User Bulletin. ABI PRISM 373 DNA Sequencer. Using the ABI PRISM 373 BigDye Filter Wheel. Contents. Introduction. February 16, 1998 (updated 06/2001)

Transcription

1 User Bulletin ABI PRISM 373 DNA Sequencer February 16, 1998 (updated 06/2001) SUBJECT: Using the ABI PRISM 373 BigDye Filter Wheel Contents The following topics are discussed in this bulletin: Topic See page Preparing to Use the ABI PRISM 373 BigDye Filter Wheel 3 Choosing a Sequencing Chemistry 3 Software Information 6 Creating a drhodamine Instrument (Matrix) File 8 Making a Matrix from Matrix Standards 8 Making a Matrix from a Sample File 20 Sequencing Reaction Setup 21 Sample Loading Recommendations 22 Quick Start Guide for the ABI PRISM 373 BigDye Filter Wheel 23 Appendix A: Part Numbers 25 Appendix B: Dye/Base Relationships 26 Appendix C: Load Chart 27 Introduction This service part will allow users of the ABI PRISM 373 DNA Sequencer to take advantage of Applied Biosystems new dichlororhodamine (drhodamine)-based chemistries: BigDye terminators drhodamine terminators BigDye primers

2 To allow 373 Sequencer users to use the drhodamine-based chemistries, a new filter wheel is now available. The band pass wavelengths of the old and new filters are listed in Table 1 below. The dye/base relationships of the rhodamine-based and drhodamine-based chemistries can be found in Appendix B: Dye/Base Relationships on page 26. Emissions from the drhodamine dyes (dr110, dr6g, dtamra, and drox) are red shifted in the spectrum, which makes them incompatible with the originally installed 373 Sequencer filter set A. This filter set was designed to work with the standard dye primers or rhodamine terminators. Emission spectra from the rhodamine and drhodamine dyes are shown in Figure 1 below. Figure 1. Emission spectra from the rhodamine and drhodamine dyes. Note that the gray bars represent the emission maxima of the four dyes in each dye set. Table 1. Wavelengths of Old and New Filters Filter Set A Color BigDye Filter Set A 531nm Blue 50 nm 560 nm Green 570 nm 580 nm Yellow 595 nm 610 nm Red 625 nm Note The unused filter in the BigDye filter wheel is 555 nm, replacing the 55-nm filter. This filter is not used for drhodamine sequencing applications. The advantage of using the drhodamine-based chemistries with the BigDye filter wheel is that the four dyes have narrower emission spectra leading to less spectral overlap (see Figure 1). This translates into reduced background noise and cleaner signals. In addition, the BigDye chemistries, with their single-molecule energy transfer Page 2 of 28 User Bulletin: ABI PRISM 373 DNA Sequencer

3 system, produce 5 times more signal to noise than the existing chemistries. The BigDye terminator and drhodamine terminator chemistries produce data with significantly more uniform peaks. This improves basecalling accuracy and leads to longer read lengths. IMPORTANT When the BigDye filter wheel is installed, the instrument will still indicate it is running filter set A with the old wavelengths. The kit contains a label that lists the new wavelengths and indicates the instrument has been upgraded to the BigDye filter wheel. At install, the service engineer will place the label on the instrument. IMPORTANT When the BigDye filter wheel is installed, the FS rhodamine terminators and FS dye primers will no longer be compatible. In addition, you cannot perform GeneScan software analysis with the BigDye filter wheel because the emissions from the GeneScan software dyes are not collected efficiently. Preparing to Use the ABI PRISM 373 BigDye Filter Wheel Choosing a Sequencing Chemistry To successfully use the new filter wheel and new sequencing chemistries, read Software Information on page 6 and Creating a drhodamine Instrument (Matrix) File on page 8. Before results can be analyzed, these important setup steps must be performed. The generally recommended chemistries are BigDye terminator and BigDye primer because of their optimal signal-to-noise characteristics. The choice between terminator and primer chemistry depends on the application. See Table 2 on page for chemistry recommendations for different applications. The drhodamine terminators are useful for templates with long homopolymer (> 25 bases) stretches or templates with GT rich motifs. However, the drhodamine terminators produce weaker signals than the BigDye chemistries; more of the sample must be loaded to ensure adequate signal is available. This is especially important for the ABI PRISM 373 DNA Sequencer with the XL Upgrade (373XL) because the 373XL has higher lane density, which requires stronger signal strength for optimal performance. User Bulletin: ABI PRISM 373 DNA Sequencer Page 3 of 28

4 Table 2. ABI PRISM 373 with BigDye Filter Wheel/310/377 Chemistry Recommendations BigDye Terminators drhodamine Terminators BigDye Primers DNA Sequencing Application de novo Sequencing High Throughput R S R de novo Sequencing Mid to Low R S S Throughput Comparative Sequencing (germline mutations 50:50 heterozygotes) R S R Comparative Sequencing (somatic mutations 30:70 heterozygotes) Comparative Sequencing (somatic mutations 10:90 heterozygotes) S N R N N R DNA Sequence Context G-C rich > 65% R S S A-T rich > 65% S R R G-T rich (repeats) N S R Homopolymer A or T > 25 bp N R R Template Plasmid (< 15 kb) R R R M13 R R R BAC, Cosmid, Lambda, XL PCR R S S Bacterial genomic DNA R N N PCR Amplicon R R R PCR Amplicon (heterozygous 50:50) R S R PCR Amplicon (heterozygous 30:70) S N R PCR Amplicon (heterozygous 10:90) N N R R S N Recommended Satisfactory Not Recommended Page of 28 User Bulletin: ABI PRISM 373 DNA Sequencer

