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ABI PRISM ® 3100 Genetic Analyzer

1. Pending Plate Records Plate Name Application Wells Status Linked Plate Records Plate Name Application Wells Status Ajmy_plate_record GS 96 pending Processed Plate Records Plate Name Application Wells Status Plate record is in the Linked Plate Records table 4 Repeat steps 1 3 to link a second plate if applicable Performing a Fragment Analysis Run 2 25 To link a plate to a plate record continued Step Action 5 Click the Run View tab to view the run schedule Note Although individual runs can be deleted the order in which the runs are scheduled cannot be altered Run scheduling depends upon a number of factors see the ABI PRISM 3100 Genetic Analyzer User s Manual for information 3100 Data Collection Software Version 1 0 1 GeneScan36_POP GeneScan36_POP 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 Records table and the plate position indicator will return to yellow 2 26 Performing a Fragment Analysis Run Starting and Monitoring the Run
2. 00 0 0 00 0000 e eee 5 14 Installing and Removing the Capillary Array 0 0 0 0 eee 5 15 Storing a Capillary Array 0 ccc eet ene 5 16 Shutting Down the Instrument 0 0 00 eee eee 5 17 Preparing Formamide Deionizing and Storing Formamide 00 00 cece cece eee A 1 Getting Help Technical Support anei n N AE 8 SY eg A E ANENA E EN R EREA B 1 Index Introduction Overview In This Chapter This chapter includes the following topics Topic See Page About This Manual 1 2 For More Information 1 2 Safety 1 3 Introduction 1 1 About This Manual Purpose The purpose of this manual is to give users basic instructions on how to Do a fragment analysis run Analyze the resulting data Calibrate and perform routine maintenance on the ABI PRISM 3100 Genetic Analyzer For More Information Procedure Where to Find More Other manuals and guides that relate to the 3100 Genetic Analyzer are listed below Information Part If you want Refer to the Number safety information and information about preparing ABI PRISM 3100 Genetic Analyzer Site Preparation 4315835 your lab for the 3100 Genetic Analyzer and Safety Guide detailed information about the 3100 Genetic ABI PRISM 3100 Genetic Analyzer User s Manual 4315834 Analyzer detailed information about analyzing and viewing ABI PRISM GeneScan Analysis Software v 3 7 4308923 fra
3. Region Telephone Fax Norway Oslo 47 23 12 06 05 47 23 12 05 75 Portugal Lisboa 351 0 22 605 33 14 351 0 22 605 33 15 Spain Tres Cantos 34 0 91 806 1210 34 0 91 806 12 06 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 392400 31 0 180 392409 or 31 0 180 392499 United Kingdom Warrington Cheshire 44 0 1925 825650 44 0 1925 282502 European Managed Territories EMT Africa English speaking Johannesburg South Africa 27 11 478 0411 27 11 478 0349 Africa French speaking Paris France 33 1 69 59 85 11 33 1 69 59 85 00 India New Delhi 91 11 653 3743 91 11 653 3744 91 11 653 3138 Poland Lithuania Latvia and Estonia 48 22 866 40 10 48 22 866 40 20 Warszawa For all other EMT countries not listed 44 1925 282481 44 1925 282509 Central and southeast Europe CIS Middle East and West Asia Japan Japan Hacchobori Chuo Ku Tokyo 81 3 5566 6230 81 3 5566 6507 Latin America Caribbean countries Mexico and 52 55 35 3610 52 55 66 2308 Central America Brazil 0 800 704 9004 or 55 11 5070 9694 95 55 11 5070 9654 Argentina 800 666 0096 55 11 5070 9694 95 Chile 1230 020 9102 55 11 5070 9694 95 Uruguay 0004 055 654 55 11 5070 9694 95 Getting Help B
4. Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free 1 800 345 5224 Fax 1 650 638 5884 Worldwide Sales Offices Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches into 150 countries on six continents For sales office locations and technical support please call our local office or refer to our web site at www appliedbiosystems com or to the Technical Support appendix in this document www appliedbiosystems com AS applied as HITACHI Applera Corporation is committed to providing the world s leading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses Printed in the USA 07 2001 Part Number 4315832C an Applera business
5. Capillary Array Usage 49 Waiting for Oven to Stabilize at Run Temperature During the run you can view the data using the Array View and Capillary View pages IMPORTANT Always exit from the Array View and the Capillary View windows Do not leave these windows open for extended periods during a run because unrecoverable screen update problems will occur Leave the Status View window open For more information about the Array and Capillary views see Viewing Raw Data on page 3 2 Performing a Fragment Analysis Run 2 27 Stopping a Run and Recovering the Data Stopping or When arun is in progress the Skip Pause and Stop buttons on the toolbar are Skipping a Run visible 3100 Data Collection Software File View Instrument Tools Service Help Stop button Pause button Skip to Next Run button To stop the current run and Click continue the other scheduled runs the Skip button stop the other scheduled runs a the Stop button b Now in the Question dialog box 22 Stop now or after current run After run Cancel If Autoextraction The auto extractor should have automatically extracted your data from the stopped Fails run If it did not use the Extract data into sample files commands as described below To recover data from a stopped run Step Action 1 From the Instrument menu
6. 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 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 Click the plate record Pending Plate Records Plate Name Application Wells Status my_plate_record GS 96 pending Place a plate into plate position B Linked Plate Records Plate Name Application Wells Status A B Processed Plate Records Click anywhere on the plate position indicator 2 24 Performing a Fragment Analysis Run To link a plate to a plate record continued Siep Action 3 Verify that the plate has been linked Once the plate has been linked the 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 3100 Data Collection Software File View Instrument Tools Service Help He gt lm M Plate View Run view Status view Array View Capilary View
7. MySample A01 Otten 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 2 9 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 Performing a Fragment Analysis Run 2 19 To enter sample information and save the plate record continued Step Action 2 Optional For each sample enter Color Info and Color Comment text 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 lt no selection 3100_Project1 Note For more information about setting up a BioLIMS project see the ABI PRISM 3100 Genetic Analyzer User s Manual 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 or select
8. Feet should be flat on the floor and the weight of the legs should be supported by the floor not the thighs 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 allows 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 ad
9. Record Step Action 1 On the Plate View page of the Data Collection software click New The Plate Editor dialog box opens In the Plate Editor dialog box a Name the plate b Select Spectral Calibration c Make sure that the appropriate plate size is selected d Click Finish Plate Name SpectralCalibration Application C Sequencing C GeneScan Spectral Calibration Plate Type os wen Comments EE Finish Cancel The Plate Editor spreadsheet opens Complete the Plate Editor spreadsheet for the wells you have loaded Type a name for the samples Select Dye Set D Select the run module Spect36_POP4DefaultModule Select the spectral parameter MtxStd GeneScan SetD par e Click OK aogop 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 seconds the entry for the plate record appears in the Pending Plate Records table of the Plate Setup page Spatial and Spectral Calibrations 4 9 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 aut
10. 7 Save the project a From the File menu select Save Project b Enter a file name for the project and click Save Double click the sample file name in the Analysis Control window to view a sample file This automatically displays the Sample Results view T 400_A03_001 fsa Of x fa m m ie re m m m m m m ig View buttons If the window looks like the one below the sample files have not been analyzed Refer to Analyzing or Reanalyzing Data in GeneScan Software on page 3 12 for information on how to analyze data 400_A03_001 fsa No Analyzed Data hs T i Peak 10 If your window does not look like either step 8 or step 9 of above click the Sample Results button in the corner of the window Sample Results Sample File Info Raw Data 3 8 Viewing and Analyzing Data To create a new project and view sample files continued Step Action 11 Use the Magnifying tool to change the scale of the plot a Click on the tool to select it E b Click on the plot to zoom in or ALT click to zoom out Alternatively use the commands on the View menu to adjust the plot scale 12 To identify a peak click it That peak s row in the GeneScan table is highlighted Viewing Multiple The procedure below introduces you to the Results Control window A complete Files description of the Results Control window is provided in
11. Spatial Calibration What a Spatial Calibration Tells You Performing a Spatial Calibration Install or replace a capillary array Temporarily remove the capillary array from the detection block A spatial calibration provides information about the position of the fluorescence from each capillary on the CCD camera It does not provide information about the performance of the capillaries To perform a spatial calibration Step Action 1 From the Tools menu select Perform Spatial Calibration The Perform Spatial Calibration dialog box opens E Perform Spatial Calibration x Spatial calibration progress Click on the Start button to initiate spatial calibration T Fill capillaries peme Stat Cancel 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 do not need to 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 2 Spatial and Spectral Calibrations To perform a spatial calibration continued Step Action 4 If the calibration Then succeeded the following dialog box opens A Perform Spatial Calibration a Click Details to view the Spatial Calibration Profile window b Con
12. 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 To begin purification and measure conductivity Step Action 1 Calibrate the conductivity meter cell and rinse the cell with distilled water 2 In a polypropylene screw cap container wash 10 g of Bio Rad AG501 X8 ion exchange resin by swirling the sample with 10 20 mL of formamide for 1 minute 3 Either decant off or filter through a course nylon or teflon filter and discard the formamide Repeat steps 2 and 3 twice Add 100 mL of formamide to the washed resin Cap the mixture ensuring that it is well sealed NI OO oO amp Stir the mixture rapidly with a magnetic stirrer or mix with an electric shaker ensuring that the resin is suspended for adequate mixing and ion exchange Stir at room temperature for approximately 2 hr Preparing Formamide A 3 To begin purification and measure conductivity continued Step Action 8 Remove a small aliquot of the mixture and measure the conductivity at room temperature 9 Rinse the conductivity cell with distilled water 10 If the conductivity is Then gt 5 uSiemens cm Return to step 7 and stir for an additional 30 min l
13. oj Analyze T Print Results m lal j 400 cos 005 fsa GS 400HD szs GS400CubicAnalysis gsp Val 400 co4 006 tsa GS 400HD szs Di SS400HDAnalysis gsp mle fall 400 D03_o07 tsa GS 400HD szs P cs4000rd2Analysis gsp m 400 D04 008 tsa GS 400HD szs Ca ery ae mlm 400 E03 009 tsa GS 400HD szs a yE p Print Setup Size Standard A Sample File GS 400HD szs a GS 400HD szs 400 403 001 fsa 400 404 002 fsa 400 B03 _003 fsa GS 400HD szs gt GS350Analysis gs 400 B04 004 tsa GS 400HD szs z yeaa v lt Analysis Parameters Collection Setting To review or edit the analysis parameters double click the parameters file name The Analysis Parameters dialog box opens Suggested values for parameters are shown below Refer to the ABI PRISM GeneScan Analysis Software User Guide for more information about analysis parameters m Analysis Range Size Call Range Full Range Full Range This Range Data Points This Range Base Pairs Stat P min P Stop i2000 Max foo m Data Processing SizeCaling Method C 2nd Order Least Squares anes CEOS 31d Order Least Squares Light Cubic Spline Interpolation Local Southern Method C Global Souther Method C Heavy m Peak Detection Baselining BaseLine Window Size B Ps m Auto Analysis Only Size Standard Min Peak Half Width E Pts ero aa GS 400HD szs w Polynomial Degree
