User Bulletin
Contents
1.2. Note The numbers in the Start at and Points boxes below are typical values Your numbers may vary Make Matrix 21 dR110 matrix std Start at E 1 dR66 matrix std Start at 2050 19 dTAMRA matrix std Start at 2000 E 23 dROH matrix std Start at dRhod Update File Instrument Comment Dye Primer Matrix Tag Terminator Matrix OT Terminator Matrix 9 a Click OK The computer makes the matrix When finished a dialog window appears with the message Make matrix successfully completed b Click OK Page 10 of 14 User Bulletin ABI PRISM dRhodamine Matrix Standards Kit To make the Dye Primer Matrix continued Step Action 10 If the computer is unable to make a matrix examine the raw data again in the Sequencing Analysis software If many peaks are off scale dilute the matrix standards and rerun them To make the Tag Terminator Matrix Step Action 1 In the Data Utility application choose Make Matrix from the Utilities menu The Make Matrix dialog box appears In the Make Matrix dialog box click the Taq Terminator Matrix button at the lower left Click on the box for each nucleotide base and enter the data file that corresponds to the correct matrix standard as shown in Table 5 on page 8 IMPORTANT The order of matrix standard data files is different from that in the Dye Primer Matrix see Ta
3. 1 Prepare a separate loading cocktail for each of the four matrix standards as shown below IMPORTANT The matrix standards can precipitate in the tube leading to very low signal strength Mix each matrix standard thoroughly before using by vortexing or pipetting up and down Component Volume pL Matrix standard 2 Deionized formamide 2 Total volume 4 WARNING CHEMICAL HAZARD Formamide is a teratogen and is harmful by inhalation skin contact and ingestion Use in a well ventilated area Use chemical resistant gloves and safety glasses when handling Heat the cocktails at 95 C for two minutes Place on ice until ready to load IMPORTANT DNA samples should not be stored in formamide for more than a few hours Load each of the four matrix standard cocktails into a separate lane of the gel as shown below Instrument Platform Loading Volume uL ABI PRISM 377 1 ABI PRISM 377XL 48 1 1 5 ABI PRISM 377XL 64 1 Perform electrophoresis according to your instrument user s manual After electrophoresis examine the raw data The matrix standards should display the following colors in the gel image Matrix Standard Color on Gel Image dR110 blue dR6G green dTAMRA yellow dROX red Check the lane tracking for the matrix standards before making the matrix Page 7 of 14 Making the Matrix You must put the correct data file
4. The new primers and terminators are labeled as follows Table 1 Dye Labels dRhodamine BigDye Base Terminators BigDye Primers Terminators A dR6G dR6G dR6G C dTAMRA dR110 dROX G dR110 dTAMRA dR110 T dROX dROX dTAMRA Using the new dRhodamine sequencing chemistries requires making instrument matrix files from the new matrix standards found in the ABI PRISM dRhodamine Matrix Standards Kit P N 403047 The new matrix standards are the following Table 2 dRhodamine Matrix Standards Color of Raw Data on Color of Raw Data on ABI Prism 310 ABI PRISM 377 Tube Label Electropherogram Gel Image dR110 Matrix Standard blue blue dR6G Matrix Standard green green dTAMRA Matrix Standard black yellow dROX Matrix Standard red red The dRhodamine matrix standards are provided in a ready to use format and are premixed with a blue dye for convenience in gel loading Matrix standards are stable for 6 months at 2 6 C Avoid freeze thaw cycles IMPORTANT The ABI PRISM dRhodamine Matrix Standards Kit is for use with the ABI PRISM 310 Genetic Analyzer the ABI PRISM 377 DNA Sequencer and the ABI PRISM 377 DNA Sequencer with XL Upgrade IMPORTANT The dRhodamine sequencing chemistries are not designed for use with the ABI 373 DNA Sequencer or the ABI 373 DNA Sequencer with XL Upgrade You must use run modules and dye set primer mobility files for virtual Filter Set E when sequencing with the dRhodamine based cycle sequencing ch
