI PRISM instruments Epson Stylus 900 Color Printer
255. t file 4 Click OK This opens the Link to dialog box CA C Disabled because no plate ce FD is currently in this position Do Not Link JEZA Note The Do Not Link button is currently enabled In the Link to dialog box select the letter that corresponds to the plate position containing the plate that you want to link to The Do Not Link option is cleared Working with Plate Records 6 33 To import a plate record and link it to its plate in a single procedure continued Step Action 6 Click OK The plt file is imported and the plate record is created Note If there is missing information in the file you may be warned by an information box This may happen for example if you make a typing error or list a module that no longer exists Depending on the problem the warning may accompany rejection of the entire plate record However in some circumstances the data will be imported despite a warning When this happens the purpose of the warning is to prompt you to examine and correct the data in the plate editor Review the plate record in the plate editor 6 34 Working with Plate Records Sequentially Importing and Linking a Plate Record Introduction Sequentially Importing and Linking a Plate Record You can sequentially import a tab delimited text file to create a plate record and then link it to its plate It takes longer to perform these steps separately
256. t for longer than 1 week Note Polymer older than 1 week may cause a transient increase in current during electrophoresis due to urea decomposition Cleaning the IMPORTANT Do not expose the polymer blocks to any organic solvents Polymer Blocks To wash the upper and lower polymer blocks Step Action 1 Remove the polymer blocks from the instrument as described previously in this section 2 Use running water or a squirt bottle to rinse the upper polymer block thoroughly with hot water 3 Visually inspect the channels for white residue dried polymer Continue washing the channels until the residue is gone Rinse the block and its channels with deionized water Remove residual water from the polymer block and fittings to ensure that the running polymer is not diluted Force air through the channels using canned compressed air until the channels are dry Cleaning the Anode To wash the anode reservoir and polymer tubing Reservoir and Polymer Tubing Step Action 1 Use a squirt bottle to flush deionized water into the polymer block tubing 2 Wash the anode reservoir with warm water and then rinse it with deionized water 3 Dry both components with compressed air Maintenance 8 27 Removing Air Bubbles from the Upper Polymer Block Clearing Air To clear air bubbles from the upper polymer block Bubbles 8 28 Maintenance
257. ta Some sequencing analysis modules are provided with the 3100 Data Collection software In the DNA Sequencing Analysis software the sequencing analysis module is called a sequencing analysis settings file Software 5 27 Viewing and Editing Analysis Modules for DNA Sequencing Viewing and Editing To view or edit an analysis module saz file Analysis Modules for 5 28 DNA Sequencing Software Step Action 1 Start the DNA Sequencing Analysis software You may have an icon for the program on the Start menu If not you can find the DNA Sequencing Analysis software SeqA exe in the following directory D AppliedBio abi SeqAnal Bin From the File menu point to Open and select Seq AZ Settings sll Sequencing Analysis 3 6 1 Edit Sample Managar Window Hep New Open Sample Cust Factura Settings Seq AZ Settings Zlose Ctrl Sawe fares Select the analysis module that you want to view or edit The analysis modules are stored in the following directory D AppliedBio abi Shared Analysis Basecaller Params Open 2x Laok in I Params z c BC 31C0_Seq0fFtOff saz a BC 31C0ORR_Seq0ffFtOff saz al BC POP5SLR_SeqQOffFtOfl saz al BC POP6_SeqDffFtOff sez Filename BC 3100_SeqUffFtOtf saz Files of type saz Settings saz x Cancel This opens the sequencing analysis setting file To view or edit an analysis module saz file continued
258. tabase All raw data plate records customized run modules spatial and spectral calibrations and instrument specific information such as polymer and capillary array information will be deleted To remove erase and reinitialize the instrument database using the utility Step Action 1 Ensure OrbixWeb Daemon is running 2 Quit the 3100 Data Collection software 3 Using Windows NT Explorer navigate to the following directory D AppliedBio abi 3100 Bin 4 Locate and double click InitDB bat 5 Locate and double click Createlndex bat 7 12 System Management and Networking Section Networking In This Section The following topics are covered in this section Topic See Page Networking Options 7 14 Networking the Computer Workstation 7 16 Requirements for a Networked Computer 7 18 System Management and Networking 7 13 Networking Options Introduction You have the option of using the ABI PRism 3100 Genetic Analyzer as a stand alone system However you will achieve optimal performance by integrating the 3100 Genetic Analyzer into your existing laboratory data flow system The 3100 Genetic Analyzer has flexible import and export capabilities that can be tailored to meet your needs Other computers can for example be used for preparing plate records providing more comprehensive analysis and storing data The networking options are configured in the 3100 Data Collection sof
