DYEnamic ET Terminator Matrix Standard for the ABI 3700
Contents
1. 0 92 26 ConditionBounds 1 0 10 0 maxScansAnalyzed 5000 6 Save the file 7 Rename the edited file SeqStd E par or SeqStd Z par as appropriate Note The instrument will only recognize the files if named exactly as described above 8 Proceed with the ET spectral calibration protocol 9 If at a later date a calibration must be performed with a sequencing chemistry other than the DYEnamic ET terminators the edited parameter file must be renamed and the OrgSeqStd E or Z par file must be renamed to its original file name 27 GE Healthcare offices GE Healthcare Bio Sciences AB Bj rkgatan 30 751 84 Uppsala Sweden GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Bio Sciences Corp 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 1327 USA GE Healthcare Bio Sciences KK Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan http www gehealthcare com lifesciences GE Healthcare regional office contact numbers Asia Pacific Tel 85 65 6 275 1830 Fax 85 65 6 275 1829 Australasia Tel 61 2 8820 8299 Fax 61 2 8820 8200 Austria Tel 01 57606 1613 Fax 01 57606 1614 Belgium Tel 0800 73 890 Fax 02 416 82 06 Canada Tel 1 800 463 5800 Fax 1 800 567 1008 Central East amp South East Europe Tel 43 1 972720 Fax 432. Edit the Run Time by clicking on the default value of 2 700 seconds and replace that with 4 000 seconds 3 Click the Save As button and enter SpecSQ1_1POP6Extended as the new module file name Click OK to save the new parameters Click Done to exit the Module Editor 4 In the Data Collection software initiate a spectral calibration run by selecting the Spectral Run command within the Run Setup page This opens the Calibration Module and Dye Set dialog window From here the appropriate type of calibration module dye set and parameter files can be selected for performing a spectral calibration 15 Note For a schematic of the procedure see pages 6 26 in the ABI 3700 user s manual 5 Using the pull down menu under Calibration Module choose the SpecSQ1_1POP6Extended file for spectral calibration for POP 6 Note For a schematic of the procedure see pages 6 26 in the ABI 3700 user s manual 6 7 10 IMPORTANT Under Dye Set choose F from the pull down menu IMPORTANT Under Parameter File choose SeqStd AnyDyeSet par from the pull down menu Click OK to accept these chosen fields The spectral run will be displayed in the run queue This action will simultaneously engage the Start Run button Activate the run by clicking on Start Run After the run is completed lt 3 hours a dialog window will display Spectral Calibration Result as shown on pages 6 29 in the ABI 3700 user s manual This di
3. 1 97272 2750 Denmark Tel 45 70 25 24 50 Fax 45 16 24 24 Eire Tel 1 800 709992 Fax 0044 1494 542010 Finland amp Baltics Tel 358 0 9 512 39 40 Fax 358 0 9 512 39 439 GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK France Tel 01 6935 6700 Fax 01 6941 9677 Germany Tel 0800 9080 711 Fax 0800 9080 712 Greater China Tel 852 2100 6300 Fox 852 2100 6338 Italy Tel 02 26001 320 Fax 02 26001 399 Japan Tel 81 3 5331 9336 Fox 81 3 53319370 Korea Tel 82 2 6201 3700 Fax 82 2 6201 3803 Latin America Tel 55 11 3933 7300 Fox 55 11 3933 7304 Middle East amp Africa Tel 30 210 9600 687 Fax 30 210 9600 693 Netherlands Tel 0800 82 82 82 1 Fax 0800 82 82 82 4 Norway Tel 47 815 65 777 Fax 47 815 65 666 imagination at work Portugal Tel 21 417 7035 Fox 21 417 3184 Russia amp other C 1S SNS Tel 7 495 956 5177 Fax 7 495 956 5176 Spain Tel 902 1172 65 Fax 935 94 49 65 Sweden Tel 018 612 1900 Fox 018 612 1910 Switzerland Tel 0848 8028 10 Fax 0848 8028 11 UK Tel 0800 515 313 Fax 0800 616 927 USA Tel 1 800 526 3593 Fax 1 877 295 8102 US84001PL Rev C 2006
4. 4 3 Select Spectral Calibration from the Application drop down list 4 4 Select 96 Well from the Plate Type drop down list 4 5 Select Heat Sealing or Septa from the Plate Sealing drop down list 4 6 Type a name for the owner and operator in the appropriate fields 5 Click OK A blank plate record opens 24 6 Complete the plate record 6 1 In the Sample Name column type a name 6 2 In the Instrument Protocol 1 column select the protocol created in the Creation of Spectral Protocol section 6 3 Select the Sample Name and Instrument Protocol 1 columns and fill down 7 Click OK 6 3 4 Adding a Plate to the Run Scheduler 1 Click the Run Scheduler icon The Run Scheduler view opens 2 In the Input Stack section click Search A search dialog box opens 3 Search for the spectral calibration record 4 Add the plate record 4 1 Select the plate you want to use in the Name column 4 2 Click Add 4 3 Click Done The plate is added to the Run Scheduler view 5 Click on the Run Instrument button 6 3 5 Evaluating Spectral Calibration Results To view the pass fail status of each capillary 1 Locate the log file at the following location E AppliedBiosystems UDC DataCollection Data ga 3730 instrument name SpectralCalMclFiles E BigDyeV1 2 Open the file in Notepad 3 View the results 25 The condition number c is a measure of the spectral overlap of the dyes As the spectral overlap on
