March 22, 2000

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1 March 22, 2000

2 ...1 run...2 Run Data Extractor Data Collection Ver Module Sample sheet(plate record) Sequencing Analysis Software Ver.3.6NT Seq file Spacing...6 Maintenance Shutdown Capillary Cuvette Flush Inline filter Sheath flow syringe Database clean-up Tank level calibration Standard sample Spectral calibration Spatial calibration...12 Troubleshooting...13 Utility softwares RenameMan WS_FTP Chromas...17 Appendix-1: 96-well plate sample sheet

3 ABI PRISM TM 3700 DNA Analyzer(Data Collection Ver.1.1) DNA Sequencing Analysis Software Ver.3.6NT DNA 1. PE Biosystems (User s Manual ) 2. ): ) 2) 3) a) b) c) - 1 -

4 run 1. Run 3700 Data Collection-Plate Setup status processed Data Collection-Run Setup status completed 2. 1 run (User s Manual: 6-5) Buffer Running Buffer with EDTA 10X (P/N ) Milli-Q 10 POP-5 polymer(p/n ) ml/run Buffer 430 Milli-Q ( ) POP-5 polymer 10 [ ] 3700 Milli-Q Run Plate setup Link Processed plate records CleanUp database(p.10 ) tip straightener autoloader tip [ ] 16 CleanUp database 3700 Data Collection ON/OFF - 2 -

5 Run 1. Milli-Q (20 L/well) 10 well 2. Aluminum foil tape(p/n 3M 439) 3. Plate record import ( ) plate name Data Collection Mobility file Run/Analysis module 4. import plate record link 5. Run Setup Run well 6. Start 7. Sample ( ) "/"( ) " "( ) "%"( ) "@"( ) "&"( ) "["( ) "]"( ) " "( ) "$"( ) Sample "+"( ) "-"( ) "#"( ) "_"( ) "("( ) ")"( ) - 3 -

1. Data Extractor a) b) "Check the database for new runs every:" 10 c) "Extraction Directory" "d:/perkin-elmer/abi/3700/dataextractor/completed Runs" d) Run color data

6 1. Data Extractor a) b) "Check the database for new runs every:" 10 c) "Extraction Directory" "d:/perkin-elmer/abi/3700/dataextractor/completed Runs" d) Run color data d:/perkin-elmer/abi/3700/dataextractor run completed extract OK 10 1 run extract "Extract Now" e) extract Database cleanup extract Start-PE Biosystems-3700 Programs-3700 Utitlities-3700DBUtils run name Extract Complete run extract Extract active (User s Manual: 10-4) f) extract d:/perkin-elmer/abi/3700/dataextractor/reextracted Runs g) run extract h) Extract d:/perkin-elmer/abi/3700/dataextractor/samplename.txt samplename.txt 3 "SAMPLEID_CapID.ab1 " CapID( capillary ) (User s Manual: 6-123) PREFIX= SUFFIX= FORMAT=SAMPLEID i) Extract d:/perkin-elmer/abi/3700/dataextractor/foldername.txt foldername.txt 2 "RUN_INSTRUMENTID_Date_RunNumIndex" INSTRUMENTID Data Collection Edit - Preference - Instrument PREFIX= SUFFIX= - 4 -

7 2. Data Collection Ver Start-PE Biosystems-3700 Programs- DataCollection (3700User password ) File-Shutdown Task manager (Ctrl+Alt+Del) d:/perkin-elmer/abi Lock3700 ( ) ) 384-well plate 10run Array view ( ) Array view Status subpage 2-2. Module 1) Instrument - Utilities - Module Editor Seq1_1POP5DefaultModule Injection Time: 15 sec Tank level error check: off Water tank level check: off Buffer tank level check: off Polymer bottle level check: off Waste tank level check: off Plenum level check: off 2) "Save As" "15secLong" Module POP5 polymer default setting Run time 7000sec Sheath flow volume 9000 counts read length Run time 10000sec Sheath flow volume counts [Sheath flow period ] Field Application ( ) Feb 25, 2000 sheath flow syringe polymer syringe 942 sec 1 ml syringe syringe syringe 942sec Data Delay Time Run Time Sheath Flow Volume 1000 counts polymer - 5 -