259. tation before the 3100 Genetic Analyzer has been installed with the 3100 Software Installing a SCSI device first will alter the drive letter assignments so that the instrument and software cannot be properly installed To install a SCSI storage device Step Action 1 Shut down the computer workstation 2 Plug the device into the external SCSI port 3 Turn the computer workstation back on 4 Ensure the drive letter assignments have not changed See page 2 14 Spatial and Spectral Calibrations Overview In This Chapter The following topics are covered in this chapter Topic See Page Section Spatial Calibration 4 3 About Spatial Calibrations 4 4 About Spatial Calibration Data 4 5 Performing a Spatial Calibration 4 6 Displaying a Spatial Calibration Profile 4 10 Evaluating a Spatial Calibration Profile 4 11 Overriding the Current Spatial Calibration Map 4 12 Section Spectral Calibration 4 15 About Spectral Calibrations 4 16 Performing a Spectral Calibration Using Default Processing Parameters 4 18 Displaying a Spectral Calibration Profile 4 25 Overriding a Spectral Calibration Profile 4 28 Section Advanced Features of Spectral Calibration 4 33 Fine Tuning a MatrixStandard Calibration 4 34 Spectral Calibration Matrices 4 36 Spectral Calibration Log Files 4 37 Spectral Calibration Parameter Files 4 38 Spectral Calibrat
260. te Editor Module Editor ictal Run Vew Change Folymer Wizard stall Capillary Array Vizard Autosampler Calibration Wizard A Perform Spatial Calibration Pending Plate RECO Display Spatial Calibration Pite nare Display Spectral Calibratior 2 Follow the directions given in the wizard to calibrate the autosampler Maintenance 8 29 Troubleshooting Overview In This Chapter The following troubleshooting topics are covered in this chapter Topic See Page Instrument Startup 9 2 Spatial Calibration 9 3 Spectral Calibration 9 4 Run Performance 9 5 Troubleshooting 9 1 Instrument Startup Observation Possible Cause Recommended Action No communication between the instrument and the computer The event viewer is blank Incorrect Ethernet configuration Check the configuration of the IP address a From the Start menu point to Programs and select Command Prompt b At the C prompt type IPconfig all c Press Enter The command prompt window displays information on the network d Ensure the IP address for Ethernet adapter 1 is set for the machine i e the motherboard Ethernet connection The correct IP address is 192 168 0 1 Note The local IT group should use Adapter 2 for networking Red light is blinking Incorrect start up procedure Start up in the following sequence a Log out of the computer ion Turn off the ins
261. te record from an Edited Plate Record Step Action 1 Open the Plate View page of the 3100 Data Collection software 2 In one of the plate record tables double click the plate record that you want to edit This opens the plate record in the plate editor Edit the plate record as required 3 From the File menu select Export This opens a browser dialog box 4 Navigate to the folder in which you want to save the file You may want to use the plate import files folder in the following directory D AppliedBio Abi Support Files Data Collection Support Files Plate Import Files Note You cannot see Network Neighborhood directories from this browser dialog box 5 In the File name dialog box type a name for the file and add the extension plt Click Save This saves the file as a tab delimited text file to the specified directory 6 If you want to use this file as a template give the file a read only status a Right click the Start icon on the Windows NT taskbar and from the pop up menu select Explore This opens Windows NT Explorer Navigate to the file that you just created Right click the file and from the pop up menu select Properties From the Attributes group box select Read only Click OK o2 ps 7 Follow the directions starting on page 6 32 for importing a tab delimited text file to create a plate record 6 30 Working with Plate Records Section Importing Plate Files a