5. Overriding the Spectral Calibration Profiles on pages 6 34 of the ABI 3700 user s manual To review the calibration profile open the Data Collection software Go to the Data Acquisition menu and open the Override Spectral Calibration function A dialog window titled Select the dye set to display will appear 12 Select F from the pull down menu IMPORTANT Click OK This action opens a dialog window titled Spectral Calibration Profile for F The fluorescence emission spectra for all four dyes in a particular capillary is displayed as shown in Figure 2 13 Examine the results for each cap and verify that the profile is similar to that shown in Figure 4 Note For additional schematic illustrations see pages 6 35 of the ABI 3700 user s manual 14 Although the software assigns values from successful caps to failed caps in some instances the profile may not be similar to the one shown in the illustration When this occurs replace the values manually by using the values from a successful adjacent matrix At the bottom left of the dialog box under Override matrix from another source click on the button From capillary which allows the selection of values from any capillary Choose any acceptable capillary 17 6 2 Spectral calibration of the ABI 3100 for DYEnamic ET Terminators The DYEnamic ET Terminator Matrix Standard is formulated for creating a spectral matrix with the ABI 3100 sequencing instrument This spectral c
6. automatically assign proper calibration values to failed caps from adjoining successful caps The capillary status bar will indicate passed caps in black and questionable caps in yellow 10 Note For further information on the significance of color coding in the capillary status bar see pages 6 29 and 5 68 in the ABI 3700 user s manual 8 Upon completion of the spectral run and data processing the quality of the spectral profile the emission spectra for all four dyes for each capillary must be examined The condition number is a measure of the spectral overlap of the dyes As the spectral overlap of a dye set decreases so does the condition number A condition number of 1 0 indicates no spectral overlap for a particular dye set The expected condition number for ET dyes analyzed on the ABI 3700 is 7 3 0 5 The Q value is a measure of how well the spectral calibration fits the data it was created from A Q value of 1 0 represents a perfect fit Any spectral calibration with a Q value less than 0 92 will automatically fail the calibration and be replaced with spectral calibration data from an adjacent capillary Those caps that have a questionable matrix should be replaced with data from a successful matrix Note For further information and a schematic see Reviewing and Overriding the Spectral Calibration Profiles on pages 6 34 of the ABI 3700 user s manual To review the calibration profile open the Data Collection softwar
7. not supplied Reagents e Water Only use deionized distilled water with the DYEnamic ET Terminator Matrix Standard Equipment e Liquid handling supplies Microcentrifuge tubes 200 ul micropipettes microcentrifuge vortex mixer e Instrument This product is designed for use with the ABI 3730 3730XL ABI 3700 and ABI 3100 sequencing instruments 6 Protocols 6 1 Spectral calibration of the ABI 3700 for DYEnamic ET Terminators 6 1 1 Preparation of DYEnamic ET Terminator Matrix Standard 1 Briefly centrifuge the tube containing the DYEnamic ET Terminator Matrix Standard to bring the contents 40 ul to the bottom of the tube 2 Add 360 ul of distilled water to the tube that contains the matrix standard u Mix the contents of the tube thoroughly by vortexing vigorously Briefly centrifuge the tube m Transfer 200 pl of the matrix standard solution into each of two 200 ul microtubes as recommended by the instrument manufacturer 6 1 2 Performing a spectral calibration See pages 6 25 of the ABI 3700 user s manual for general guidelines for performing a default calibration To create a spectral matrix for DYEnamic ET terminator chemistry follow the protocol below 1 Firmly place the two tubes containing the ET terminator matrix standard into the right bar in slot positions 9 and 10 as shown in Figure 1 and on pages 6 25 in the ABI 3700 user s manual Left Bar Right Bar oooo0oo0oo0oo0o0 00000