14. preparing A 3 electrical shock warning 1 6 e mail address for technical support B 1 F Field Service in North America contacting B 3 Fill Down command 2 20 formamide deionizing and storing A 1 to A 4 sample preparation 2 4 G GeneScan Analysis software 3 12 to 3 14 starting 2 31 H hard drive space checking 5 6 Hi Di Formamide 2 4 I instrument operating 2 27 shutting down 5 17 Internet address customer training information B 7 Documents on Demand B 6 L laser warning 1 6 Linkage Mapping Sets pooling ratios for 2 4 linking a plate 2 24 loading volumes See sample volumes M maintenance discussed 5 2 to 5 18 manuals 3100 set 1 2 MSDS how to order 1 4 N naming convention for sample files 2 19 O OrbixWeb starting 2 6 P panels of data displaying 3 10 part numbers manuals 1 2 parts and consumables See 3100 user s manual Pause button 2 28 peak identification GeneScan Analysis software 3 9 plate assembly placing on autosampler 2 17 plate record creating 2 18 to 2 23 creating for spectral calibration 4 9 linking a plate 2 24 Plate View tab 2 18 2 24 polymer blocks Index 1 cleaning 5 11 removing air bubbles 5 4 removing from instrument 5 10 polymer adding and changing 5 12 pooling ratios 2 4 preferences 2 8 R raw color data viewing in Data Collection software 3 2 to 3 4 Raw Data button GeneScan Analysis software 3 8 reanalyzing data See data analyzing and reanalyzing reservo
15. with 8 cross linkage The minimum wet capacity is 1 5 meq mL with 20 50 dry mesh size AG501 X8 molecular biology grade mixed bed resin Available from Bio Rad Laboratories P N 143 6424 or equivalent Conductivity meter A commercial conductivity meter or pH meter with an external conductivity cell is sufficient to measure the conductivity of formamide if it has a cell constant of 1 0 Na EDTA Dihydrate M 372 2 ACS reagent 99 purity or greater Available from Sigma P N E4884 or equivalent Container for storing formamide Use a polypropylene screw cap container Note Glass containers are not recommended because of potential contamination from minerals Ion Exchange Resin The raw formamide is deionized with cationic and anionic mixed resins to remove impurities such as ammonium and formate ions Deionization occurs at a slow mass transfer rate in the equilibrium ion exchange kinetics due to A 2 Preparing Formamide Physical changes in the resin in the presence of formamide Differences in molecular size and selectivity between the impurity ions and the H and OH counterions Therefore the conductivity of formamide must be monitored over time to determine the extent of deionization by the resin Calibrating the A conductivity meter and cell are needed to measure the effectiveness of the Conductivity Meter deionization process The more deionized the formamide the lower it
16. 5 To Reach Technical At the Applied Biosystems web site you can search through frequently asked Support Through questions FAQs or a solution database or you can submit a question directly to the Applied Technical Support Biosystems Web Site Search FAQs To search for FAQs Step Action 1 Go to http www appliedbiosystems com 2 Click SERVICES amp SUPPORT at the top of the page then click Frequently Asked Questions 3 Click you geographic region for the product area of interest 4 Follow the instructions under the Frequently Asked Questions section 1 to display a list of FAQs for your area of interest Search the Solution Database To search for solutions to problems using the Solution Database Step Action 1 Go to http www appliedbiosystems com 2 Click SERVICES amp SUPPORT at the top of the page then click Frequently Asked Questions 3 Follow the instructions under the Search the Solution Database section 2 to find a solution to your problem Submit a Question To submit a question directly to Technical Support Go to http www appliedbiosystems com 2 Click SERVICES amp SUPPORT at the top of the page then click Frequently Asked Questions 3 In the Personal Assistance E Mail Support section 3 click Ask Us RIGHT NOW In the displayed form enter the requested information and your question then click Ask Us RIGHT NOW
17. 5 11 Putting Fresh Polymer on the Instrument When to Change the We recommend that you change the polymer weekly The polymer is good at 25 C for Polymer about 7 days Adding or Changing Determine whether to add or change the polymer on the instrument before proceeding Polymer with instrument preparation CHEMICAL HAZARD POP polymers may cause eye skin and respiratory tract irritation Please read the MSDS for the polymer you are using and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Use for research and development purposes only If polymer on the instrument is Then less than 1 week old Ensure there are no air bubbles and then proceed with and sufficient in instrument preparation quantity to complete N F d bubbi your runs ote For a procedure to remove any air bubbles see ABI PRISM 3100 Genetic Analyzer User s Manual greater than 1 week Fill the syringes and the upper polymer block with polymer by old or insufficient in following the instructions under Change Polymer Wizard quantity to complete your runs Fie View Instrument UGRIEM Service Help Plate Editor Module Editor Change Polymer izard Install Capillary Array Wizard Autosampler Calibration Wizard Perform Spatial Calibration Display Spatial Calibration Display Spectral Calibration Changing Polymer To put fresh polymer on the instrument
18. B Peak Window Size 9 Pts Slope Threshold for bo Peak Start Slope Threshold for po Peak End If you make changes to the analysis parameters a Select Save As from the File menu b Choose a new name for the analysis parameters gsp file Viewing and Analyzing Data 3 13 To analyze or reanalyze sample files continued Step Action 8 Select all dye lanes by clicking in the upper left corner of the dye color fields bar Click here to select all lanes and all colors Demo prj Analysis Control Demo prj Analysis Control T Print Results Analyze Print Results B Sample File Sample File E Ml 400 403 001 fsa 2 i A 400 404 0024sa 400 404 002 fsa Before clicking After clicking Click the Analyze button The analyzed data is automatically saved to the sample files If analyzed data already existed in the sample file it is overwritten As the lanes are analyzed they are deselected in the dye color fields 3 14 Viewing and Analyzing Data Spatial and Spectral Calibrations Overview In This Chapter This chapter includes the following topics Topic See Page Performing a Spatial Calibration 4 2 Performing a Spectral Calibration 4 6 Spatial and Spectral Calibrations 4 1 Performing a Spatial Calibration When to Doa A spatial calibration must be performed each time after you
19. Sequencing Systems 1 800 831 6844 press 3 then press 2a 1 650 638 5981 Sequence Detection Systems Real Time PCR and PCR 1 800 762 4001 then press 1 for PCRa 2 for TaqMan applications and Sequence Detection Systems including ABI Prism 7700 7900 and 57004 6 for the 6700 Automated Sample Prep Systema or 1 800 831 6844 then press 5a 1 240 453 4613 Mariner ESI TOF Mass Spectrometry Workstations Voyager MALDI TOF Biospectrometry Workstations MassGenotyping Solution 1 MGS1 Systems Proteomics Solution 1 PS1 Systems ICAT Reagent 1 800 899 5858 press 1 then press 3 1 508 383 7855 Getting Help B 3 To Contact Technical Support by Telephone or Fax Outside North America B 4 Getting Help Product Product Area Telephone Fax Biochromatography BioCAD SPRINT VISION and INTEGRAL Workstations and POROS Perfusion Chromatography Products 1 800 899 5858 press 1 then press 4 1 508 383 7855 Expedite 8900 Nucleic Acid Synthesis Systems 1 800 899 5858 press 1 then press 5 1 508 383 7855 Pioneer Peptide Synthesizers 1 800 899 5858 press 1 then press 5 1 508 383 7855 PNA Custom and Synthesis 1 800 899 5858 press 1 then press 5 1 508 383 7855 FMAT 8100 HTS Systems CytoFluor 4000 Fluorescence Plate Reader 1 800 899 5858 pre
20. be adjusted to ensure an appropriate detection of all the loci Suggested Loading Volumes IMPORTANT Follow the suggested protocol for each application See the Linkage Mapping Sets example below Pooling Ratios for the ABI PRISM Linkage Mapping Sets For Linkage Mapping Sets LD20 MD10 and HD5 a ratio of 1 1 1 6 FAM HEX NED labeled products gives acceptable balance across most loci For each Linkage Mapping Set panel pool 1 uL of each PCR product in a microcentrifuge tube If necessary bring the total volume to 10 20 uL with deionized water 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 Prepare the formamide size standard mix using 1000 uL of Hi Di Formamide P N 4311320 or similar quality formamide 50 uL of GS500 ROX GS500 LIZ GS120 LIZ or GS400 HD Note We recommend that you use Hi Di Formamide but if you prefer to prepare your own formamide see Appendix A Preparing Formamide for important information 2 4 Performing a Fragment Analysis Run Note Use these ratios of pooled PCR products and size standards as a starting point only Optimize these ratios as necessar
21. briefly in a microcentrifuge 3 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 Prepare the Matrix Standard Set DS 30 for Dye Set D by combining the following in a labeled 1 5 mL microcentrifuge tube Reagent Volume uL 6FAM 2 5 HEX 2 5 NED 2 5 ROX 2 5 Hi Di Formamide P N 4311320 190 Final Volume 200 4 Vortex thoroughly 5 Spin the mixture briefly in a microcentrifuge 4 6 Spatial and Spectral Calibrations continued To prepare the Matrix Standards Dye Set D Matrices as an example Action Heat the standard tube at 95 C for 5 min to denature the DNA Immediately place the tubes on ice for 2 min Step 6 7 Loading the To load the standards Dispense 10 uL of the denatured matrix standard into a 96 well plate wells A1 through H2 as shown below rOOO00000 OO000000 OOOO00000 GR1315b 384 well plate wells A1 A3 C1 C3 E1 E3 etc as shown below xooooo0o00000000000 zoooo000000000000 aooooooo0000000000 aoooooooo0o00000000 goooooooo0o00000000 2Z000000000000000
22. create Every week delete records in the database The procedures that follow tell you How to check the available space on the hard drive typically located on drive D for the extracted sample files How to check the available space in the instrument database typically located on Drive E for the raw data Where to find the procedures for deleting database records 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 the D drive and select Properties A My Computer Iofx Fils Edit View Help My Conputer z Sl le lale JE E 3 Floppy A 3100Files D Properties General Tools Sharing Security 1 1x Label 3100Files Type Loca Disk File system NTFS Tl Used space ff Free space 1 644 572 672 bytes 5 305 786 358 bytes 1 53GB 4 94GB 6 950 359 040 bytes Capacity 6 47GB Diive D T Compress D m e 5 6 Maintaining the Instrument To check the hard drive for space for sample files continued Step Action 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 fragment analysis 500 Unanalyzed sample file 100 a The values provided are estimates only The a
23. detection cell To clean the detection cell Step Action 1 Put one drop of methanol on the front surface of the 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 Front surface of detection cell 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 NM NN 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 5 14 Maintaining the Instrument 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 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