262. tep Action 1 Press CTRL ALT DELETE This opens the Windows NT Security dialog box The user name is displayed in the Logon Information group box in the following message Name is logged on as name instrument serial number 7 16 System Management and Networking Viewing the The computer name is set during installation using the following format Computer Name 3100 instrument serial number To see the computer name and network domain Step Action 1 From the Start menu point to Settings and select Control Panel This opens the Control Panel window 2 In the Control Panel window double click Network This opens the Network property sheet The Identification tabbed page displays the computer name and domain System Management and Networking 7 17 Requirements for a Networked Computer Introduction Minimum Requirements Hard Disk Space Software Required for Remote Extraction This section describes Hardware requirements for a networked computer on which you intend to perform Remote data extraction and analysis Additional data analysis using DNA Sequencing or GeneScan Analysis software Components of the 3100 software that must be installed on the computer The minimum requirements for running Auto Extractor and either DNA Sequencing Analysis or GeneScan Analysis software are Intel Pentium processor 400 MHz or faster Microsoft Windows NT 4 0 oper
263. th Data collected during a previous run on the same capillary array if the detection cell has not been moved A spatial calibration map used to process any previous sample run still stored in the database When overriding the data with data from a previous run if possible use data that was collected during a run performed Onthe same capillary array Since the capillary array was last moved IMPORTANT Overriding calibration data is only allowed if the capillary array has not been removed and the detection cell has not been moved do not use calibration data collected from another capillary array Overriding the To override the current spatial calibration map Current Spatial Calibration Profile __StP_ Action 1 From the File menu select Override Spatial Calibration The Select file dialog box appears Look in E Spatial Cal Logs al l ex a SpatialCal 3100_Rabbit 08 Jun 2000 11 19 28 scl a SpatialCal 3100_Rabbit 08 Jun 2000 11 21 43 scl a SpatialCal 3100_Rabbit 09 Jun 2000 1 4 42 00 scl a SpatialCal 3100_Rabbit 09 Jun 2000 1 4 48 36 scl a SpatialCal 3100_Rabbit 09 Jun 2000 16 14 45 scl a SpatialCal 3100_Rabbit 09 Jun 2000 16 18 01 sel a SpatialCal 3100_Rabbit 09 Jun 2000 16 55 05 scl a SpatialCal 3100_Rabbit 09 Jun 2000 16 57 17 scl sa SpatialCal 3100_Rabbit 1 2 Jun 2000 15 41 03 scl SpatialCal 3100 Rabbit 12 Jun 2000 15 43 49 scl xl Filename R Files of type S
264. th any chemicals or hazardous materials Minimize contact with and inhalation of chemicals Wear appropriate personal protective equipment when handling chemicals e g safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Do not leave chemical containers open Use only with adequate ventilation Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended on the MSDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal rte CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury illness or death Read and understand the material safety data sheets MSDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste Handle chemical wastes in a fume hood Minimize contact with and inhalation of chemical waste Wear appropriate personal protective equipment when handling chemicals e g safety glasses gloves or protective clothing After emptying the waste container seal it with the cap provided Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or national environmental and health regulations A site preparation and
265. the format file name saz Note You can check that the analysis module was saved by examining a plate record in the plate editor as described below Ensuring the To check that the analysis module was saved Analysis Module Was Saved Step Action 1 Open the 3100 Data Collection software 2 In the Plate View page double click a plate record This opens the plate editor If the plate record is already open close it and then re open it 3 Scroll horizontally to the Analysis Module 1 column 4 Click in a cell that lists a sequencing analysis module The list of sequencing analysis modules drops down Analysis Module 1 lt no selection lt no selection BC 3100RR_SeqOttFtOft saz BC 3100_SeqOffFtOff saz 5 Make sure that the sequencing analysis module you just created is listed Note If itis not listed you may have saved the sequencing analysis module in the wrong folder Software 5 35 5 36 Software Section Working with GeneScan Analysis Modules In This Section The following topics are covered in this section Topic See Page Viewing and Editing Analysis Modules for GeneScan 5 38 Creating a GeneScan Analysis Module 5 40 Introduction GeneScan analysis modules provide Auto Extractor with the parameters needed for analyzing data from fragment analysis Software 5 37 Viewing and Editing Analysis Modules for GeneScan Viewing and Editing To view or edit a