8. 000 9 10 Fig 1 Matrix standard loading position 9 6 1 3 Performing a spectral calibration run for POP 5 1 Open the ABI 3700 Data Collection software Initiate a spectral calibration run by selecting the Spectral Run command under the Run Setup page This command opens the Calibration Module and Dye Set dialog window from which the appropriate calibration module dye set and parameter files are selected Note For a schematic of the procedure see pages 6 26 in the ABI 3700 user s manual 2 Using the pull down menu under Calibration Module choose the SpecSQ1_1POP5DefaultModule file for spectral calibration for POP 5 Note For a schematic of the procedure see pages 6 26 in the ABI 3700 user s manual 3 IMPORTANT Under Dye Set choose F from the pull down menu 3 4 IMPORTANT Under Parameter File choose SeqStd AnyDyeSet par from the pull down menu 5 Click OK to accept these chosen fields 6 The spectral run will be displayed in the run queue This action will simultaneously engage the Start Run button Activate the run by clicking on Start Run 7 After electrophoresis is completed lt 3 hours a dialog window displays Spectral Calibration Result as shown on pages 6 29 in the ABI 3700 user s manual This display indicates the number of capillaries caps that passed spectral calibration For a schematic see pages 6 29 in the user s manual Accept the result by clicking OK The software will
9. 648 and 5 707 804 2006 General Electric Company All rights reserved GE Healthcare reserves the right subject to any regulatory and contractual approval if required to make changes in specification and features shown herein or discontinue the product described at any time without notice or obligation Contact your GE Healthcare representative for the most current information and a copy of the terms and conditions http www gehealthcare com lifesciences GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK 2 Handling 2 1 Safety warnings data sheet s and or safety and precautions statement s for specific advice Warning For research use only Not recommended or intended for diagnosis of disease in humans or animals Do not use internally or externally in humans or animals 2 2 storage Store at 15 C to 30 C All chemicals should be considered as potentially hazardous We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice Wear suitable protective clothing such as laboratory overalls safety glasses and gloves Care should be taken to avoid contact with skin or eyes In the case of contact with skin or eyes wash immediately with water See material safety 3 Introduction The DYEnamic ET Terminator Matrix Stand
10. GE Healthcare DYEnamic ET Terminator Matrix Standard for the ABI 3700 For use with ABI 3100 3700 and 3730 3730xXL Product Booklet Code US84001 Page finder 1 Legal 2 Handling 2 1 Safety warnings and precautions 2 2 Storage Introduction Components of the kit Materials not supplied Oy Oe Ee iy Protocols 6 1 Spectral calibration of the AB13700 for DYEnamic ET Terminators 6 1 1 Preparation of DYEnamic ET Terminator Matrix Standard 6 1 2 Performing a spectral calibration 6 1 3 Performing a spectral calibration run for POP 5 6 1 4 Performing a spectral calibration run for POP 37 6 1 5 Performing a spectral calibration run for POP 6 6 2 Spectral calibration of the ABI 3100 for DYEnamic ET Terminators 6 2 1 Preparation of DYEnamic ET Terminator Matrix Standard 6 2 2 Performing a spectral calibration 6 3 Spectral calibration of the ABI 3730 3730xXL for DYEnamic ET Terminators 6 3 1 Preparation of DYEnamic ET Terminator Matrix Standard 6 3 2 Creation of Spectral Protocol 6 3 3 Creating a Plate Record 6 3 4 Adding a Plate to the Run Scheduler 6 3 5 Evaluating Spectral Calibration Results 6 3 6 Editing Spectral calibration Files 2 O ON ONNA WN 18 18 19 22 22 23 24 25 25 26 1 Legal GE and GE monogram are trademarks of General Electric Company Amersham and DYEnamic are trademarks of GE Healthcare companies This kit is sold pursuant to Authorization from PE Applied Bios
11. a Source x cancer Fig 4 Spectral calibration profile for F using POP 6 with the ABI 3100 6 3 Spectral Calibration of the ABI 3730 3730XL for DYEnamic ET Terminators DYEnamic ET Terminator Matrix Standard for the ABI 3700 US84001 is formulated for performing a spectral calibration and creating a spectral matrix for the ABI 3730 sequencing instrument 6 3 1 Preparation of DYEnamic ET Terminator Matrix Standard 1 Briefly centrifuge the tube containing the DYEnamic ET Terminator Matrix Standard to bring the contents 40 yl to the bottom of the tube 2 tubes for 3730XL 2 Add 460 ul of distilled water to the tube containing the matrix standard Mix the contents of the tube thoroughly by vigorous vortexing Briefly centrifuge the tube 22 Note Combine 2 tubes for 3730XL 3 Dispense 10 ul of the DYEnamic ET Terminator matrix standard into every other column of a 96 well plate all wells for 3730XL It is essential to centrifuge the plate to position the samples at the bottom of each well 6 3 2 Creation of Spectral Protocol 1 Expand the view in the tree pane 1 1 Click the box next to the GA intruments icon 1 2 Click the box next to the ga3730 icon 2 Click the Protocol Manager icon 3 In the Instrument Protocols section click New The Protocol Editor dialog box opens 4 Create a spectral protocol 4 1 Type ET_Spectral or a similar name in the Name Field 4 2 Select SPECTRAL from the Type drop dow