8 2-3. Sample sheet(plate record) User s Manual: 5-46, Appendix-1 a) Plate name 32 b) Sample name (P.3 ) 59 c) Dye Set "E" d) Mobility file "b" e) Comment 1 f) Project Name "3700Project1" g) Run Modules 1 "15secLong" h) Analysis Module 1 BC-POP5LR_FacOffSeqOff.saz " i) Well No. Sample Name Well No. (A1 A2 A3 A1 B1 C1 ) well j) ( txt plt) k) Data Collection Plate Setup import l) Import plate record Open ( ) 3. Sequencing Analysis Software Ver.3.6NT User s Manual 3-1. Seq file (*.seq) Data Collection Sequencing Analysis Software Edit - Preference - Sequence File Formats Write.Seq file Sample Manager Defaults Analysis 3-2. Spacing ( N ) Spacing (default: 16) 18 1) Sample manager 2) A(analysis) F(factura) P(print) ( ON/OFF Cntr+D) 3) "Start" Spacing spacing "0(zero)" "Start" - 6 -

9 Maintenance ABI3700 Data Collection Ver.1.1 User s Manual 1. Shutdown ( ) Data Collection 3700 reset 1) computer Windows computer 2) ) ) 2 5) Milli-Q ( ) 6) 7) POP5 polymer polymer 8) ) 10) Polymer 11) Computer ) Data Collection Instrument - Wizards - Change polymer 2 User s Manual 9-26 shutdown

10 2. Capillary User s Manual: 9-16 : run : 4 : 1 mol/l Nitric acid( )40mL Milli-Q 1 run OrbixWeb Daemon, Data Collection 1) Data Collection Instrument - Wizard - Change Array "Clean the array" NEXT 2) Inline filter ( ) 3) NEXT Part-1 (70min) 4) NEXT 5) filter Milli-Q 6) NEXT Part-2 (140min) 7) FINISH 8) Cuvette flash : capillary run (User s Manual: 11-24) run polymer cuvette flush 3. Cuvette Flush : Data Collection Array view User s Manual: cuvette Service Tools Data Collection Start - PE Biosystems Service - Service Tools v1.1 CCD Calibration Run Calibration Mod CCD image : 1 1) Data Collection Instrument - Utilities - Run Service Module 2) Select Module CuvetteFlush.mod(D:/Perkin-elmer/Abi/Support Files/Data Collection Support Files/Service Modules ) Run Method - 8 -

11 4. Inline filter User s Manual:9-13 : 30 run : 1 : Filter(Gelman Science, Acrodisc TM Syringe Filter, 0.2 m pore size, 32mm Product No.4652) 1) Milli-Q filter (20mL 2 ) 2) Filter polymer filter (polymer 5mL ) 3) filter polymer filter connector male luer-lock fitting polymer 4) OrbixWeb Daemon Data Collection 5) Data Collection Instrument - Utilities - Wizards - Change Polymer 6) "Replace polymer with polymer from the same lot" Next 2 7) 1 ("Finish" ) 8) "Finish" Inline filter capillary Sheath flow syringe User s Manual: 9-15 : polymer (User s Manual: 8-10) : 65 1) (P/N ) Milli-Q 2) Data Collection Instrument - Wizards - Change syringe 3) Wizard ( 200mL ) - 9 -

12 6. Database clean-up User s Manual:10-3 : 30 run : ( ) 1) run extract Data Collection D:/Perkin-Elmer/Abi/3700/bin/CleanUpDB.bat 2) 3700 tip straightener autoloader tip(9-21) ( Database ) CleanUp Data Collection Default Module import D:/Perkin-Elmer/Abi/3700/Bin/NewMethodImportUtlity.bat NewMethodImportUtlity.bat (edit) -import "%ABIHOME%/Support Files/Data Collection Support Files/Method Files/ "Sequencing1.mtd" 7. Tank level calibration User s Manual: : Reservoir level error message : 1) Start - PE Biosystems Service - Service Tools v1.1 2) Calibration click 3) Bottle Levels click 4) Isopropanol polymer buffer water (waste ) 5) SETLIQCAL click 6) GETLIQCAL click 7) 4 8) GETLIQCAL click 9) RETURN - RETURN - EXIT Reservoir check Module editor OFF 8. Standard sample Package insert BigDye Terminator Sequencing Standard with AmpliTaq FS Polymerase (P/N ) 1) Milli-Q 500 L/tube 2) well 10 L/well 3) 95 2 denature 4) 3700 Default Module run 5) Signal intensity - 10-