5. Enter the same numbers for each matrix standard sample in the Start at and Points boxes as were used in the Dye Primer Matrix and Taq Terminator Matrix Click Update File A dialog window appears Choose dRhod from the ABI folder within the System folder and click Save The Make Matrix dialog box should look like that shown below Make Matrix C 1 dR6G matrix std Start at 2050 19e dTAMRA matrix std Start at 2000 G 23 dROH matrix sta Start at T 21 dR110 matrix sta Start at Points fae Update File Instrument Comment Dye Primer Matrix Tag Terminator Matrix T Terminator Matrix User Bulletin ABI PRISM dRhodamine Matrix Standards Kit To make the T7 Terminator Matrix continued Step Action 7 a Click OK The computer makes the matrix When finished a dialog window appears with the message Make matrix successfully completed b Click OK To check the instrument file Step Action 1 From the Utilities menu choose Copy Matrix 2 Under Source select Instrument file and choose dRhod from the ABI folder within the System folder The three matrix files within the dRhod instrument file appear as shown below Copy Matrix Source dRhod Instrument Comment Destination No Destination File Instrument Comment EJ Copy Primer Matrix J Copy Ta
6. User Bulletin ABI PRISM 310 377 377 with XL Upgrade August 15 2000 updated 01 2001 SUBJECT ABI PRISM dRhodamine Matrix Standards Kit Introduction New Dyes Matrix standards are used to generate the multicomponent matrix required for four color fluorescence detection on the Applied Biosystems ABI PRISM 310 Genetic Analyzer the ABI PRISM 377 DNA Sequencer and the ABI PRISM 377 DNA Sequencer with XL Upgrade ABI PRISM 377XL Sequencing Analysis software uses this multicomponent matrix to analyze samples that are labeled with four different fluorescent dyes but are run in a single capillary injection or gel lane A set of four matrix standards only needs to be run once to generate a matrix file that is used with all samples run under similar conditions For more information on the use of matrix standards refer to the user s manual for your instrument Note Matrix files are called instrument files in the ABI PRISM 377 Collection software versions 2 0 and 2 1 and in the Sequencing Analysis software Applied Biosystems has designed four new dichlororhodamine dRhodamine fluorescent dyes dichloro R1 10 dR110 dichloro R6G dR6G dichloro TAMRA dTAMRA and dichloro ROX dROX They are used with the following new cycle sequencing chemistries dRhodamine Terminators BigDye Primers BigDye Terminators Applied KS Bibsystems dRhodamine Matrix Standards Filter Set E Page 2 of 14
7. ble 5 on page 8 Enter the same numbers for each matrix standard sample in the Start at and Points boxes as were used for the Dye Primer Matrix Click Update File A dialog window appears Choose dRhod from the ABI folder within the System folder and click Save The Make Matrix dialog box should look like that shown below Make Matrix 23edROH matrix std Start at 1 dR66 matrix std Start at G 21 dR110 matrix sta Start at 19 dTAMRA matrix std Start at Points tor Update File Instrument Comment Dye Primer Matrix Taq Terminator Matrix T Terminator Matrix User Bulletin ABI PRISM dRhodamine Matrix Standards Kit Page 11 of 14 Page 12 of 14 To make the Taq Terminator Matrix continued Step Action 7 a Click OK The computer makes the matrix When finished a dialog window appears with the message Make matrix successfully completed b Click OK To make the T7 Terminator Matrix Step Action 1 In the Data Utility application choose Make Matrix from the Utilities menu The Make Matrix dialog box appears 2 In the Make Matrix dialog box click the T7 Terminator Matrix button at the lower left 3 Click on the box for each nucleotide base and enter the data file that corresponds to the correct matrix standard as shown in Table 5 on page 8 note the order of the matrix standard files 4
8. ds Standards on the ABI PRISM 310 Page 6 of 14 Step Action 1 Prepare a separate loading cocktail for each of the four matrix standards as shown below IMPORTANT The matrix standards can precipitate in the tube leading to very low signal strength Mix each matrix standard thoroughly before using by vortexing or pipetting up and down Component Volume uL Matrix standard 1 Deionized formamide 12 Total volume 13 WARNING CHEMICAL HAZARD Formamide is a teratogen and is harmful by inhalation skin contact and ingestion Use in a well ventilated area Use chemical resistant gloves and safety glasses when handling Heat each sample at 95 C for 2 minutes Place on ice until ready to load Run each matrix standard sample on the ABI PRISM 310 ina separate injection Refer to the ABI PRISM 310 Genetic Analyzer User s Manual or User Bulletin 1 P N 904261 for instructions on setting up the instrument Examine the electropherogram of the raw data The matrix standards should display the following colors Matrix Standard Color in Electropherogram dR110 blue dR6G green dTAMRA black dROX red continued on next page User Bulletin ABI PRISM dRhodamine Matrix Standards Kit Running To run standards Standards on the ABI PRISM 377 or ABI PRISM 377 with XL Upgrade User Bulletin ABI PRISM dRhodamine Matrix Standards Kit Step Action