12. alibration must be performed prior to analysis of samples labelled with any dye set not previously used with the instrument It is strongly recommended that the user read and thoroughly understand the section of the ABI 3100 user s manual copyright 2001 titled Performing a Spectral Calibration pages 4 15 to 4 49 before attempting to create a matrix for DYEnamic ET terminators 6 2 1 Preparation of DYEnamic ET Terminator Matrix Standard 1 Briefly centrifuge the tube containing the DYEnamic ET Terminator Matrix Standard to bring the contents 40 ul to the bottom of the tube 2 Transfer 5 ul of the DYEnamic ET Terminator Matrix Standard into a tube containing 195 ul of distilled water WG Mix the contents of the tube thoroughly by vigorous vortexing S Briefly centrifuge the tube and dispense 10 ul of the DYEnamic ET Terminator Matrix Standard into a 96 well plate as shown below Figure 3 It is crucial to centrifuge the plate to position the samples at the bottom of each well s Assemble the plate for loading onto the ABI 3100 see pages 3 9 of the ABI 3100 user s manual 18 e00e0e0e0 00000 OO0O000 OO0O000s gt mn pa ee Fe 6 OOO0O00O0O H OOOOCO0CO0O Fig 3 Matrix standard loading positions Oo0o000000 OOQOOO000O OOQOOOO0O0OS 6 2 2 Performing a spectral calibration For general guidelines on performing a default calibration see pages 4 17 of the ABI 3100 user s manual To
13. ard for the ABI 3700 ABI3730 ABI 3730XL US84001 is formulated to create a spectral matrix with the ABI 3700 and ABI 3100 sequencing instruments This spectral calibration must be performed prior to analysis of samples labelled with any dye set not previously used with the instrument It is strongly recommended that the user read and thoroughly understand the section of the ABI 3100 user s manual copyright 2001 titled Spectral Calibration pages 4 15 to 4 50 ABI 3730 3730x1 User s guide copyright 2002 titled Spectral Calibration pages 4 11 to 4 28 ABI 3700 user s manual copyright 1999 titled Performing a Spectral Calibration pages 6 21 to 6 56 before attempting to create a matrix for DYEnamic ET terminators 4 Components of the kit This reagent has been tested extensively and its concentration adjusted to meet rigorous standards It is strongly recommended that the reagent be used exactly as described in this protocol The product consists of the following solution DYEnamic ET Terminator Matrix Standard for the ABI 3700 3730 3730XL 40 ul Each tube contains enough matrix standard to perform one spectral calibration on the ABI 3700 1 calibration on 3730 1 2 calibration on 3730XL sequencing instrument or eight calibrations on the ABI 3100 The product should be stored at 15 C to 30 C not in a frost free freezer When not in a freezer keep the reagent on ice prior to use 5 Materials
14. create a spectral matrix for DYEnamic ET terminator chemistry follow the protocol below 1 Place the plate on the autosampler Note For a schematic of the procedure see pages 4 24 in the ABI 3100 user s manual 2 Open the 3100 Data Collection software and initiate a spectral calibration run by selecting New within the Plate View page This opens up the Plate Editor dialog box Name the plate select Spectral Calibration and select 96 Well plate type Click Finish This opens the Plate Editor spreadsheet 3 Within the Plate Editor spreadsheet complete the following for the 16 samples in the sample plate 3 1 Name the samples 3 2 IMPORTANT Select Dye Set F 3 3 Select the run module appropriate for your capillary array size 36 cm Spect36_POP6DefaultModule 50 cm Spect50_POP6DefaultModule 80 cm Spect80_POP4DefaultModule 3 4 IMPORTANT Select the spectral calibration parameters SeqStd AnyDyeSet par 3 5 Click OK 4 The newly created plate record then appears in the Pending Plate Records table of the Plate Setup page In the Plate Setup page select the newly created plate from the Pending Plate Records table 5 Click on the graphic that corresponds to the plate on the autosampler The plate then moves from the Pending Plate Records table to the Linked Plate Records table For a pictorial representation see pages 3 39 in the ABI 3100 user s manual 6 Click the Run Instrument button on the toolbar