24. display the panels and table O Demo prj Display 3 O x lo 4 0 120 160 200 240 280 320 360 400 440 480 520 560 600 640 2400 1800 blu wil LIL 3B 400_B03_003 fsa GH 36 400_p03_003 tsa OI 37 400_803_003 tsa BE 3 400_B03_003 tsa LULL 4B 400_B04_004 fsa 4G 400_B04_004 fsa Om 4 400_B04_004 fsa BE 4R 400_804_004 tsa Z X F Dye Sample Peak DDD m m m m m m m m E 3 10 Viewing and Analyzing Data To view multiple data sets using the Results Control window continued Step Action 8 To print the display from the File menu choose Print 9 Use the Clear Panel or Clear All buttons on the Results Control window to clear panels Viewing and Analyzing Data 3 11 Analyzing or Reanalyzing Data in GeneScan Software Introduction Note For more information about analyzing data using GeneScan Analysis software see the ABI PRISM GeneScan Analysis Software User Guide When to Analyze Data with GeneScan Analysis Software The sample file will not contain analyzed data if you did not specify an analysis module in the plate record If the sample file does not contain analyzed data you need to analyze the file as described in the procedure below When to Reanalyze Data with GeneScan Analysis Software Reanalyze the sample files using GeneScan Analysis software when you Chose the wrong analysis module file in the plate
25. 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 documents by automated telephone service 1 From the U S or Canada dial 1 800 487 6809 or from outside the U S and Canada dial 1 858 712 0317 2 Follow the voice instructions to order documents for delivery by fax Note There is a limit of five documents per fax request To order documents by telephone In the U S Dial 1 800 345 5224 and press 1 To order in English dial 1 800 668 6913 and press 1 then 2 then 1 In Canada To order in French dial 1 800 668 6913 and press 2 then 2 then 1 From any other country See the specific region under To Contact Technical Support by Telephone or Fax Outside North America To view download or order documents through the Applied Biosystems web site Step Action 1 Go to http www appliedbiosystems com 2 Click SERVICES amp SUPPORT at the top of the page click Documents on Demand then click MSDS 3 Click MSDS Index search through the list for the chemical of interest to you then click on the MSDS document number for that chemical to open a pdf of the MSDS Instrument Safety Labels About Waste Disposal Befo
26. plate record continued Step Action 8 Make sure the plate record is correct and then click OK my_plate_record _Dye set A1 GeneScan_sample1 3100 _Project1 D HTGS36_PO GS400HDAnalysis gsp B1 GeneScan_sample2 3100_Project1 D HTGS36_PO GS400HDAnalysis gsp C1 GeneScan_sample3 3100_Project1 D HTGS36_PO GS400HDAnalysis gsp D1 GeneScan_sample4 3100_Project1 D HTGS36_PO GS400HDAnalysis gsp E1 GeneScan_sample5 3100_Project1 D HTGS36_PO GS400HDAnalysis gsp This is an example plate record Note It may take a moment for the new plate record to be saved to the database and added to the Pending Plate Records table as shown below Note The plate record must be deleted from the database first in order to use the same name for another plate record 3100 Data Collection Software S Mil im al Performing a Fragment Analysis Run 2 23 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
27. the ABI PRISM GeneScan Analysis Software User Guide To view multiple data sets using the Results Control window Step Action 1 If not already open start the GeneScan Analysis software 2 Create a project as described in steps 2 to 6 beginning on page 3 6 Or open an existing project by selecting Open from the File menu From the Windows menu select Results Control From the of Panels menu select 8 of Panels v4 Dye Sample 5 oo Mog 8 JE E 5 mm Viewing and Analyzing Data 3 9 To view multiple data sets using the Results Control window continued Step Action 5 Click on the project sample number to select all colors for that sample Project sample number J P Panel number Dye color fields Electropherogram button Table button Number of panels 400 C03 005 fsa 400 C04 006 fsa 400 D03 007 fsa 3B 400_B03_003 fsa 3G 400_B03_003 fsa 3Y 400_B03_003 fsa 3R 400_803_003 fsa amp 400 D04 008 fsa nee 400 E03 009 tsa 400 E04 010 tsa 400 F03 011 fsa 400 F04 012 tsa 400 C03 013 fsa 400 C04 014 fsa 400 H03 015 fsa 400 HO4 016 fsa elf of of f EE a Quick Tile On Off Display Configure the next panel by clicking the panel number button for panel 2 and repeating step 5 above Click the display button to
28. your runs Note To remove any air bubbles see page 5 4 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 5 12 CHEMICAL HAZARD POP polymers may cause eye skin and respiratory tract irritation Please read the MSDS for the polymer you are using and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Use for research and development purposes only 3100 Data Collection Software File View Instrument GTS Service Help Hels Em Pte Eator Module Editor Plate View l Run View ETT Instrument Condition insufficient in quantity to complete your runs Install Capillary Array Wizard Autosampler Calibration Wizard Perform Spatial Calibration Display Spatial Calibration Display Spectral Calibration a A run uses 50 80 uL of polymer depending on the length of the array This is equivalent to 60 100 runs from one 5 mL syringe A minimum of 100uL 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 and lower polymer blocks before proceeding To remove any air bubbles see page 5 4 Replace the 1X Genetic Analyzer buffer in the anode buffer reservoir and the cathode buffer reservoir daily or before each batch of runs IMPORT
29. 0 20000000000000000 ooo0000000000000 2 0o000000000000000 20000000000000000 zoo00000000000000 20000000000000000 sooooo000000000000 oo00000000000000 20O000000000000000 0000000000000000 2 0000000000000000 o0o00000000000000 20o000000000000000 0000000000000000 0o0o00000000000000 c 0000000000000000 o soooooo0oo0000000000 fia 5 00000000 0000000000000000 S lt o o A w a O F lt moowuor s izz00 fr lt 6 a o Nn oo ic N Spatial and Spectral Calibrations 4 7 To load the standards continued Step Action 2 Centrifuge the plate so that the each standard is positioned at the bottom of its well Your samples should Look like this Not look like this Not look like this The sample is positioned correctly in the bottom of the well A 5 U The sample lies on the side wall because the plate was not centrifuged J U An air bubble lies at the bottom of the well because the plate was not Centrifuged with enough force or Centrifuged for enough time Preparing the Plate Follow the instructions on pages 2 11 through 2 17 to and Instrument 4 Assemble the plates Check and refill the fluids on the instrument Place the plate on the autosampler 4 8 Spatial and Spectral Calibrations Creating a Plate To create a plate record for the denatured matrix standards
30. 5 Performing a Spectral Calibration Introduction Performing a spectral calibration can be divided into three main tasks Setting up the standards Starting the spectral calibration Checking the spectral calibration Note This section describes spectral calibration using the Matrix Standard Set DS 30 of Dye Set D For information about performing spectral calibration for another dye set see the ABI PRISM 3100 Genetic Analyzer User s Manual A spectral calibration is performed to create a matrix to correct for the overlapping of fluorescence emission spectra of the dyes Application of this matrix to the raw data is called multicomponenting The multicomponenting occurs as the data is collected therefore it is important to make good quality matrices for each capillary and ensure each is unique For a more detailed explanation of spectral calibration see the ABI PRISM 3100 Genetic Analyzer User s Manual When to Performa A spectral calibration must be performed Spectral Calibration 4 Whenever you use a new dye set on the instrument After the laser or CCD camera has been realigned by a service engineer Ifyou begin to see a decrease in spectral separation pull up and or pull down peaks Instructions for To prepare the Matrix Standards Dye Set D Matrices as an example Preparing the Matrix Standards SteP_ Action 1 Thaw and mix thoroughly the four DS 30 P N 4316100 matrix standard tubes 2 Spin the tubes
31. 6 WF 18 19 20 Intensity vs Bin Number 3000 4000 5000 Intensity vs Scan Number Capillary Number 1 K 2 Condition Number 5 18 Q Value 0 994 Data Source ok Ce 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 Condition number from 4 7 Note This condition number is for dye set D only The condition number is specific for each dye set Click Cancel to close the dialog box Note The software automatically overrides failed capillaries However if you are dissatisfied with a particular profile you can override it with a preferred profile For more information see the ABI PRism 3100 Genetic Analyzer User s Manual Spatial and Spectral Calibrations 4 13 Maintaining the Instrument Overview In This Chapter This chapter contains information about the things you should do to maintain your ABI PRism 3100 Genetic Analyzer Topic See Page Maintenance Task Lists 5 2 Removing Air Bubbles from the Upper Polymer Block 5 4 Checking the Available Disk Space 5 6 Cleaning and Inspecting Syringes 5 8 Removing the Polymer Blocks 5 10 Cleaning the Polymer Blocks 5 11 Putting Fresh Polymer on the Instrument 5 12 Before Installing a Previously Used Capillary Array 5 14 Installing and Removing the Capillary Array 5 15 Storing a Capillary Array 5 16 Shutting Do
32. ABI PRISM 3100 Genetic Analyzer Quick Start Guide for Fragment Analysis AS Applied s HITACHI Copyright 2001 Applied Biosystems All rights reserved For Research Use Only Not for use in diagnostic procedures Information in this document is subject to change without notice Applied Biosystems assumes no responsibility for any errors that may appear in this document This document is believed to be complete and accurate at the time of publication In no event shall Applied Biosystems be liable for incidental special multiple or consequential damages in connection with or arising from the use of this document FOR LIMITED LICENSE INFORMATION PLEASE SEE THE ABI PRISM 3100 GENETIC ANALYZER USER S MANUAL 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 GeneMapper Genotyper and MicroAmp are registered trademarks of Applera Corporation 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 Applera Corporation 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 Corp
33. ANT Failing to replace buffer may lead to loss of resolution precision 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 Performing a Fragment Analysis Run 2 13 Making Buffer fora To prepare 50 mL of 1X Genetic Analyzer buffer Single Run Step Action 1 Add 5 0 mL of 10X Genetic Analyzer buffer with EDTA into a graduated cylinder NAI CHEMICAL HAZARD Genetic Analyzer Buffer with EDTA may cause eye skin and respiratory tract irritation Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Add deionized water to bring the total volume up to 50 mL Mix well Filling the Water IMPORTANT Wear gloves while performing the following procedure and any other time you and Cathode Buffer andle the capillary array glass syringes septa or buffer reservoirs 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 position Tray button Wait until the autosampler has stopped moving then open the instrument doors Remove th
34. BI PRISM 3100 Genetic Analyzer in conjunction with the BioLIMS database system you may want to refer to the ABI PRISM GeneScan Analysis Software User Guide P N 4308923 for information about accessing the database using the analysis program Viewing and Analyzing Data 3 1 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 correction has not been applied There are two formats for viewing the raw data within the ABI PRism 3100 Data Collection Software Inthe Array View page the review data accesses all 16 capillaries Inthe Capillary View page the data is accessed 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 The Select the run to display dialog box opens ee the run to display x Run_demo_3100_2000 04 06_23 x
35. Edit Fill Down Note Press CTRL D or select Edit Fill Down whenever a field is the same for all samples in the plate record For each sample select the appropriate Dye Set from the drop down list 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 This is called a chemometric process 2 20 Performing a Fragment Analysis Run 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 GeneScan36_POP4DefauttModule The table below shows the run module to select based on your run type Analysis Type Run Module GeneScan GeneScan36_POP4DefaultModule SNP SNP36_POP4DefaultModule Note If you need to view or edit a run module file see page 2 29 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 IMPORTANT Runs are scheduled alphanumerically by run module name not by the order indicated in the plate record nor by sample name Performing a Fragment Analysis Run 2 21 To enter sample information and save the plate record continued Step Acti