266. three letter extensions in their file names The common file types and their extensions are listed below Extension File Type Directory If Applicable ab1 ABIF sample file for sequencing analysis D AppliedBio abi 3100 DataExtractor ExtractedRuns bat Batch file initiates a series of software events e g 3100Collection bat bcp Basecaller parameter file exe Executable program fsa ABIF sample file for fragment analysis D AppliedBio abi 3100 DataExtractor ExtractedRuns sf Factura settings file D AppliedBio abi Shared Analysis Factura Settings gsp Analysis module for GeneScan D AppliedBio abi Shared Analysis Sizecaller Params ini Initialization file log Log file in text file format mcl Spectral calibration file D AppliedBio abi 3100 DataCollection Spectral Cal Logs Spectral Cal mob Mobility file D AppliedBio Abi Shared Analysis Basecaller Mobility mod Run module Ti modexp Exported run module file mtd Method file D AppliedBio abi Support Files Data Collection Support Files Method Files par Spectral calibration parameter files D AppliedBio abi Support Files Data Collection Support Files Calibration Data Spectral Calibration Param Files pdf Portable document format file that can be read by Adobe Acrobat Reader plt Plate file tab delimited text file for import D AppliedBio Abi Support Files Data Collection Support into the instr
267. tion When you are performing a calibration See the procedure run with Use dataType on page Matrix Standard Set DS 30 for Dye SetD matrixStandard 4 34 standard or Matrix Standard Set DS 01 standard for Dye Set E A sequencing reaction that uses Dye Set E sequenceStandard 4 34 A sequencing reaction that uses a four color sequenceStandard dye set other than Dye Set D or E Matrix standards not using Dye Set D or E matrixStandard If you are using either dataType parameter you can determine whether the calibration for a particular capillary will pass or fail by specifying the values for the parameters minQ conditionBounds If for your particular samples you want spectral calibration matrices that are very close to the perfect theoretical matrix you can specify the parameter values so that only very high quality matrices will pass Alternatively you can select less stringent parameter values which may give you calibrations that are reliable enough for your particular application and also result in more passing capillaries Note If you make the stringency very high there will be more failed capillaries If you use a failed capillary the matrix may be overridden by a matrix from a distant capillary The overriding matrix may give poorer results when applied to the new capillary You must consider this when assigning the parameters particularly for fragment analysis Spatial and
268. tion Tray button GR1770 3 Wait until the autosampler has stopped moving and then open the instrument doors Remove the cathode buffer reservoir and water reservoirs from the instrument 5 Dispose of remaining fluids and rinse out the reservoirs with deionized water Note The waste is very dilute however you should follow your company s waste disposal practices for appropriate disposal procedures 6 Rinse the cathode reservoir with 1X running buffer and then fill to the line with 1X running buffer about 16 mL To fill the water and cathode buffer reservoirs continued Step Action 7 Fill the water reservoirs to the line with quality deionized water about 16 mL 8 Place a clean septa strip on each reservoir and dry the outside of the reservoirs using a lint free wipe PNIA Be sure that the septa fit snugly and flush on the tops of the reservoirs in order to prevent damaging the capillary tips Septa is lying flat on the reservoir Fill line Filling the Anode Change the anode buffer Buffer Reservoir Before each run or at least every 24 hours Every time you fill the polymer block with new polymer To fill the anode buffer reservoir to the fill line with 1X running buffer Step Action 1 Remove the anode buffer reservoir by firmly pulling down and twisting slowly Discard the used buffer appropriately
269. tions An external LIMS database can be used to assemble all of the data needed to create plate records Once a LIMS database has been set up correctly and the data has been entered into the LIMS database the creation of plate records in the database becomes completely automatic With the BioLIMS database system data is collected on the computer workstation and written to a BioLIMS database on a networked server The data can later be analyzed using Auto Extractor and viewed and reanalyzed using DNA Sequencing Analysis software or GeneScan Analysis software These programs can either be on the computer workstation which is used to collect the data or on a different computer that has access to the BioLIMS database The data can also be viewed and edited but not analyzed using DNA Sequencing Analysis software or GeneScan Analysis software on a Macintosh computer with access to the BioLIMS database With the stand alone option all operations including the creation of plate records collection of data and review of data with GeneScan Analysis software or DNA Sequencing Analysis software are carried out on the computer workstation System Management and Networking 7 15 Networking the Computer Workstation Introduction The 3100 Genetic Analyzer fully supports connections to local area networks LANs Your network system must be planned and set up by a systems administrator who is familiar with the Windows NT operating system If you pla