15. e Go to the Data Acquisition menu and open the Override Spectral Calibration function A dialog window titled Select the dye set to display will appear 9 Select F from the pull down menu IMPORTANT Click OK This action opens the dialog window titled Spectral Calibration Profile for F The fluorescence emission spectra for all four dyes in a particular capillary will be displayed as shown in Figure 2 10 Examine the results for each cap and verify that the profile is similar to that shown in Figure 2 11 Note For additional schematic illustrations see pages 6 35 of the ABI 3700 user s manual 11 Although the software assigns values from successful caps to failed caps in some instances the profile may not be similar to the one shown in the illustration When this occurs replace the values manually by using the values from a successful adjacent matrix At the bottom left of the dialog box under Override matrix from another source click on the button From capillary which allows the choice of values from any capillary Choose any acceptable capillary Spectral Calibration Profile for F E 1000 3000 000 6000 Intensity vs Soan Number Capillary Number 52 zi d z Condition Number 7 41 Q Value 0 966 Data Source From capillary 51 Override matrix trom another source __ From capillary From data file E Cea Fig 2 Spectral calibration profile for F using POP 5 with the ABI 3700 6 1 4 Performing a sp
16. ectral calibration run for POP 37 1 Open the ABI 3700 Data Collection software Initiate a spectral calibration run by selecting the Spectral Run command under the Run Setup page This command opens the Calibration Module and Dye Set dialog window from which the appropriate calibration module dye set and parameter files are selected Note For a schematic of the procedure see pages 6 26 in the ABI 3700 user s manual 2 Use the pull down menu under Calibration Module and choose the SpecSQ1_2POP37DefaultModule file for spectral calibration for POP 37 Note For a schematic of the procedure see pages 6 26 in the ABI 3700 user s manual 3 IMPORTANT Under Dye Set choose F from the pull down menu 4 IMPORTANT Under Parameter File choose SeqStd AnyDyeSet par from the pull down menu 5 Click OK to accept these chosen fields 6 The spectral run will be displayed in the run queue This action will simultaneously engage the Start Run button Activate the run by clicking on Start Run 7 After electrophoresis is complete lt 3 hours a dialog window displays Spectral Calibration Result as shown on pages 6 29 in the ABI 3700 user s manual This display indicates the number of capillaries caps that passed spectral calibration For a schematic see pages 6 29 in the user s manual Accept the result by clicking OK The software will automatically assign proper calibration values to failed caps from adjoining succes
17. he calibration profile using the following steps Open the Data Collection software select Override Spectral Calibration from the File Menu A dialog window titled Select the dye set to display appears Select F from the pull down menu IMPORTANT Click OK This action opens the dialog window Spectral Calibration Profile for F The fluorescence emission spectra for all four dyes in a particular capillary is displayed as shown in Figure 4 Examine the results for each cap and verify that it is similar to the one shown in this example and as illustrated on page 4 29 of the ABI 3100 user s manual Although the software assigns values from successful caps to failed caps in some instances the profile may not be similar to the one shown in the illustration When this happens manually replace the values with the data from a successful adjacent matrix At the bottom left of the dialog box under Override matrix from another source click on the button From capillary These commands allow you to select a value from any acceptable capillary 10 Examine the results for each cap and verify that the profile is similar to that shown in Figure 4 Note For additional schematic illustrations see pages 4 29 of the ABI 3100 user s manual 21 Matrices for dye set F x 41 09 o7 29 2 a SS FR AN RO te AS we Ne AS we ao Intensity vs Bin Number 4000 Intercity vs Sean Number Capillary Number 16 E Zi Condition Number 7 43 QValue 0 957 Dat