36. 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 data in the instrument database Note It may take a few moments to retrieve the data 3 2 Viewing and Analyzing Data To view raw data from a completed run continued Step Action 4 Use the scroll features on the Array View page to view the data 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 Capillary Color Raw electropherogram Data display display for selected capillary 3100 Data Collection Software File View Instrument Tools Service Help 12 12 4 122 Intensity vs Run Time mins i 2 4 6 8 10 12 14 16 18 20 2 4 6 8 10 12 14 16 Capillary a 9 Intensity vs Spectral Bin Intensity vs Cap Number Selected capillary to be displayed Use this scroll box to in the center plot view data Viewing and Analyzing Data 3 3 To view raw data from a completed run continued Step Action 5 Alternatively to view electropherogram data from several capillaries at once click the Capillary View tab to display the Capillary View page IMPORTANT Always exit from the Array View and the Capillary View windows During a run do not leave these pa
37. PeBe oo 3 The default name of the run folder is Run_ lt lnstrument name gt _ lt date gt _ lt runID gt An example of a run folder name is shown below J Run_3100_Rabbit_2000 06 09_3 e Run Index number Instrument Year month day name Note The counter for the Run Index number does not reset each day Viewing and Analyzing Data 3 5 Viewing Individual Note All the features of the software are described in the ABI PRISM GeneScan Analysis Sample Files Software User Guide To create a new project and view sample files 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 GeneScan Analysis program GeneScan exe in the following directory D appliedbio Genescan Bin Genedcan exe From the File menu select New Edit Project Sampl Open Ctr O0 Wose Project Close Cti Gaye FS Saye ES Erpa Print Setup CtrltJ Print One Print Ctrl P Exit Click the Project icon to create a new project alt x Create New ay A l L Project Analysis Parameters An untitled Analysis Control window opens From the Project menu select Add Sample Files to open the Add Sample Files dialog box Wi GeneScan Eile Edit untitle ET Bt Adak Renove Semple ries Eid Missing cempe ries Find He
38. Start F Min F Data Processing Size Calling Method C 2nd Order Least Squares Sea Options 3rd Order Least Squares Light Cubic Spline Interpolation oe Local Southern Method C Global Southern Method m Peak Detection p Baselining Peak Amplitude Thresholds BaseLine Window Size 251 Pts m Auto Analysis Only Size Standard Min Peak Half Width 7 Pts Polynomial Degree B Peak Window Size fia Pts Slope Threshold for po Peak Start Slope Threshold for po Peak End GS 400HD szs v want to If you have made changes to the analysis module and you Then save the changes as a new analysis module we recommend that you select Save As from the File menu assign a new name and click OK Note The analysis modules must be stored in the following folder D appliedbio Shared Analysis Sizecaller Params discard the changes click the Close button to close the window Performing a Fragment Analysis Run Viewing and Analyzing Data Overview In This Chapter This chapter includes the following topics Topic See Page Viewing Raw Data from a Completed Run in the Data Collection Software 3 2 Viewing Analyzed Data in GeneScan Software 3 5 Analyzing or Reanalyzing Data in GeneScan Software 3 12 Note This chapter assumes that run data has been extracted into sample files If you are using the A
39. Starting a Run To start a run Step Action 1 Click the green Run Instrument button to begin the scheduled runs 3100 Data Collection Software File View Instrument Tools Service Help HS gt i Run Instrument button A run using GeneScan_POP4DefaultModule takes approximately 45 min Monitoring a Run To monitor a run Step Action 1 Click the Status View tab to monitor the status of the instrument during the run 3100 Data Collection Software x Fie View Instrument Tools Service Help mje Ma me Plate View Run View Status View array view Capilary view r Instrument Condition r Events Time Remaining this run 00 42 06 Wed Apr 12 16 58 24 PDT 2000 a E Laser On E Set run status to STARTING Wed Apr 12 16 58 24 PDT 2000 E oft Started a Gene Scan Run Run_demo_3100_2000 04 12_46 Wed Apr 12 16 58 23 PDT 2000 Sending run module to the instrument E oven On Wed Apr 12 18 37 15 PDT 2000 dis connect data communication port k Wed Apr 12 17 02 00 PDT 2000 MES trout Doors csod 96 RUN PARAMETERS commend reply Wed Apr 12 17 02 03 PDT 2000 Oven Door Closed BEGIN command reply Wed Apr 12 17 02 36 PDT 2000 OVEN TEMPERATURE TOLERANCE 3 0 Oven temperature ti W oAutosampler Return Capillary Array Serial Number demo 4 gt p Errors Wed Apr 12 16 58 29 PDT 2000 Instrument offline
40. Step Action 1 From the Tools menu select Change Polymer Wizard 3100 Data Collection Software File View Instrument BGS Service Help fal Eal ee Plate Editor Module Editor Plate View l Run View Change Polymer Wizard p Instrument Condition Install Capillary Array Wizard Autosampler Calibration Wizard Perform Spatial Calibration Display Spatial Calibration Display Spectral Calibration A warning message displays 5 12 Maintaining the Instrument To put fresh polymer on the instrument continued Step Action 2 If the length of the array on the instrument is the same as the length given in the warning message click OK to begin the Change Polymer Wizard The currently installed capillary array is 36cm 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 i Cancel 3 Follow the directions given in the wizard to put fresh polymer on the instrument Maintaining the Instrument 5 13 Before Installing a Previously Used Capillary 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
41. Within 24 to 48 hours you will receive an e mail reply to your question from an Applied Biosystems technical expert To Obtain Technical You can obtain technical documents such as Applied Biosystems user documents Documents MSDSs certificates of analysis and other related documents for free 24 hours a day You can obtain documents By telephone Through the Applied Biosystems web site B 6 Getting Help Ordering Documents by Telephone To order documents by telephone 1 From the U S or Canada dial 1 800 487 6809 or from outside the U S and Canada dial 1 858 712 0317 2 Follow the voice instructions to order documents for delivery by fax Note There is a limit of five documents per fax request Obtaining Documents Through the Web Site To view download or order documents through the Applied Biosystems web site Step Action 1 Go to http www appliedbiosystems com 2 Click SERVICES amp SUPPORT at the top of the page then click Documents on Demand 3 In the search form enter and select search criteria then click Search at the bottom of the page 4 In the results screen do any of the following Click the pdf icon to view a PDF version of the document Right click the pdf icon then select Save Target As to download a copy of the PDF file Select the Fax check box then click Deliver Selected Documents Now to have the document faxed to you Select th
42. 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 table below 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 Clean the syringe thoroughly by rinsing the inside and outside of the syringe barrel and the syringe tip with warm water For more information about cleaning syringes see the ABI PRISM 3100 Genetic Analyzer User s Manual F NOTA Do not use hot water Hot water can distort the Teflon in the syringe tip IMPORTANT Be sure there is no dried polymer left in the syringes 5 8 Maintaining the Instrument To clean a syringe continued Step Action 2 Rinse the syringe barrel and tip with deionized water 3 Blow dry with compressed air 4 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 du
43. ation for the plate assembly components Component P N 384 Well P N 96 Well Plate Retainer 4317240 4317241 Plate Septa 4315934 4315933 MicroAmp Reaction Plate 4305505 N801 0560 Plate Base 4317236 4317237 Preparing a Plate To prepare a plate assembly Assembly Step Action 1 Secure a clean and dry septa strip on the micro amp optical 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 3 Snap the plate retainer onto the plate and plate base Performing a Fragment Analysis Run 2 11 To prepare a plate assembly continued Step Action 4 Ensure the plate retainer holes are aligned with the holes in the septa strip IMPORTANT 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 2 12 Performing a Fragment Analysis Run Checking and Refilling Fluids Adding or Changing Determine whether to add or change the polymer on the instrument before proceeding Polymer With instrument preparation When to Replace the Buffer If polymer on the instrument is Then less than 1 week old and Ensure there are no air bubbles and then proceed n i i with instrument preparation sufficient in quantity to complete prep
44. ck 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 inside the syringe around the plunger Daily and capillary array ferrule Check database space Delete plate records from the Daily page 5 6 instrument database and archive sample files 5 2 Maintaining the Instrument Weekly Tasks Perform tasks listed below at least once per week Maintenance Task Frequency See Clean the syringes Weekly page 5 8 Clean the water and buffer reservoirs with warm water Weekly Clean the upper and lower polymer blocks Weekly page 5 11 Replace the polymer in the syringes upper polymer Weekly page 5 12 block and capillary array Check the storage conditions of the used arrays Weekly As Needed Tasks Perform tasks listed below as needed Maintenance Task Frequency See Clean the drip trays As needed E Change the array As needed page 5 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 User s Manual Maintaining the Instrument 5 3 Removing Air Bubbles from the Upper Polymer Block Removing Air Note For information about the Change Polymer wizard see the ABI PRISM 3100 Genetic Bubble
45. ctual file size depends on the run module selected For example 400 runs x 16 samples equals approximately 6 4 MB 4 If there is insufficient space a Archive the sample files to another volume b Delete the original files from the drive Checking Database Note The instrument database automatically expands from 2 to 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 the ABI PRISM 3100 Genetic Analyzer User s Manual 2 If the used space is more than 8 GB purge the database of some or all data For instructions see the ABI PRISM 3100 Genetic Analyzer User s Manual Maintaining the Instrument 5 7 Cleaning and Inspecting Syringes When to Clean Thoroughly clean the syringes Syringes 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 Note For more information about cleaning syringes see the ABI PRISM 3100 Genetic Analyzer User s Manual IMPORTANT Wear gloves while performing the following procedure and any other time you handle the capillary array glass syringes septa or buffer reservoirs Removing Syringes To remove the syringes from the instrument Step Action 1 Remove the syringe guard 2 Grasp the polymer reserve syringe just
46. e 30 __ float 1 15 kvots Injection Time o __int 1 600 sec is les36_POP4_1 2kvDefaultModule IGS36_POP4_12 2kvDefaultModule 7 Run Voltage Data Delay Time 1 Run Time 1500 _ int 300 14000 sec float 15 15 kVolts Oven Temperature throughout the run Performing a Fragment Analysis Run 2 29 Editing or Creating To edit an existing run module or to create a new run module a Run Module Step Action 1 Click the Module Editor button on the toolbar EA The Module Editor dialog box opens 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 Click Save As to create a new run module Enter a unique descriptive name and click OK Biswas ES Enter Name of New Module Jry_new_module OK Cancel Note Save cannot be applied to default run modules Modules are saved in the database and are accessed through the module editor for viewing When you are finished click the Close button XJ to exit the Module Editor 2 30 Performing a Fragment Analysis Run About Viewing and Editing Modules for GeneScan Analysis Introduction Viewing and Editing Modules for GeneScan Analysis The analysis module specifies how the raw data is autoanalyzed at the end of the run e g analysis range a