13 9. Spectral calibration User s Manual: 6-21 Matrix Standard Set DS-01 (P/N ) : ) 40 L Matrix Standard 360 L loading buffer 2) 95 5 denature 3) MicroAmp Reaction tube(2 ) (P/N N ) 200 L 4) (9 10) tube 5) Data Collection Run Setup tab Spectral Run 6) Dialog box Dye Set "E" OK Run table spectral run start 7) Run OK 8) Data Collection Data acquisition - Override Spectral Calibration 9) Dye Set "E" OK profile box 10) "From data file" D:/Perkin-Elmer/Abi/3700/Data Collection/SpectralCalLogs Matrix file(50 ) OK 11) profile box OK User s Manual: ) "Override all capillaries" capillary data file "Override Cap X" - 11-

14 10. Spatial calibration CCD pixel position 2 dye(red&blue) User s Manual: 6-8 ABI PRISM TM 3700 Spatial Calibration Standard(P/N ) : 2.5 1) Spatial calibration buffer urea 2) 20 L Standard-Red 180 L buffer 3) R MicroAmp Reaction tube(2 ) (P/N N ) 4) 2 3 Standard-Blue 1 5) 4 6) Red 3 4 Blue 7) Data Collection Run Setup tab Spatial Run 8) Dialog box "Spart1_POP5DefaultModule" OK 9) Run Setup page spatial run toolbar Start 10) Run OK 11) Spatial Calibration profile box User s Manual: OK 12) Data Collection Data acquisition - Override Spatial Calibration 13) D:/Perkin-Elmer/Abi/3700/Data Collection/SpatialCalLogs *.scl file OK 14) Profile from box OK - 12-

15 Troubleshooting ( :AutoLoader) 3700 ( :Galil control error) 3700 ( :database disc space ) PC 3700 ( : calibration file Module ) database disc space database clean-up 3700 reset User s Manual -11-Troubleshooting Reset Data Collection ( text ) PE biosystems [ ] PE Biosystems (9:00-19:00) : : Machine Serial No.(3700 ) (Data collection Run Log ) Status-Errors [Websites]

Utility softwares 3700 software 1. RenameMan : : Windows98/NT shareware (http://shareware.cnet.

16 Utility softwares 3700 software 1. RenameMan : : Windows98/NT shareware ( 1) RenameMan folder C: C: program files 2) RenameMan.exe Registration 3) 1) 2) "Number aware" 3) folder 4) file list exclude - 14-

17 5) (New Name ) Change Name Box 6) To: ( ) 7) Case "Leave unchanged" Length "Truncate" Maximum Length "0" 8) file list Rename : folder file WindowsNT Task manager (Ctrl+Alt+Del) - 15-

18 2. WS_FTP95 : FTP : Windows98/NT free ware 1) WS_FTP95 folder C: C: program files 2) 1) 2) Session Properties General New Profile Name Host Name/Address Host Type User ID Password (2 profile name ) 3) OK 4) window local window host - 16-

5) Options "Save Current Directories As Connection Directories" default 6) file/folder folder 1: Exit 2: file folder FFFTP(free ware) ( ) 3: directory list

19 5) Options "Save Current Directories As Connection Directories" default 6) file/folder folder 1: Exit 2: file folder FFFTP(free ware) ( ) 3: directory list 3. Chromas : (Mac EditView ) : Windows98/NT Ver.1.56 shareware Ver.1.45 free ware 1) ( 2) Windows ".ab1" - 17-

20 Appendix-1 Appendix-1: 96-well plate sample sheet 1 SeqPlate96 SQ 96-Well 3700User 96 well sequencing plate Well Sample Name Dye Set Mobility File Comment Project Name Sample Tracking Id Run Module 1 Analysis Module 1 A1 E A2 E A3 E A4 E A5 E A6 E A7 E A8 E A9 E A10 E A11 E A12 E B1 E B2 E ( ) H12 E SeqPlate96 = 3700User = Comment Sample Tracking Id 1 Dye Set Project Name

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