18. n list 4 3 Select the appropriate run module from the drop down list i Spect36_SeqStd_POP7_ET for 36 cm array with POP 7 Note Prior to this step the Spect36_SeqStd_POP7 module must be edited to include a run time of 2000 seconds The module can be edited in the Protocol Manager by creating a new module based on the Spect36_SeqStd_POP7 module ii Spect50_SeqStd_POP7 for 50 cm array with POP 7 5 Select E BigDyeV1 or Z BigDyeV3 from the Dye Set drop down list Note Select a Dye Set not in use An ET spectral calibration will overwrite any previous spectral calibrations per formed on the Dye Set selected 6 Select SeqStd Any4DyeSet par from the Params drop down list 25 Note If the SeqStd Any4DyeSet par is not available an existing parameter file must be edited to be compatible with the DYEnamic ET terminators See the Editing Spectral Calibration Section at the end of this protocol 7 Click OK The module is saved and displayed in the Instrument Protocols section of the Protocol Manager view 6 3 3 Creating a Plate Record 1 Expand the view in the tree pane 1 1 Click the box next to the GA Instruments icon 1 2 Click the box next to the ga3730 icon 2 Click the Plate Manager icon The Plate Manager view opens 3 Click New The New Plate Dialog opens 4 Complete the plate information 4 1 Type a name for the plate ID in the ID barcode field 4 2 Type a name for the plate in the Name field
19. sful caps The capillary status bar will indicate passed caps in black and questionable caps in yellow 13 Note For further information on the significance of color coding in the capillary status bar see pages 6 29 and 5 68 in the ABI 3700 user s manual 8 Upon completion of the spectral run and data processing the quality of the spectral profile the emission spectra for all four dyes for each capillary must be examined The condition number is a measure of the spectral overlap of the dyes As the spectral overlap of a dye set decreases so does the condition number A condition number of 1 0 indicates no spectral overlap for a particular dye set The expected condition number for ET dyes analyzed on the ABI 3700 is 7 3 0 5 The Q value is a measure of how well the spectral calibration fits the data it was created from A Q value of 1 0 represents a perfect fit Any spectral calibration with a Q value less than 0 92 will automatically fail the calibration and be replaced with spectral calibration data from an adjacent capillary Those caps that have a questionable matrix should be replaced with data from a successful matrix Note For further information and a schematic see Reviewing and Overriding the Spectral Calibration Profiles on pages 6 34 of the ABI 3700 user s manual 9 To review the calibration profile open the Data Collection software Go to the Data Acquisition menu and open the Override Spectral Calibration func
20. splay indicates the number of caps that passed spectral calibration For a schematic see pages 6 29 in the user s manual Accept the result by clicking OK The software automatically assigns proper calibration values to failed caps from the adjoining successful caps The capillary status bar indicates passed caps in black and questionable caps in yellow Note For further information on the significance of color coding in the capillary status bar see pages 6 29 and 5 68 in the ABI 3700 user s manual 11 Upon completion of the spectral run and data processing the quality of the spectral profile the emission spectra for all four dyes for each capillary must be examined The condition number is a measure of the spectral overlap of the dyes As the spectral overlap of the dyes decrease so does the condition number A condition number of 1 0 indicates no spectral overlap for a 16 particular dye set The expected condition number for ET dyes on the ABI 3700 is 7 3 0 5 The Q value is a measure of how well the spectral calibration fits the data it was created from A Q value of 1 0 represents a perfect fit Any spectral calibration with a Q value less than 0 92 will automatically fail the calibration and be replaced with spectral calibration data from an adjacent capillary Those caps that have a questionable matrix should be replaced with data from a successful matrix For further information and a schematic see Reviewing and
21. the dyes decreases so does the condition number A condition number of 1 0 would indicate no spectral overlap for a particular dye set The expected condition number for ET dyes on the ABI 3730 is 6 7 0 5 The Q value is a measure of how well the spectral calibration fits that data it was created from A Q value q of 1 0 represents a perfect fit Any spectral calibration with a Q value less than 0 92 will automatically fail the calibration and be replaced with spectral calibration data from an adjacent capillary 6 3 6 Editing Spectral calibration Files If the SeqStd Any4DyeSet par is not available when setting up the ET terminator spectral calibration protocol on the instrument an existing parameter file must be edited to be compatible with the DYEnamic ET terminators 1 Locate the Spectral Calibration ParamFiles folder in the following Path E Appliedbiosystems UDC ga3730 CalibrationData SupportFiles SpectralCalibration ParamFiles Note Search for files with a par extension if the ParamFiles folder cannot be located 2 Edit the SeqStd E par if you are performing the ET spectral calibration on Dye Set E and edit the SeqStd Z par if the calibration to be performed on Dye Set Z 3 Rename the existing SeqStd E or Z par file OrgSeqStd E or Z par 4 Make a copy of the file to be edited by copying and pasting the file into the ParamFiles folder 5 Edit the copied parameter file to include the following minQ