47. e generated If necessary refer to Chapter 5 of this guide 2 2 Performing a Fragment Analysis Run ABI PRISM 3100 Genetic Analyzer User Flowchart for Fragment Analysis User Flowchart Turn on computer OrbixWeb Daemon automatically launches Turn on instrument Launch ABI Prism 3100 Data Collection Software Present autosampler and place fresh deionized water and 1X GA buffer in positions 1 to 4 Place lower polymer block and anode buffer jar on the instrument Clean the capillary array detection window with ethanol if necessary Place cleaned array on ABI 3100 using Install Array Wizard The Install Array Wizard takes you through the steps to Install the array Filla 5 mL reserve syringe and a 250 uL array syringe with ABI 3100 POP 4 polymer Remove bubbles Open Change Polymer Wizard Log the lot number information in the database e Perform spatial calibration e Prepare spectral standards and perform spectral calibration e Combine PCR product with Hi Di formamide internal lane standard i e GeneScan 400HD ROX e Heat denature for 5 minutes at 95 C and immediately chill on ice for 2 minutes e Add plate septa tray cover and plate base and place on autosampler e Complete plate record and link to plate assignment e Press the green arrow gt e Review extracted sample files in GeneScan or GeneMapper software Fail Repeat spatial calibration withou
48. e Email check box then click Deliver Selected Documents Now to have the document PDF format e mailed to you Note There is a limit of five documents per fax request but no limit on the number of documents per e mail request To Obtain Customer To obtain Applied Biosystems training information Training Information Step Action 1 Go to http www appliedbiosystems com 2 Click SERVICES amp SUPPORT at the top of the page then click Training Getting Help B 7 Index Numerics 1X running buffer making fora single run 2 14 A air bubbles removing from upper polymer block 5 4 analysis module viewing and editing 2 31 Analysis Parameters dialog box 3 13 Array View page 3 3 autoextration failure 2 28 autosampler placing plates 2 17 reservoir positions 2 15 B BioLIMS Project field in plate record 2 20 buffer making fora single run 2 14 C capillary array installing removing storing 5 14 to 5 16 chemical safety 1 3 cleaning syringes 5 8 computer checking database space 5 7 checking hard drive space 5 6 creating projects in GeneScan Analysis software 3 6 customer support See technical support B 1 D data analyzing or reanalyzing 3 12 extracting 2 28 viewing analyzed 3 5 viewing raw 3 2 Data Collection software starting 2 6 denaturing samples 2 5 detection cell cleaning 5 14 Display Run Data command 3 2 Documents on Demand B 6 dye set selecting 2 20 supported 2 4 E EDTA
49. e cathode buffer reservoir and water reservoirs from the instrument 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 Rinse the cathode reservoir with 1X Genetic Analyzer buffer and fill to the line with 1X Genetic Analyzer buffer about 16 mL Fill the water reservoirs to the line with quality deionized water about 16 mL 2 14 Performing a Fragment Analysis Run To fill the water and cathode buffer reservoirs continued Step Action 8 Place a clean septa strip on each reservoir and dry the outside of the reservoirs using a lint free wipe Note We suggest labeling the reservoirs to prevent mixing them up F NOTA le 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 9 Place the reservoirs into position on the autosampler as shown below Water reservoir waste Water reservoir Buffer Reservoir Before each batch of runs or at least every 24 hours Filling the Anode Change the anode buffer Every time you fill the polymer block with new polymer To fill the anode buffer reservoir to the fill line with 1X Genetic Analyzer buffer Step Actio
50. e end of the run while the data is being analyzed the Spectral Calibration Result dialog box opens to indicate which capillaries have passed and which have failed The example below for Dye Set D shows failed capillaries which are represented by x and passed capillaries which are represented by dots Passed capillary Failed capillary X E Spectral Calibration Result Found 15 possible spectra for dye set D Please view and edit the spectra x To acknowledge the completed calibration run Step Action 1 In the Spectral Calibration Result 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 the ABI PRISM 3100 Genetic Analyzer User s Manual If a capillary fails it is automatically assigned the spectral profile of its nearest passing capillary to the left If there are no passing capillaries to the left it will be assigned the profile of the nearest passing capillary to the right These capillaries are marked yellow instead of green in the Array View e g page 3 3 IMPORTANT For applications where pull ups and pull downs will cause critical errors we recommend that you repeat the spectral calibration and use a unique spectral for each capillary If the spectral calibration failed or if you do not like the appearance of the passed calibration try on
51. e 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 Spatial and Spectral Calibrations 4 11 Examining a After completing a spectral calibration it is good practice to check the quality of the spectral data for each capillary To display a current spectral calibration profile stored for a dye set Action From the Tools menu select Display Spectral Calibration 3100 Data Collection Software Version 1 0 1 Tools Spectral Calibration The Question dialog box opens Question 2 Spectral Calibration Profile for Dye Set D Step 1 2 4 12 Click Dye set The Select the source to display dialog box opens Fes Select the source to display D j Drop down list of dye sets ox o Spatial and Spectral Calibrations To display a current spectral calibration profile stored for a dye set continued Step Action 3 From the drop down list select Dye Set D and click OK The Matrices for dye set D dialog box opens E Matrices for dye set D x o 4 3 4 5 6 7 8 9 10 11 12 13 14 15 1
52. eee eee 2 27 Stopping a Run and Recovering the Data 0 0 eee eee eee eee 2 28 Viewing Editing or Creating a Run Module 00 00 eee eee eee 2 29 About Viewing and Editing Modules for GeneScan Analysis 2 000 2 31 Viewing and Analyzing Data OVERVIEW daa elit Ud sine ar Mists Materia Mastek eda 3 1 Viewing Raw Data from a Completed Run in the Data Collection Software 3 2 Viewing Analyzed Data in GeneScan Software 0 2 0 0 eee 3 5 Analyzing or Reanalyzing Data in GeneScan Software 0 0000002 eee 3 12 4 Spatial and Spectral Calibrations OVERVIEW 5 ines told hoes seve ke E E ee heed ee ee ge eee PM wba eed 4 1 Performing a Spatial Calibration 22 0 0 ec eee eee 4 2 Performing a Spectral Calibration 2 0 0 cee cence eee 4 6 5 Maintaining the Instrument QVELVIEW he oi tik Sood aed Pow hoe bebe Gaal e a eat ba ede 5 1 Maintenance Task Lists ei iu ea eit ie Sed as alt dee deat eal eae 5 2 Removing Air Bubbles from the Upper Polymer Block 00 0 0 0005 5 4 Checking the Available Disk Space 0 0 00 cee cena 5 6 Cleaning and Inspecting Syringes 0 0 0 0 eee cee eee nee 5 8 Removing the Polymer Blocks 0 0 0 c eee cect eens 5 10 Cleaning the Polymer Blocks 0 0 0 eect nets 5 11 Putting Fresh Polymer on the Instrument 00 00 00 5 12 Before Installing a Previously Used Capillary Array
53. eral safety practices for laboratories Received instruction in specific safety practices for the instrument Read and understood all related MSDSs 7 eT NUE 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 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 Introduction 1 5 Electrical Shock Warnings Laser Warning 1 6 Introduction
54. fety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation e g fume hood For additional safety guidelines consult the MSDS Introduction 1 3 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 Site Preparation and A site preparation and safety guide is a separate document sent to all customers who Safety Guide have purchased an Applied Biosystems instrument Refer to the guide written for your instrument for information on site preparation instrument safety chemical safety and About MSDSs Ordering MSDSs 1 4 Introduction 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
55. form a short term shutdown 5 16 Maintaining the Instrument Shutting Down the Instrument When to Perform Perform the appropriate shut down procedure as follows Each Shut Down Procedure If the instrument will be unattended for Perform this shut down procedure no more than 1 week with a full buffer reservoir Short term IMPORTANT The key to a successful short term shutdown is keeping the capillary array tip in 1X Genetic Analyzer 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 using manual control commands 2 Push the Tray button to move the autosampler forward 3 Fill the buffer reservoir with 1X Genetic Analyzer buffer to the black line of the reservoir 7 Nor NU ale CHEMICAL HAZARD Genetic Analyzer Buffer with EDTA may cause eye skin and respiratory tract irritation Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Fill the other reservoirs with fresh deionized water Secure a septa onto each reservoir and place on the autosampler Close the instrument doors The autosampler will move to position 1 leaving the capillary tips in the buffer reservoir Shut down the computer and turn off the in
56. from gray to yellow Check to ensure this has happened Plate placed in position A Place a plate into No plate in position B alsa S 5 finch o SO 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 Fragment Analysis Run 2 17 Creating a Plate Record About Plate Records Plate records are data tables in the instrument database that store information about the plates and the samples they contain Note A plate record is similar to a sample sheet or an injection list that you may have used with other ABI PRISM instruments Using the Plate Follow the two procedures below to create a plate record with the Plate Editor Editor to Create a See the ABI Prism 3100 Genetic Analyzer User s Manual P N 4315834 for other Plate Record ways to create plate records and for information about importing and exporting plate records Entering Plate Note You cannot create a plate record while a run is in progress Record Information eee 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 In the Plate View page click New Or double click the Plate Editor button on the toolbar San EEE HH The Plate Ed
57. ges open for extended periods This may cause unrecoverable screen update problems Leave the Status View window open File View Instrument Tools Service Help els 1 Plate View Run View Status View Array View Capilary view Selec copia a adder Select check boxes of the A ri Seiren m Ran Time ia be displayed EEE eee 2 124 122 en vs Run Time mins aE ae 3 12 4 12 2 icone vs Run Time mins EE 4 12 4 122 Em vs Run Time mins al T gt l 3 4 Viewing and Analyzing Data Viewing Analyzed Data in GeneScan Software Introduction Locating Sample Files Run Folder Default Name After a run has been extracted to sample files you can use the ABI PRISM GeneScan Analysis Software to view the electropherogram data both raw and analyzed Refer to the ABI PRISM GeneScan Analysis Software User Guide 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 following directory D appliedbio 3100 DataExtractor ExtractedRuns An example of the run folder and its contents is shown below amp D appliedbio 31 00 D ataE xtractor E xtractedRuns Run_demo_3100_2001 06 04_70 Mi Ea File Edit View Help 6 3_CO1_05 fsa E 4_D01_07 fsa E 5_E01_09 tsa E 6_F01_11 fsa E 7_G601_13 fsa E 8_H01_15 fsa E 9 402_02 fsa Run_demo_3100_2001 06 04_70 log PERE
58. gment data using the ABI PRISM GeneScan User Guide Analysis Software or the ABI PRISM GeneMapper Analysis Software an abbreviated procedure for how to do a typical ABI PRISM 3100 Genetic Analyzer Quick Start 4315833 sequencing run view and analyzer run data and Guide for Sequencing perform common maintenance operations information on a procedure for block cleaning ABI PRISM 3100 Genetic Analyzer Block Cleaning 4322930 1 2 Introduction Safety Documentation User Attention Words Chemical Hazard Warning Chemical Waste Hazard Warning 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 Nor Ne 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 ONY ela 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 NAY NSE CHEMICAL HAZARD Some of the chemicals used with Applied Biosystems instruments and protocols are potentially ha