270. to the right These capillaries are marked yellow instead of green in the Array View e g Array View Page on page 3 53 For applications where pull up and pull down peaks will cause critical errors we recommend that you repeat the spectral calibration and use a unique spectral for each capillary When the If the spectral calibration failed or if you do not like the appearance of the passed Calibration Fails calibration try one or more of the following Verify that the correct parameter file and run module were selected If not correct and then repeat the run Verify the freshness of the reagents used Verify that all peaks were detected A slow running system can result in the blue peak being partially or totally cut off Add time to the run or change the reagents if they are suspect and then repeat the run 4 24 Spatial and Spectral Calibrations Displaying a Spectral Calibration Profile Introduction At any time you can display the Current spectral calibration profile for a specified dye set The current profile is the one that was created when the last spectral calibration was performed and which is stored in the instrument database The current profiles can be examined only if a spectral calibration has been performed for this dye set Spectral calibration profiles used to process any of the runs currently stored in the instrument database Examining a_ To display a current spectral calibration profile
271. tore the capillary array upright 11 Check the 1X running buffer level in the reservoir and tube weekly Maintenance 8 17 8 18 Maintenance Section Syringes In This Section The following topics are covered in this section Topic See Page Syringe Maintenance 8 20 Cleaning and Inspecting Syringes 8 21 Priming and Filling Syringes 8 22 Installing and Removing Syringes 8 23 Maintenance 8 19 Syringe Maintenance Syringe Types The following table lists the name volume and function of the two syringes Caring for the Syringes When to Replace the Syringes 8 20 Maintenance Name Volume Function Array fill syringe 250 uL High pressure syringe that displaces polymer into the capillary array Polymer reserve syringe 5 mL Stores polymer for multiple sequential runs IMPORTANT To extend the lifetime of the syringe plunger s Teflon fitting do not insert a dry plunger into the barrel of the syringe Place a small drop of deionized water on the plunger s end before inserting it into the syringe Pump the plunger slowly IMPORTANT Do not mix the barrels and plungers from different syringes Mixing and matching is a common cause of leaks IMPORTANT Wear gloves while handling the glass syringes To maintain optimal performance we recommend that you replace syringes about every 3 months Cleaning and Inspecting Syringes When to Clean Thoroughly clean th
272. trument Boot up the computer After the computer has booted completely turn the instrument on Wait for the green status light to come on e Launch the Data Collection software aoa Data Collection software will not launch Did not launch Orbixweb Daemon first Relaunch application following Orbixweb Daemon Computer screen is frozen Communication error This may be due to leaving the user interface in the Capillary View or Array View window There will be no loss of data However if the instrument is in the middle of a run wait for the run to stop Then exit the Data Collection software and restart as described above Autosampler does not move to the forward position Possible communication error Restart the system and then press the Tray button Oven or instrument door is not closed a Close and lock the oven door b Close the instrument doors c Press the Tray button 9 2 Troubleshooting Spatial Calibration Observation Possible Cause Recommended Action Unusual peaks or a flat line for the spatial calibration The instrument may need more time to reach stability An unstable instrument can cause a flat line with no peaks in the spatial view Check or repeat spatial calibration Improper installation of the detection window Reinstall the detection window and make sure it fits in the proper position Broken capillary res
273. tware Overview Diagram The following diagram summarizes the relationships among the different elements of the software and the options for networking with external computers External Computers 3100 Instrument Se LIMS database 3100 NT External computer Import A Workstation with sample records data 3100 or in a spreadsheet to create plate records Detector E E BioLIMS Instrument database database ABIF sample files External computer with Export unanalyzed o e GeneScan Analysis software or analyzed data DNA Sequencing Analysis software External computer with customized software prepared with ABIF Sample File Toolkit GeneScan Analysis DNA Sequencing Analysis software software 7 14 System Management and Networking Using an Additional Networked Computer LIMS Database Option BioLIMS Option Stand Alone Option Using an additional networked computer makes more efficient use of the 3100 Genetic Analyzer While the instrument is performing a run you cannot create plate records review data from past runs or reanalyze data By using another computer to perform these functions you can perform more runs in a day The networked computer can run with a Microsoft Windows NT or Macintosh operating system however if Macintosh versions of analysis applications are used you can only view and edit the data To reanalyze the data you must use the Windows NT versions of analysis applica