9. emistries User Bulletin ABI PRISM dRhodamine Matrix Standards Kit Installing Run Modules and Dye Set Primer Files Overview Run Modules Run modules and dye set primer mobility files are found on the diskette supplied with the dRhodamine Matrix Standards Kit They can also be obtained from the Applied Biosystems site on the World Wide Web www appliedbiosystems com techsupport or from your local Field Applications Specialist call Applied Biosystems Technical Support or your local sales office for more information Use the appropriate run module for your run parameters on your instrument as shown in Table 3 Table 3 Run Modules Instrument Configuration Run Module ABI PRISM 310 DNA Sequencing Polymer Seq Run 250 uL E 250 uL syringe POP 6 polymer Seq POP6 1 mL E 1 mL syringe POP 6 polymer Seq POP6 1 mL Rapid E 1 mL syringe Rapid Sequencing ABI Prism 377 2X 36 cm wtr 36 well Seq Run 36E 1200 4X 36 cm wtr 36 well Seq Run 36E 2400 48 cm wtr 36 well Seq Run 48E 1200 ABI PRISM 377 2X 36 cm wtr 36 48 or Seq Run 36E 1200 with XL Upgrade 64 well 4X 36 cm wtr 36 48 or Seq Run 36E 2400 64 well 48 cm wtr 36 48 or Seq Run 48E 1200 64 well a The DNA Sequencing Polymer is not supported for use with the BigDye Primers and BigDye Terminators b Use any plate check and prerun module on the ABI PRISM 377 DNA Sequencer and ABI PRISM 377 DNA Sequencer with XL Upgrade continued o
10. for each matrix standard into the correct box in the Data Utility application Table 5 Table 5 Placement of Standards in the Data Utility Application Dye Primer Taq Terminator T7 Terminator Box Matrix Matrix Matrix C dR110 dROX dR6G A dR6G dR6G dTAMRA G dTAMRA dR110 dROX Mss dROX dTAMRA dR110 IMPORTANT You need to make all three matrix files even if you are only using one dRhodamine based chemistry The Collection software will not run with only a terminator matrix in the file An error message will appear saying Tag not found Cannot start the run To make the Dye Primer Matrix Step Action 1 Set the analysis start point and the number of data points to analyze a Inthe Sequencing Analysis software examine the raw data for one of the matrix standard samples as shown below b Select a starting point where there are no peaks and the baseline is flat c Select a number of data points to analyze such that no peaks in the range are off scale i e above 4000 relative fluorescence units RFU and that the baseline at the end of the range is flat A typical number of data points is 1500 23edROH matrix std ae hn ie Page 8 of 14 User Bulletin ABI PRISM dRhodamine Matrix Standards Kit To make the Dye Primer Matrix continued Step Action 2 Repeat step 1 on page 8 for each matrix standard sample Record the results for later
11. n next page User Bulletin ABI PRISM dRhodamine Matrix Standards Kit Page 3 of 14 Dye Set Primer Use the correct dye set primer mobility file for your instrument as Files shown in Table 4 Table 4 Dye Set Primer Files Sequencing Chemisiry Instrument Dye Set Primer File dRhodamine Terminators ABI PRISM 310 DNA Sequencing Polymer ABI PRISM 310 POP 6 polymer ABI PRISM 310 POP 6 polymer Rapid Sequencing ABI PRISM 377 ABI PRISM 377 with XL Upgrade DT DSP dR Set AnyPrimer DT POP6 dR Set Any Primer DT POP6 dR Set Any Primer DT dR Set Any Primer DT dR Set Any Primer BigDye Primers ABI PRISM 310 POP 6 polymer 21 M13 primers ABI PRISM 310 POP 6 polymer M13 Reverse primers ABI Prism 377 ABI PRISM 377 with XL Upgrade DP POP6 BD Set 21M13 DP POP6 BD Set M13 Reverse DP5 LR BD M13 FWD amp REV DP5 LR BD M13 FWD amp REV BigDye Terminators ABI PRISM 310 POP 6 polymer ABI PRISM 310 POP 6 polymer Rapid Sequencing ABI PRISM 377 ABI PRISM 377 with XL Upgrade DT POP6 BD Set Any Primer DT POP6 BD Set Any Primer DT BD Set Any Primer DT BD Set Any Primer a DSP DNA Sequencing Polymer The DNA Sequencing Polymer is not supported for use with the BigDye Primers and BigDye Terminators b The dye set primer file can be used with 5 and 5 5 Long Ranger gels and 4 and 4 25 polyacrylamide gels 19 1 acrylamide bis IMPORTANT Mobili