59. he View menu select Preferences or click the Preferences button on the toolbar EA 3100 Data Collection Software The dialog box has two pages and are described below Setting Preferences 2 8 Performing a Fragment Analysis Run The table below 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 Note Avoid spaces between words or any special characters Data Analysis Page Setting Preferences Eg Data Collection Data Analysis AutoAnalysis IV AutoAnalysis On BioLIMS Enable BioLIMS User Name F er Password assword Database Name Biotin Server Name Fer er Sample File Name Prefix Format Sample Name z fiven Position x fenone z lt none The table below describes the preferences that can be set within this page Data Analysis Page Settings Preference Description AutoAnalysis On Select AutoAnalysis ON to have samples automatically analyzed by the analysis software after the run Note You will still be able to reanalyze your sample data at a later time BioLIMS Use these settings to have data extracted to a BioLIMS database instead of to sample files on the hard drive Performing a Fragment Analysis Run 2 9 Data Analysis Page Setti
60. ifting or moving the instrument Safety training for proper lifting techniques is recommended Do not attempt to lift or move the instrument without the assistance of others Depending on the weight of the instrument this action may require two or more people Introduction 1 7 Performing a Fragment Analysis Run Overview In This Chapter This chapter includes the following topics Topic See Page Before You Begin 2 2 ABI Prism 3100 Genetic Analyzer User Flowchart for Fragment Analysis 2 3 Sample Preparation 2 4 Starting the Data Collection Software 2 6 Setting Software Preferences 2 8 Working with Plate Assemblies 2 11 Checking and Refilling Fluids 2 13 Placing the Plate onto the Autosampler 2 17 Creating a Plate Record 2 18 Linking and Unlinking a Plate 2 24 Starting and Monitoring the Run 2 27 Stopping a Run and Recovering the Data 2 28 Viewing Editing or Creating a Run Module 2 29 About Viewing and Editing Modules for GeneScan Analysis 2 31 Performing a Fragment Analysis Run 2 1 Before You Begin Assumptions The procedures in this chapter make the following assumptions The computer and the instrument have been correctly configured The instrument has been calibrated spatial and spectral calibrations have been successfully run If necessary refer to Chapter 4 of this guide There is sufficient space on the computer hard drive to store the data that will b
61. ing procedure and any other time you handle the capillary array glass syringes septa or buffer reservoirs To store the capillary array off of 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 5 15 6 Replace the cover over the detection cell 7 a Fill a buffer reservoir with fresh 1X Genetic Analyzer buffer and cover with a septa strip b Insert the capillary tips into the buffer 8 a Fill a storage cap from a new array or a 5 mL ABI 310 buffer vial P N 401955 with septa and caps P N 401956 with 1X Genetic Analyzer buffer b Insert the rod end of the capillary array into the cap vial 9 Wrap the tube with laboratory film such as Parafilm to prevent evaporation 10 Store the capillary array upright 11 Check the 1X Genetic Analyzer buffer level in the reservoir and tube weekly Store the capillary array on the instrument when the capillary array will be unused for less than one month To store the capillary array on the instrument follow the instructions to per
62. ions In addition formamide is often supplied in glass bottles which when opened exposes the formamide to the air and allows it to absorb water Water reacts slowly with formamide to produce formic acid methanoic acid and ammonia The ionic products of this reaction cause two problems They compete significantly with the larger DNA ions for injection into the capillary resulting in weaker signals They react with the DNA causing degradation of the sample The figure below shows the effect of the ionic products from formamide on electrokinetic injection Samples prepared using high Samples prepared using quality deionized formamide commercial formamide Deionized formamide containing an alkaline stabilizer prevents these problems Preparing Formamide A 1 Materials Required The following materials are recommended for this procedure Material Description Formamide The raw prior to deionization formamide should be 99 5 purity or greater with low water content Packed under an inert gas Have a conductivity of approximately 100 uSiemens cm or less Note Siemens formerly called mho are the units of measurement for specific conductance or conductivity lon exchange resin Mixed bed resin containing the following strong ion exchange functional groups R SO as H form cation R CH N CH3 3 as OH form anion These groups are attached to a styrene divinylbenzene matrix
63. irs filling 2 13 to 2 16 positions on autosampler 2 15 Results Control window GeneScan Analysis Software 3 9 to 3 11 run monitoring 2 27 skipping pausing stopping 2 28 starting 2 27 run module selecting 2 21 viewing editing and creating 2 29 S safety discussed 1 3 to 1 7 manual 1 2 sample files default naming convention 2 19 maximum length for 2 19 viewing 3 6 viewing in GeneScan Analysis software 3 9 to 3 11 Sample Info button GeneScan Analysis software 3 8 sample preparation 2 4 Sample Results button GeneScan Analysis software sample volumes required for a run 2 4 Select the run to display dialog box 3 2 size standard szs files selecting 3 12 Skip to Next Run button 2 28 software setup 2 8 to 2 26 software preferences 2 8 spatial calibration discussed 4 2 to 4 5 spectral calibration discussed 4 6 to 4 13 starting GeneScan Analysis software 3 6 syringes discussed 5 8 3 8 T technical support B 1 to B 7 e mail address B 1 Internet address B 6 regional sales offices B 4 to B 5 telephone fax North America B 3 B 4 training obtaining information B 7 Index 2 U upper polymer block removing air bubbles 5 4 V viewing raw color data in Data Collection software 3 2 to 3 4 sample files in GeneScan Analysis software 3 9 to 3 11 volume of sample required for a run 2 4 W waste safety 1 3 water and cathode buffer reservoirs filling 2 13 to 2 16 Y yellow capillary in Array View 4 11
64. itor dialog box opens Plate Editor x Plate Name my_plate_record Application Sequencing GeneScan C Spectral Calibration Plate Type fo6 Welr z Comments This is an example plate record 2 18 Performing a Fragment Analysis Run To enter plate record information continued Step Action 3 Use the Plate Editor dialog box to name your plate and to specify the application and plate type Entering comments is optional In the Plate Editor dialog box a Name your plate b Specify the application c Select the plate type d Enter any comments 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 displays Plate Editar File Edit Plate Name KPA wel Sample Neme Dyes color Into Color Comnent BIoLIMS Project Dye Set Runt At B1 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 Use Edit Copy and Edit Fill Down whenever a field is the same for all samples in the plate record Note Inthe default naming convention the sample name you type is incorporated into the sample file name For example
65. justment 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 legroom for adequate movement ELECTRICAL SHOCK HAZARD Severe electrical shock which could cause physical injury or death can result from working on an instrument when the high voltage power supply is operating To avoid electrical shock disconnect the power supply to the instrument unplug the power cord and wait at least 1 minute before working on the instrument NONIN 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 NZ Tie LASER BURN HAZARD An overheated laser can cause severe burns if it comes in contact with the skin DO NOT operate the laser when it cannot be cooled by its cooling fan Always wear laser safety goggles Moving and Lifting PXA PHYSICAL INJURY HAZARD Improper lifting can cause painful and the Instrument Sometimes permanent back injury Use proper lifting techniques when l
66. n 1 Remove the anode buffer reservoir by firmly pulling down and twisting slowly 2 Discard the used buffer appropriately 3 Clean and rinse the reservoir with deionized water and then rinse with buffer 4 Fill the reservoir to the fill line with fresh 1X Genetic Analyzer buffer about 9 mL Fill line GR1878 5 Put the anode buffer reservoir on the instrument Note The meniscus should line up with the fill line Performing a Fragment Analysis Run 2 15 To fill the anode buffer reservoir to the fill line with 1X Genetic Analyzer Step Action 6 If the reservoir fills with fluid repeat this procedure to discard and replace the Genetic Analyzer buffer Note The reservoir could fill during bubble clearing 2 16 Performing a Fragment Analysis Run 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 When the plate is correctly positioned the plate position indicator on the Plate View page changes
67. nd size standard parameters To view or edit a GeneScan analysis module gsp file 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 GeneScan Bin From the File menu select Open Select the Analysis Parameters icon all x Open Existing BA Bo Bee L Project Sample Analysis Size Parameters Standard Select the analysis module you want to view or edit The analysis modules are stored in the following directory D appliedbio Shared Analysis Sizecaller Params Look in I Params 7 l c Bgl a GS3504nalysis gsp a GS400CubicAnalysis gsp a GS400HDAnalysis gsp a GS4000rd2Analysis gsp a GS5004nalysis gsp Fiename Files of type Analysis Params File GSP z Cancel Click Open This opens the analysis module Performing a Fragment Analysis Run 2 31 2 32 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 User Guide m Analysis Range Size Call Range Full Range Full Range This Range Data Points This Range Base Pairs
68. ng Formamide Getting Help Technical Support Contacting You can contact Applied Biosystems for technical support Technical Support 4 By e mail By telephone or fax Through the Applied Biosystems web site 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 Technical Documents following the telephone information below To Contact Technical You can contact Applied Biosystems Technical Support by e mail for help in the Support by E Mail following product areas Product Product Area E mail address Genetic Analysis DNA Sequencing galab appliedbiosystems com Sequence Detection Systems Real Time pcrlab appliedbiosystems com PCR and PCR Protein Sequencing Peptide and DNA corelab appliedbiosystems com Synthesis Getting Help B 1 B 2 Getting Help Product Product Area E mail address o gt gt gt gt o Biochromatography BioCAD SPRINT VISION and INTEGRAL Workstations and POROS Perfusion Chromatography Products Expedite 8900 Nucleic Acid Synthesis Systems MassGenotyping Solution 1 MGS1 Systems PNA Custom and Synthesis Pioneer Peptide Synthesizers Proteomics Solution 1 PS1 Systems ICAT Reagent FMAT 8100 HTS Sys
69. ngs continued Preference Description 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 and contains the capillary number and the date Sample Name Taken from the Plate Editor spreadsheet entry See page 2 18 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 This is also called the Run ID Therefore in the Data Analysis page example shown above the sample name will be Sample Name_Well Position_Capillary Number fsa IMPORTANT Using additional filters will create very long file names which may affect down stream software analysis 2 10 Performing a Fragment Analysis Run Working with Plate Assemblies Plate Assembly Components The plate assembly components are assembled as follows Plate Retainer Plate Septa MicroAmp Reaction Plate Plate Base The table below contains ordering inform