274. ty Diskspace utility InitDB utility ABI Sample File Toolkit OrbixWeb 3 2 Professional Edition Orbix Desktop 2 3 software Persistence Powertier 4 321 gt gt gt gt HH H o o o o o Java Runtime Environment 1 1 7b Adobe Acrobat Reader with Search 3 01 GeneScan Applications optional ABI PRISM GeneScan Analysis software including the GeneScan program and sizecaller Sequencing Analysis Applications optional ABI Prism DNA Sequencing Analysis software including the Sequencing Analysis program basecaller and Factura Software Oracle Software Oracle 8 0 5 database standard edition Microsoft Windows NT Image software This software prepares the computer hard disks for installing the 3100 software Diagnostic software This software consists of diagnostic utilities for use by Applied Biosystems service engineers only Determining the To determine the ABI PRISM 3100 firmware and the ABI PRISM 3100 Data Collection Software Versions on Software versions installed on your system click the About Data Collection button on Your System the toolbar Lic 5 4 Software 3100 Genetic Analyzer Software Suite Introduction This section contains overviews of the software provided on the ABI PRISM 3100 Firmware 3100 Data Collection Software Genetic Analyzer software CD ROMs Introduction to Firmware Firmware controls the most basic operat
275. ulting in a bad polymer fill Check for a broken capillary particularly in the detection window area If necessary replace the capillary array using the Install Array Wizard Dirty detection window Place a drop of methanol onto the detection window and dry with compressed air Use only light air force Persistently bad spatial calibration results Bad capillary array Replace the capillary array and then repeat the calibration Call Technical Support if the results do not improve Troubleshooting 9 3 Spectral Calibration Observation Possible Cause Recommended Action No signal Incorrect preparation of sample Replace samples with fresh samples prepared with fresh HiDi Formamide Air bubbles in sample tray Centrifuge samples to remove air bubbles Autosampler not correctly aligned The capillary tips may be hitting the bottom of the wells or they may not be touching the samples Check the autosampler calibration If necessary recalibrate the autosampler using the Autosampler Calibration Wizard If the spectral calibration fails or if a message displays No candidate spectral files found Clogged capillary Refill the capillaries using manual control Look for clogged capillaries during capillary fill on the cathode side Incorrect parameter files and or run modules selected Correct the files and rerun the calibration Insuff
276. umber mcl Examining a If you open a matrix file in an accessory application such as WordPad or Notepad you Spectral Calibration can see that it is a matrix of numbers Matrix The top two numbers define the dimensions of the matrix Left to right the columns represent the 20 spectral bins Top to bottom the numbers represent the relative fluorescence intensity of each of the dyes across a bin in the following order blue green yellow red fifth dye E Cap01 mel WordPad ofx File Edit View Insert Format Help oles alal al fel 2 ko 0 001685 0 002258 0 069567 0 767317 1 000000 0 811798 0 517854 0 373225 0 306652 0 235608 0 156547 0 002109 0 001360 0 002342 0 014515 0 154655 0 628248 1 000000 0 902877 0 607860 0 393029 0 293759 0 002832 0 002235 0 002925 0 007867 0 011204 0 031481 0 127013 0 427612 0 862388 1 000000 0 767486 0 001343 0 001926 0 001454 0 003841 0 004242 0 005029 0 004127 0 009794 0 028196 0 178376 0 598727 0 050000 0 050000 0 050000 0 050000 0 050000 0 050000 0 050000 0 050000 0 050000 0 050000 0 050000 condition number 3 39 quality value 0 992 machine lt gt method lt matrixStandard gt source lt CapO1 tmp gt creator lt Data Collection 3100 gt algorithm lt spcall vers 1 2 a3 gt comment lt gt For Help press F1 NUM 4 4 36 Spatial and Spectral Calibrations Spectral Calibration Log Files Introduction At the end of a spect
277. ument database to create a Files Plate Import Files plate record saz Analysis module for sequencing analysis D AppliedBio abi Shared Analysis Basecaller Params Scl Spatial calibration file D AppliedBio abi 3100 DataCollection SpatialCalLogs SCP Sizecaller parameter file SZS Size standard file D AppliedBio abi Shared Analysis SizeCaller SizeStandar ds tmp Temporary run or calibration data file written in code txt Text file that can be read by Notepad Software 5 9 5 10 Software Section Setting the Format for the Displayed Dye Colors In This Section The following topics are covered in this section Topic See Page Using the Edit Dye Display Information Dialog Box 5 12 Using the Set Color Dialog Box 5 13 Software 5 11 Using the Edit Dye Display Information Dialog Box Introduction The formats for the dye colors shown in the electropherogram and capillary displays are set in the Edit Dye Display Information dialog box You may use the Edit Dye Display Information dialog box to View the current settings for the displayed dye colors e g the blue plots may represent the base cytosine Change the names of the dye Change the color intensity Opening the Edit Dye Display Information Dialog Box Using the Edit Dye Display Information 5 gt gt gt Color Dialog Box on page 5 13 To open the Edit Dye Display Information dialog box Hide the data for part