22. tion A dialog window titled Select the dye set to display will appear 10 Select F from the pull down menu IMPORTANT Click OK This action opens the dialog window titled Spectral Calibration Profile for F The fluorescence emission spectra for all four dyes in a particular capillary will be displayed as shown in Figure 2 11 Examine the results for each cap and verify that the profile is similar to that shown in Figure 2 14 Note For additional schematic illustrations see pages 6 35 of the ABI 3700 user s manual 12 Although the software assigns values from successful caps to failed caps in some instances the profile may not be similar to the one shown in the illustration When this occurs replace the values manually by using the values from a successful adjacent matrix At the bottom left of the dialog box under Override matrix from another source click on the button From capillary which allows the choice of values from any capillary Choose any acceptable capillary 6 1 5 Performing a spectral calibration run for POP 6 1 Open the ABI 3700 Data Collection software and then open the Module Editor listed under the Instrument Utilities menu Select the Others tab under the module menu within the Module Editor Load the SpectSQ1_1POP6DefaultModule parameters into the Module Editor by clicking on its listing within the Others tab 2 Scroll down to the Run Time parameter number 15 listed in the Module Parameters window
23. to begin the run 7 After completing the run 40 65 minutes a dialog window displays Spectral Calibration Result as shown on pages 4 23 in ABI 3100 user s manual indicating the number of caps that passed spectral calibration Accept the result by clicking OK The software then automatically assigns proper calibration values to failed caps from the adjoining successful caps 8 Upon completion of the spectral run and data processing the quality of the spectral profile the emission spectra for all four dyes for each capillary must be examined Any capillary that generated a questionable matrix should be carefully examined and replaced with data from a successful matrix Instructions for 20 matrix replacement are provided in the ABI 3100 user s manual on pages 4 27 Overriding a Spectral Calibration Profile 9 The condition number is a measure of the spectral overlap of the dyes As the spectral overlap of a dye decreases so does the condition number A condition number of 1 0 indicates no spectral overlap for a particular dye set The expected condition number for ET dyes on the ABI 3100 is 7 3 0 5 The Q value is a measure of how well the spectral calibration fits the data it was created from A Q value of 1 0 represents a perfect fit Any spectral calibration with a Q value less than 0 92 will automatically fail the calibration and be replaced with spectral calibration data from an adjacent capillary Review t
24. ystems under one or more of the following U S Patents 4 849 513 4 855 255 5 015 733 5 118 800 5 118 802 5 161 507 5 171 534 5 242 796 5 306 618 5 332 666 and 5 366 860 and corresponding foreign patents and patent applications The purchase of this kit includes limited non transferable rights without the right to resell repackage or further sublicense under such patent rights to use this kit for DNA sequencing or fragment analysis solely when used in conjunction with an automated instrument for DNA sequencing or analysis which have been authorized for such use by Applied Biosystems or for manual sequencing Purchase of this product does not itself convey to the purchaser a complete license or right to perform automated DNA sequence and fragment analysis under the subject patents No other license is hereby granted for use of this kit in any other automated sequence analysis instrument The rights granted hereunder are solely for research and other used that are not unlawful No other license is granted expressly impliedly or by estoppel Further information on purchasing licenses to perform DNA sequence and fragment analysis may be obtained by contacting the Director of Licensing at Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 GE HEALTHCARE IS LICENSED AS A VENDOR FOR AUTHORIZED SEQUENCING AND FRAGMENT ANALYSIS INSTRUMENTS Energy Transfer dyes and primers US Patent numbers 5 654 419 5 688
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