70. on 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 2 9 Analysis Module 1 sno selection GS3504nalysis gsp GS400CubicAnalysis qsp GS400HDAnalysis gsp GS4000rd24nalysis qsp GSs004nalysis gsp The table below shows which analysis module to select based on the number of fragments in your size standard If using size standard Select this analysis module GS120 GS120Analysis gsp GS400HD GS400HDAnalysis gsp GS350 GS350Analysis gsp GS500 GS500Analysis gsp GS500 GS400CubicAnalysis gsp GS4000rd2Analysis gsp a These modules are for advanced users with specific sizing needs See the AB PRISM GeneScan Analysis Software User Guide 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 GeneScan Analysis Software User Guide P N 4308923 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 2 22 Performing a Fragment Analysis Run To enter sample information and save the
71. or buffer reservoirs CHEMICAL HAZARD POP polymers may cause eye skin and respiratory tract irritation Please read the MSDS for the polymer you are using 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 3100 Data Collection Software Version 1 0 1 Toos The Install Replace Capillary Array Wizard opens Install Replace Capillary Away Wizard 3 Follow the directions given in the wizard to replace or install an array Maintaining the Instrument 5 15 Storing a Capillary Array When to Store off the Instrument Storing the Capillary Array off the Instrument When to Store on the Instrument Storing a Capillary Array on 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 reuse the capillary array do not let the capillaries dry out Store the capillary array with both ends in 1X Genetic Analyzer buffer IMPORTANT Wear gloves while performing the follow
72. oration 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 Printed in the USA 07 2001 Part Number 4315832 Rev C Contents 1 Introduction OVEIVIEW oo einen bce deed ak oie ibe wet we hgh haa eid 1 1 About This Manival s dees dees iepen eeoa Seed ee RE Ga EGG atte EN egda Peed s 1 2 For More ntormation 3 3 o sik a a a e as ie el dete PE ee 1 2 Safety roae discs Rovio eye ew AE PW ee Ena e E he ace 1 3 2 Performing a Fragment Analysis Run OVEIVIEW aea E wade ais Weta ares RPO go Hhk eeu Garey ete oe as 2 1 Before YOu Begin is os bees het pent BS Gack beak eine ated tae an ee Sl 2 2 ABI PrisM 3100 Genetic Analyzer User Flowchart for Fragment Analysis 2 3 Sample Prepatationt c bade ibe te Me ee oneal Wainy SGM E cokes ey 2 4 Starting the Data Collection Software 2 0 eee cee 2 6 Setting Software Preferences 2 0 00 cee eee eee eee nea 2 8 Working with Plate Assemblies 0 0 cece eee nee eee n ees 2 11 Checking and Refilling Fluids 0 0 0 0 ee eee eens 2 13 Placing the Plate onto the Autosampler 20 e cece cece teen nee 2 17 Creating a Plate Record eoira casi e nai ete ee Se ee Gee ee eto 2 18 Linking and Unlinking a Plate 2 eee eens 2 24 Starting and Monitoring the Run 2 2 ec
73. ory 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 2 6 Performing a Fragment Analysis Run Starting the Data To start the Data Collection software Collection Software Step Action 1 From the Start menu point to Applied Biosystems and select 3100 Data Collection Software Note To create an Applied Biosystems shortcut a Navigate to 3100Collection bat in the following directory D appliedbio 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 Plate View window displays as shown below fi 3100 Data Collection Software TE all sim cal 2 un view Status View array View Capitry view Performing a Fragment Analysis Run 2 7 Setting Software Preferences Introduction The Data Collection software preferences are set during instrument installation however you can view or change these preferences in the Setting Preferences dialog box Viewing the Setting To view the Setting Preferences dialog box Preferences Dialog Box Data Collection Page Step Action 1 From t
74. osampler 3100 Data Collection Software x Fie View Instrument Tools Service Help maaa m e l Plate View Run View Status View Array View Capilary view Pending Plate Records Plate Name Application Wells Status Place a plate into plate position B Linked Plate Records Plate Name Application SpectralCalibrati Spectral Wells Status A 96 pending Processed Plate Records Plate Name Application Wells Status 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 To review the run schedule before beginning the run click the Run View tab 2 Click the Run Instrument button on the toolbar to begin the run The spectral calibration run takes approximately 30 min Run Times The table below 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 4 10 Spatial and Spectral Calibrations Spectral Calibration Result Box When a Capillary Fails When the Calibration Fails At th
75. oss 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 the ABI PRISM 3100 Genetic Analyzer User s Manual If the calibration continues to provide unsatisfactory results see If the Calibration Fails on page 4 5 Click OK to close the Perform Spatial Calibration window and to send the passing calibration to the instrument The Question dialog box opens Q Save spatial calibration data Ifyou choose Yes spatial calibration data will be saved in the database and sentto the instrument 4 4 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 Data Collection software database delete this data and use datafroma a Click No previous run b Override the current spatial calibration map 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 Reposition the array window in the detection cell and then repeat the calibration Spatial and Spectral Calibrations 4
76. point to Data Acquisition and select Extract data into sample files 3100 Data Collection Software Version 1 0 1 File View Toos Service Help Rony Skip IG MEX Ronn Plate view pouse Array View Capilary View Stay Manual Control Data Acquisiticn d Set Cobr Flate Name Applicat GOCE RUT Status toamne Display Ruri Dala Display EPT Data Fytrart data intn sample fles Look for the message Sample Files Successfully Extracted in the Status bar Note The extracted data is unanalyzed Use GeneScan Analysis software to analyze the sample files 2 28 Performing a Fragment Analysis Run Viewing Editing or Creating a Run Module Introduction The run module specifies information about how the sample is run e g the duration of the run the run temperature and the injection time Viewing a Run To view a run module Module Step Action 1 Click the Module Editor button on the toolbar The Module Editor dialog box opens 2 In the Modules group box click the GeneScan tab 3 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 Module Editor G536_POP4 10kvDefaultModule Run Te ure int 18 65 Deg C G536_POP4_13kvDefaultModule il Cap Fil Volume 184 __ int 4 200 steps PreRunVotage t5 float 0 15 kvots Pre Run Time tso int 1 1000 sec Injection Votag
77. re Operating the Instrument Safe and Efficient Computer Use For chemicals not manufactured or distributed by Applied Biosystems call the chemical manufacturer 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 which 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 gen
78. record Want to see the effect of changing analysis parameters on your data Analyzing or To analyze or reanalyze sample files Reanalyzing 5 aa Sample Files tep cuon 1 If not already open start the GeneScan Analysis software 2 Create a project as described in steps 2 to 6 beginning on page 3 6 Or open an existing project by selecting Open from the File menu 3 CTRL click the dye color field to set the size standard color Demo prj Analysis Control Analyze I Print Results N Sample File SI oo 403 oo1fsa 2 BE aj 400 404 oo2tsa The diamond lozenge in the red color field indicates that the red size standard will be used for the analysis 4 From the Size Standard pop up list select the correct size standard szs file for the sample files in the table Nore File Edit Project Sample Settings View wi lt Collection Setting gt GS 950977 ss 3 GS 350 Allszs Analyze I Print Results GS 350 250 ses GS 400HD szs GS 500 377 szs GS 500 All szs v GS 500 250 szs Sample File P43 502 016 381 p27 fsa P43 B03 012 376 p28 fsa 3 12 Viewing and Analyzing Data To analyze or reanalyze sample files continued Step Action 5 From the Parameters pop up list select the correct analysis parameters gsp file for the sample files in the table Wi GeneScan Eile Edit Project Sample Settings View Windows Help Demo prj Analysis Control
79. reme Ww isplayo RemenuenWispiaie Previous Displays 3 6 Viewing and Analyzing Data To create a new project and view sample files continued Step Action 5 In the Add Sample Files dialog box a Select the folder containing the sample files of interest b Click Add All to add all the files in the folder to the project list or Add to add only selected files c Use the Add All Remove or Remove All buttons as necessary to list all the files that you want in the File Name list box These are the files that will be added to the Sample Manager Note If you hold down the Suirt key and press ENTER you can select all continuous files i e 1 2 3 4 etc If you hold down the ControL key and press ENTER you can select files out of order i e 1 3 7 12 etc Add Sample Files 2 x o Lookin Sy Run_demo_3100_2000 0323 14 E E JAnalyzed Failed 400_c03_005 E 400_Fo4_012 Analyzed Success 400_co4_006 400_c03_013 Select the folder Unanalyzed 400_p03_007 E 400_co4_o14 containing 400_A03_001 3 400_po4_o08 E 400_Ho3_o15 3 2 400_A04_002 5 400_E03_009 E 400 _H04_016 sample files 3 400_B03_003 400_E04_o10 3 400_B04_004 400_Fos_o11 File name 3 fsa 400_404_002 fsa 400_403_001 fsa Files of type all Readable files fsa z TENG ce 400_A04_002 400_B03_003 TEA Add the Remove All selected files to this list box Fini
80. ring 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 i 0 05 0 1 0 15 02 0 25 2 Verify that the ferrule is firmly seated in the end of the syringe Maintaining the Instrument 5 9 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 5 8 3 Disconnect the capillary array from the polymer block Press the Tray button Open the instrument oven and detection block doors Loosen the capillary array nut Pull out the polymer block part way Remove the detection cell from the detection block 929 5 p Remove the capillary array sleeve from the polymer block g If the capillary array is to be reused store it as described on page 5 16 Disconnect the lower polymer block by unscrewing the polymer block tube fitting on the upper polymer block s under right side Grasp the upper polymer block with two hands and pull it straight out 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 Step Action 1 Remove the anode reservoir and properly dispose of the b
81. s 4alyzer User s Manual P N 4315834 To clear air bubbles from the upper polymer block use the Change Polymer Wizard in the steps below 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 o D N c o Polymer block tube 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 GR1877 5 4 Maintaining the Instrument To clear air bubbles from the upper polymer block use the Change Polymer Wizard in the steps below continued Step Action 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 Maintaining the Instrument 5 5 Checking the Available Disk Space Introduction Before a run or a batch of runs check the available space to ensure there is sufficient space to store the data you will
82. s conductivity Preparing EDTA Preparing the Formamide Within the range or measurement the conductivity meter should be routinely calibrated to 10 uSiemens cm or less Calibrate the meter using standard potassium chloride solutions that are traceable to the National Institute of Standards and Technology NIST Because temperature affects conductivity samples must be brought to room temperature before measuring the conductivity Alkaline EDTA ethylenediaminetetraacetic acid is added to the deionized formamide to stabilize it and to facilitate the electrokinetic injection of DNA To minimize the amount of water added to the formamide a concentrated 200 mM stock solution of the EDTA is added NAAI CHEMICAL HAZARD EDTA may cause eye skin and respiratory tract irritation Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves To prepare the 200 mM EDTA stock solution Step Action 1 Add 7 44 g of Na EDTA to 70 mL of deionized water and stir 2 While stirring slowly adjust to pH 8 0 8 8 by dropwise addition of a concentrated solution of sodium hydroxide Note This helps the EDTA to dissolve over time because the EDTA has a limited solubility until the pH is increased 3 Dilute to 100 mL with deionized water Store at 4 C CHEMICAL HAZARD Formamide is harmful if absorbed through the skin and may cause irritation to the eyes skin