278. ure below to perform a basic default spectral calibration for both DNA sequencing and fragment analysis To prepare the equipment and supplies Step Action 1 Start the computer and the instrument 2 Prepare the instrument for a run as described starting on page 3 19 3 Prepare an ice bucket with wet ice There are two types of samples from which matrices can be made Matrix standard BigDye sequencing sample The procedures for preparing both sample types are covered in the tables below To prepare the matrix standards for Dye Set E Matrices Step Action 1 Thaw and mix thoroughly the DS 01 P N 4315974 matrix standard tube 2 Spin the tube briefly in a microcentrifuge 3 Prepare the Matrix Standard Set DS 01 for Dye Set E by combining the following in a labeled 1 5 mL microcentrifuge tube Reagent Volume pL Matrix Standard Set DS 01 dROX dTAMRA dR6G 5 dR110 Hi Di Formamide P N 4311320 195 Final Volume 200 PAEAN CHEMICAL HAZARD Formamide is harmful if absorbed through the skin and may cause irritation to the eyes skin and respiratory tract It may cause damage to the central nervous system and the male and female reproductive systems and is a possible birth defect hazard Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves 4 Vortex thoroughly 5 Spin the mixture briefly
279. ures Check Boxes Electropherogram Displays 3 56 Performing a Run Click the Capillary View tab to examine the quality of electropherogram data for several capillaries at once IMPORTANT Always exit from the Array View and the Capillary View windows During a run do not leave these pages open for extended periods This may cause unrecoverable screen update problems Leave the Status View window open This is an example of the Capillary View page for a GeneScan run Select check boxes of capillaries to be displayed 6 3100 Data Collection Software File View Instrument Tools Service Help eS Ems Plate View Run View Status view Array View Capillary View 41 10 15 Select capilaries to display f lt M o v w m m E e e 800 400 400 11 6 117 11 8 11 9 12 12 1 12 2 12 3 12 4 12 4 12 Intensity vs Run Time mins a 1200 2 SS nN 600 11 6 abby 11 8 11 9 12 12 1 12 2 123 12 4 12 4 12 Intensity vs Run Time mins in _ Electro pherogram displays 1200 a omc w 600 11 6 abe 11 8 11 9 12 12 1 122 123 12 4 12 4 12 Intensity vs Run Time mins in 1200 600 600 11 6 447 11 8 11 9 12 12 1 122 123 12 4 12 4 12 Intensity vs Run Time mins 4 2l in Select the check boxes of the capillaries for which you want electropherograms displayed Note Only four capillary electropherograms will fit on t
280. utility and import the new method 7 10 System Management and Networking Removing Run Modules from the Instrument Database The Remove Run Modules Utility Function File Name and Directory Removing Run Modules The Remove Run Modules utility removes all modules and associated information from the instrument database This utility is used to quickly delete all old modules before you import new ones The Remove Run Modules utility is named RemoveRunModules bat and is located in the following directory D AppliedBio abi 3100 Bin To remove run modules using the utility Step Action 1 Ensure OrbixWeb Daemon is running 2 Quit the 3100 Data Collection software 3 Using Windows NT Explorer navigate to the following directory D AppliedBio abi 3100 Bin 4 Locate and double click RemoveRunModules bat System Management and Networking 7 11 Reinitializing the Instrument Database The Initialize Database Utility Function The Initialize Database utility completely erases and reinitializes the instrument File Name and Directory Erasing and Reinitializing the Instrument Database database The Initialize Database utility is named InitDB bat and is located in the following directory D AppliedBio abi 3100 Bin IMPORTANT Do not run this utility unless instructed to do so by a Applied Biosystems representative FNO The Initialize Database utility completely erases the instrument da