12. q Term Matrix 1 000 0 12 0 011 0 000 1 000 0 12 0 011 0 000 0 455 1 000 0 183 0 000 0 455 1 000 0 183 0 000 0 248 0 483 1 000 0 151 0 248 0 483 1 000 0 151 0 115 0 282 0 529 1 000 0 115 0 282 0 529 1 000 K Copy T Term Matrix 1 000 0 12 0 011 0 000 0 455 1 000 0 183 0 000 0 248 0 483 1 000 0 151 0 115 0 282 0 529 1 000 Make sure that all three matrix files have numbers that range from 0 1 The numbers on the diagonals from top left to bottom right should be 1 If not then repeat the matrix making procedure starting with To make the Dye Primer Matrix on page 8 Note The corresponding numbers for all three matrix files will be the same Click Cancel User Bulletin ABI PRISM dRhodamine Matrix Standards Kit Page 13 of 14 To check the instrument file continued Step Action 5 Restart the Sequencing Analysis software and use dRhod as the instrument file to analyze your sequencing data a For Research Use Only Not for use in diagnostic procedures Copyright 2000 Applied Biosystems Printed in the U S A ABI PRISM and the ABI PRISM design and Applied Biosystems are registered trademarks of Applera Corporation or its subsidiaries in the U S and certain other countries ABI is a trademark of Applera Corporation or its subsidiaries in the U S and certain other countries All other trademarks are the sole property of their respective owners P N 904917D
13. ty shifts and dye set primer file names for the dRhodamine Terminators are similar to those for the BigDye Terminators Their respective mobility files can be mistaken for each other easily without noticeably affecting the base spacing in the data If a mobility file for the wrong sequencing chemistry is used some bases will be miscalled because of differences in which terminators are labeled with which dyes see Table 1 on page 2 and because of the mobility shifts Page 4 of 14 continued on next page User Bulletin ABI PRISM dRhodamine Matrix Standards Kit Installing Run To install the run modules and dye set primer mobility files Modules and Dye Set Primer Files Step Action 1 Copy the run modules for your instrument into the Module folder within the Collection software folder The modules are on the diskette supplied with the dRhodamine Matrix Standards Kit Copy the mobility files for your instrument into the ABI folder within the System folder The mobility files are on the diskette supplied with the dRhodamine Matrix Standards Kit Relaunch the Collection and or Sequencing Analysis software if either was open while the files were installed Note Sometimes it is necessary to restart the Macintosh to use the new run modules and dye set primer files User Bulletin ABI PRISM dRhodamine Matrix Standards Kit Page 5 of 14 Making Instrument Matrix Files Running To run standar
14. use IMPORTANT The number of data points analyzed is the same for each matrix standard Choose starting points for each sample such that all peaks are less than 4000 RFU and that both the starting and ending points have flat baselines and no peaks Launch the Data Utility software From the Utilities menu choose Make Matrix The Make Matrix dialog box appears as shown below Verify that the Dye Primer Matrix button at the lower left is selected Make Matrix C Start at m Start at c Start at m Start at Update File Dye Primer Matrix Taq Terminator Matrix T Terminator Matrix Click on the box for each nucleotide base and enter the data file that corresponds to the correct matrix standard as shown in Table 5 on page 8 Enter the analysis start point for each matrix standard sample as determined in step 1 on page 8 User Bulletin ABI PRISM dRhodamine Matrix Standards Kit Page 9 of 14 To make the Dye Primer Matrix continued Step Action 7 Click New File A dialog window appears as shown below Name the file dRhod and save it in the ABI folder within the System folder J ABI Folder Y Shadow Generic Matrix 2 Eject Koshka s Matrix Seq Analysis Command File Desktop Seq Analysis Error File Shadow s Matrix Name for new matrix file dRhod 8 The Make Matrix dialog box should look like that shown below
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