83. sh Cancel 6 Click Finish to close the Add Sample Files dialog box The sample files are added to the Analysis Control window Note Only the first 20 characters of the sample file name are displayed in the Analysis Control window untitled Analysis Control D Analyze T Print Results Sample File Size Standard Parameters 400 403 001 fsa 2GS 400HD szs gt lt GS400HDAnalysis gsp gt E 400 404 002 tsa isGS 400HD szs gt Js GS400HDA nalysis gsp gt E 400 B03 003 fsa 2GS 400HD szs gt Js GS400HDA nalysis gsp gt E 400 B04 004 fsa 2GS 400HD szs gt Js GS400HDA nalysis gsp gt E 400 c03 005 fsa 2GS 400HD szs gt Js GS400HDA nalysis gsp gt 400 C04 006 fsa 2GS 400HD szs gt is GS400HDA nalysis gsp gt 00 D03 007 fsa 2GS 400HD szs gt Js GS400HDA nalysis gsp gt 400 D04 003 fsa isGS 400HD szs gt Is GS400HDA nalysis gsp gt 400 03 009 fsa isGS 400HD szs gt is GS400HDAnalysis gsp gt 400 E04 010 fsa 2GS 400HD szs gt isGS400HDAnalysis gsp gt 400 FO3_011 fsa GS 00HD szs gt 1s GS400HDA naiysis gsp 400 G03 _013 fsa SGS 00HD szs gt p lt GS400HDAnaiysis gsp gt 00 G04 014 fsa SGS 00HD szs gt p lt GS400HDAnaiysis gsp gt ma ZS z 2 cn co ra CEAAL E H2200 400 H03 015 fsa 2GS 400HD szs gt lt GS400HDAnalsis gsp gt Viewing and Analyzing Data 3 7 To create a new project and view sample files continued Step Action
84. ss 1 then press 6 1 508 383 7855 Chemiluminescence Tropix 1 800 542 2369 U S only or 1 781 271 0045 1 781 275 8581 LC MS Applied Biosystems MDS Sciex 1 800 952 4716 1 508 383 7899 a 5 30 AM to 5 00 PM Pacific time b 8 00 AM to 6 00 PM Eastern time c 9 00 AM to 5 00 PM Eastern time To contact Applied Biosystems Technical Support or Field Service outside North America use the telephone or fax numbers below Region Telephone Fax Eastern As ia China Oceania Australia Scoresby Victoria 61 3 9730 8600 61 3 9730 8799 China Beijing 86 10 64106608 or 86 10 64106617 86 800 8100497 Hong Kong 852 2756 6928 852 2756 6968 India New Delhi 91 11 653 3743 3744 91 11 653 3138 Korea Seoul 82 2 593 6470 6471 82 2 593 6472 Malaysia Petaling Jaya 60 3 79588268 60 3 79549043 Singapore 65 896 2168 65 896 2147 Taiwan Taipei Hsien 886 2 2358 2838 886 2 2358 2839 Thailand Bangkok 66 2 719 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 2 532 4484 32 0 2 582 1886 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 Italy Milano 39 0 39 83891 39 0 39 838 9492
85. strument Performing a To perform a long term shutdown Long Term Shutdown Step Action 1 Follow the procedure on page 5 16 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 5 8 Upper polymer block For instructions see page 5 10 Lower polymer block For instructions see page 5 10 3 Remove from the autosampler Plate assemblies Reservoirs Wipe the autosampler 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 Maintaining the Instrument 5 17 To perform a long term shutdown continued Step Action 7 Wash the syringes polymer blocks and reservoirs with warm water Rinse with deionized water IMPORTANT Make sure all parts of the array are completely dry before long term storage 5 18 Maintaining the Instrument Preparing Formamide Deionizing and Storing Formamide About Formamide Denaturation Agent Problems with Commercial Formamide Formamide is used to denature the DNA samples before placing them on the ABI PRISM 3100 Genetic Analyzer IMPORTANT High quality formamide is essential for reproducible data Formamide purchased from commercial suppliers is often contaminated with variable amounts of water and undesirable organic and inorganic
86. t 5 uSiemens cm Continue with To complete purification of deionized formamide below Note If the conductivity is not lt 5 uSiemens cm after about 4 5 hr of mixing repeat the entire procedure using a new lot of formamide and new resin Note Starting formamide with a higher purity and lower conductivity deionizes more efficiently To complete purification of deionized formamide Step Action 1 Vacuum filter the deionized formamide using a 0 2 or 0 4 um nylon or teflon filter 2 Measure the final volume of deionized formamide 3 Add the required volume of 200 mM EDTA to the deionized formamide to achieve a final concentration of approximately 0 3 mM EDTA Note After adding the EDTA the final conductivity of the formulation is increased to approximately 30 uSiemens cm Use the equation below to calculate the volume of EDTA to add Vepta uL 1 5V Form mL Where VEDTA uL Volume of EDTA to add in microliters Veorm miy Measured volume of formamide in milliliters Sample calculation with a final volume of 90 mL formamide Veptau 1 5 x 90 135 uL 4 Immediately aliquot the formamide into smaller polypropylene tubes and store at 15 to 20 C for up to about 6 mos Using the When ready for use thaw and completely use one tube at a time before opening and Formamide exposing another tube Store the tubes at 4 C during the day for intermittent use Otherwise refreeze them A 4 Prepari
87. t filling the capillary Remove capillary array and clean capillary window Inspect window for damage i e scratches or cracks Refill capillary with fresh polymer and rerun spatial calibration If the spatial calibration continues to be unsuccessful refer to the ABI Prism 3100 User Manual troubleshooting chapter Performing a Fragment Analysis Run 2 3 Sample Preparation Dye Set The ABI PRISM 3100 Data Collection Software version 1 0 1 supports Dye Set DS 30 DS 31 DS 32 DS 33 DS 02 and the ABI PRism Linkage Mapping Sets LD20 MD10 and HD5 Dye Sets Dye Chemistry and Applications Dye Set Dye Chemistry Application Kit DS 30 D 6 FAM blue HEX Linkage Mapping green NED yellow Sets LD20 MD10 and ROX red HD5 Custom Oligos DS 31 D New 4 Dye Chemistry Linkage Mapping Custom 6 FAM blue VIC green Oligos NED yellow ROX red Mouse Mapping Markers Custom Oligos DS 32 F 5 FAM JOE NED AmpFSTR products ROX Stockmarks DS 33 G5 6 FAM VIC NED PET AmpFSTR Identifiler 5 dye chemistry for high Liz Custom Oligos throughput genotyping DS 02 E5 dR110 dR6G dTAMRA SNAPshot Multiplex Kit dROX LIZ Pooling Ratios The fluorescent dyes are detected with different efficiencies The pooling ratio or amount of each dye labeled product added with respect to the other products in the pool should
88. tems Mariner ESI TOF Mass Spectrometry Workstations Voyager MALDI TOF Biospectrometry Workstations CytoFluor 4000 Fluorescence Plate Reader tsupport appliedbiosystems com LC MS Applied Biosystems MDS Sciex support sciex com Chemiluminescence Tropix tropix appliedbiosystems com To Contact Technical To contact Applied Biosystems Technical Support in North America use the telephone Support by Telephone or Fax North America or fax numbers in the table below Note To schedule a service call for other support needs or in case of an emergency dial 1 800 831 6844 then press 1 Product Product Area Telephone Fax ABI PRism 3700 DNA Analyzer 1 800 831 6844 then press 8a 1 650 638 5981 DNA Synthesis 1 800 831 6844 press 2 then press 14 1 650 638 5981 Fluorescent DNA Sequencing 1 800 831 6844 press 2 then press 2 1 650 638 5981 Fluorescent Fragment Analysis including GeneScan applications 1 800 831 6844 press 2 then press 3a 1 650 638 5981 Integrated Thermal Cyclers ABI PRism 877 and Catalyst 800 instruments 1 800 831 6844 press 2 then press 4a 1 650 638 5981 ABI PRISM 3100 Genetic Analyzer 1 800 831 6844 press 2 then press 64 1 650 638 5981 Peptide Synthesis 433 and 43x Systems 1 800 831 6844 press 3 then press 1a 1 650 638 5981 Protein Sequencing Procise Protein
89. tinue on to Viewing Successful Results and Saving the Data below failed an error message box opens providing some information about the reason for the failure Perform Spatial Calibration 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 5 Spatial and Spectral Calibrations 4 3 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 evaluating the profile see the ABI PRISM 3100 Genetic Analyzer User s Manual P N 4315834 Spatial Calibration Profile x 400007 300007 200007 100007 0 10000 o 20 a0 60 80 100 120 140 160 180 200 220 240 260 Intensity vs Pixel Number Capillary Number 1 2 3 4 5 6 a eal 7 8 g yl esa by EN br eo Be SAIE Capillary Position fiz 27 2 58 f3 88 f103 119 134 fas 165 180 figs R10 225 241 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 Reposition one or more of the red crosses To move a cr
90. uffer Inspect the O rings Grasp the lower polymer block and pull it straight out 2 3 4 Disconnect the polymer block tube fitting 5 10 Maintaining the Instrument Cleaning the Polymer Blocks When to Clean the Clean the upper and lower polymer blocks once a week Polymer Blocks Before replacing the polymer on the instrument When the polymer has been on the instrument 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 on page 5 10 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 5 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 Note To purchase a 3100 Polymer Cleaning Kit P N 4322931 visit our web site at http www appliedbiosystems com and select Store Maintaining the Instrument
91. wn the Instrument 5 17 Maintaining the Instrument 5 1 Maintenance Task Lists Overview This section lists common tasks required to maintain your Genetic Analyzer in good working condition The lists are divided into tables based on how often you should perform each task IMPORTANT Wear gloves anytime you handle the capillary array glass syringes septa or buffer reservoirs Daily Tasks Perform tasks listed below at least once per day Maintenance Task Frequency See Ensure the reservoir septa are firmly seated and flat Before each run DnE Ensure the plate assemblies were put together properly Before each run page 2 11 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 Genetic Analyzer buffer Daily page 2 13 reservoirs on the instrument Check for bubbles in the polymer block and polymer Daily page 5 4 block channels and remove IMPORTANT Ensure that all bubbles have been pushed through the peek tubing Check the loading end header to ensure the capillary tips Daily are not crushed or damaged Check the level of polymer in the polymer reserve Daily or before syringe to ensure there is at least 1 mL each run Che
92. y based on your experimental results For loading mix 1uL of pooled PCR products with 10 uL of formamide size standard mix Denaturing Samples To denature the samples Step Action 1 Heat samples at 95 C for 3 5 min There are several acceptable options for covering samples during denaturation MicroAmp Clear Adhesive Films P N 4306311 MicroAmp Caps 12 Strip P N 801 0534 MicroAmp Caps 8 Strip P N 801 0535 MicroAmp Optical 96 well Reaction Plates P N N801 0560 MicroAmp 384 well Reaction Plates P N 4305505 2 Place immediately on ice for at least 5 min before loading gt gt gt OH Performing a Fragment Analysis Run 2 5 Starting the Data Collection Software Before You Begin Before starting the ABI PRiISM Data Collection Software Step Action 1 Ensure the computer and monitor are powered on IMPORTANT The computer must be powered on before the instrument The default user name is 3100User and the default password is blank Ensure the ABI PRiSM 3100 Genetic Analyzer is powered on and the green status light is on solid not flashing Ensure OrbixWeb Daemon is running by finding its button on the Windows NT taskbar A Start E OrbixWeb Daemon If OrbixWeb Daemon is not running go to the Start menu point to Applied Biosystems and select OrbixWeb Daemon Note To create a shortcut a Navigate to orbixd exe in the following direct
93. zardous 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 with any chemicals or hazardous materials Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals e g safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation e g fume hood For additional safety guidelines consult the MSDS 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 PNG Ie 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 chemicals Wear appropriate personal protective equipment when handling chemicals e g safety glasses gloves or protective clothing For additional sa

ABI PRISM ® 3100 Genetic Analyzer

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ABI PRISM &reg; 3100 Genetic Analyzer

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ABI PRISM ® 3100 Genetic Analyzer