281. ventions the version number will change and you will be notified Plate Header The plate header is a sequence of five cells or tokens separated by tabs These cells or tokens must always be typed in the same order across the plate header Cell or Token Function Plate Name Identifies a specific plate The plate name you assign must not exceed 32 characters Note This is the same as the Plate ID listed in the plate record tables of the Plate View page Application Identifies a plate as containing samples for either GeneScan analysis GS or DNA sequencing SQ IMPORTANT Do not mix samples for sequencing analysis and fragment analysis in the same plate Plate Type Defines the type of plate The codes used for the two plate types are 96 well plates 96 Well 384 well plates 384 Well Owner Identifies the name of the person who loaded the samples onto the plate and or created the spreadsheet Comment Allows you to enter comments about the plate 6 14 Working with Plate Records Column Header for The column header for sequencing analysis contains up to eight cells or tokens that Sequencing Analysis provide headings for the columns that will contain the sample data The order in which the column cells or tokens are listed is important and is noted below Token Function Well Position Identifies the well in which the sample is located e g A1 G6 O18 etc For 96 well plates the well
282. vides a network connection to the computer workstation Chassis fan Pulls air out of the instrument Laser fan Cools the laser Power cord Supplies power to the instrument 2 12 System Overview Section Computer and Software Overview In This Section The following topics are covered in this section Topic See Page Computer Workstation 2 14 Software 9 15 System Overview 2 13 Computer Workstation Overview The 3100 Genetic Analyzer is shipped with a computer workstation running the Microsoft Windows NT operating system An optional color printer is available This manual is written with the assumption that you know how to use a computer workstation running the Windows NT operating system If you are not familiar with this computer refer to the Windows NT workstation documentation shipped with this system for specific operating information Function The computer workstation collects and analyzes data from the 3100 Genetic Analyzer System The following table lists the minimum requirements for the computer workstation Requirements Item Minimum Requirements Hard drive storage 2 drives 9 GB each Memory 256 MB RAM Monitor 17 in SVGA Operating system Microsoft Windows NT v 4 0 with Service Pack 5 Printer Optional Processor Intel Pentium III 550 MHz Hard Drive During installation the hard drives of your computer workstation were
283. wing table Note The many lenses and mirrors integral to detection are not covered in this section Part Function Laser Excites the attached dye labels as the DNA fragments pass through the detection cell Transmission grating Disperses the light by wavelength and a second set of lenses focuses the resulting light spectrum onto the CCD camera CCD camera Converts the incident fluorescence into digital information that is processed by the 3100 Data Collection software Note More information on each of the components follows this section Laser Overview Laser Type Emission Wavelengths Interlock When a dye labeled DNA fragment moves into the path of the laser beam some electrons in the dye are excited to higher energy levels as the laser light is absorbed Shortly afterwards the electrons return to their ground states and emit fluorescence light energy The light emitted from each dye has a different spectral profile color The laser used to excite the dyes is an argon ion laser The primary emission lines are at 488 nm and 514 5 nm For your safety an interlock switch shutters the laser and shuts off the electrophoresis power supply if the doors of the instrument are opened For more information on laser safety refer to the ABI PRISM 3100 Genetic Analyzer Site Preparation and Safety Guide P N 4315835 7 WANS NINE LASER HAZARD Exposure to direct or reflected laser light at 40 mW
284. xt file it compares the relevant tokens with lists of run modules analysis modules etc stored in the database or hard drive The Data Collection software recognizes the data only if it can make a match If an illegal value is typed into a cell in certain columns the typed data will be deleted and the field will be blank in the imported plate record If the sample name contains restricted characters the entire plate will not be imported IMPORTANT When naming the plate you can use letters numbers and the following punctuation only _ Do not use spaces When importing data from a LIMS database an error will be logged and no plate record will be created if the file contains a typing error Working with Plate Records 6 13 Spreadsheet or Tab Delimited Text File Information Introduction Four types of information are contained in a spreadsheet or tab delimited text file intended for import into the 3100 Data Collection software Version number Plate header Column header Sample data See the spreadsheet examples in Using Spreadsheets to Create Tab Delimited Text F iles on page 6 12 Version Number The version number is the only cell or token on the first row of a spreadsheet or tab delimited text file It specifies the version of the formatting conventions used for importing plate records Currently the version that must be entered into all spreadsheets is 1 0 If there are changes to the con

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