5 Suggested Gel Types The BigDye filter wheel has been tested with the following gel types: 5.75% Long Ranger, 3-cm WTR.75% 19:1 acrylamide:bis acrylamide, 3-cm WTR 5% Long Ranger, 8-cm WTR % 19:1 acrylamide:bis acrylamide, 8-cm WTR 5% 29:1 acrylamide:bis acrylamide, 3-cm WTR Mobility files were developed using these gel types. However, mobility files may work for other gel types: 5.75% PagePlus, 3-cm WTR 5.25% PagePlus, 8-cm WTR User Bulletin: ABI PRISM 373 DNA Sequencer Page 5 of 28

6 Software Information Required Files Before samples can be analyzed, two files that interface with the software must be in place: a drhodamine instrument (matrix) file and dye set/primer (mobility) files for the BigDye filter wheel. Instructions for creating the drhodamine instrument (matrix) file begin on page 8. Instructions for installing the mobility files are given in Installation of Mobility Files on page 7. The mobility files are: 373BDP Rev 373BDP -21M13 373dRhod 373BDT Use with ABI PRISM BigDye Primer Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase, FS, M13Rev. Use with ABI PRISM BigDye Primer Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase, FS, -21M13. Use with ABI PRISM drhodamine Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase, FS. Use with ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase, FS. All other software requirements are unchanged; follow the user s manual for your ABI PRISM 373 DNA Sequencer model. Sources The diskette supplied with the BigDye filter wheel includes the mobility files needed to analyze data with the new sequencing chemistries. These can also be obtained from the following sources: Applied Biosystems site on the World Wide Web ( Your local field applications specialist (call your local sales office for more information) Page 6 of 28 User Bulletin: ABI PRISM 373 DNA Sequencer

7 Installation of Mobility Files Perform the following steps to install the mobility files. Step Action 1 In your Macintosh system folder, locate the ABI folder. 2 Install the following files in the ABI folder: 373BDP Rev 373BDP -21M13 373dRhod 373BDT IMPORTANT Dye set/primer (mobility) 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. In addition, if a mobility file for the wrong sequencing chemistry is used, bases will be miscalled. 3 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 computer to use the dye set/primer files. Software Settings To set up a run with the BigDye filter wheel, the following software settings must be made. Select filter set A. Select the appropriate dye set/primer mobility file. Select the drhodamine instrument (matrix) file. If you are running the BigDye filter wheel for the first time, refer to Creating a drhodamine Instrument (Matrix) File on page 8 and Installation of Mobility Files above. To set up all other software settings, refer to the user s manual for your ABI PRISM 373 DNA Sequencer model. IMPORTANT When the BigDye filter wheel is installed, the instrument will still indicate it is running filter set A with the old wavelengths. The kit contains a label that lists the new wavelengths and indicates the instrument has been upgraded to the BigDye filter wheel. At install, the service engineer will place the label on the instrument. User Bulletin: ABI PRISM 373 DNA Sequencer Page 7 of 28

8 IMPORTANT When the BigDye filter wheel is installed, the FS rhodamine terminators and FS dye primers will no longer be compatible. In addition, you cannot perform GeneScan software analysis with the BigDye filter wheel because the emissions from the GeneScan software dyes are not collected efficiently. Creating a drhodamine Instrument (Matrix) File Making a Matrix from Matrix Standards Using the new drhodamine sequencing chemistries requires a new instrument (matrix) file. The new instrument file is made from the matrix standards found in the ABI PRISM drhodamine Matrix Standards Kit (P/N 0307). A set of four matrix standards only needs to be run once to generate an instrument (matrix) file that is used with all three of the drhodamine-based chemistries. An alternative method for preparing an instrument (matrix) file is described in Making a Matrix from a Sample File on page 20. The drhodamine matrix standards are provided in a ready-to-use format and are premixed with a blue dye for convenient gel loading. Matrix standards are stable for six months at 2 6 C. The drhodamine matrix standards are listed below. Table 3. drhodamine Matrix Standards Dye Tube Label Color in Gel Image for Filter Set A Color in Electropherogram dichloro[r110] dr110 Matrix Std Blue Blue dichloro[r6g] dr6g Matrix Std Green Green dichloro[tamra] dtamra Matrix Std Yellow Black dichloro[rox] drox Matrix Std Red Red Page 8 of 28 User Bulletin: ABI PRISM 373 DNA Sequencer

9 Running Standards To run matrix standards on the instrument: Step Action 1 Mix the matrix standards well. 2 Prepare a separate loading cocktail as shown below for each of the four matrix standards: 3 µl of matrix standard 3 µl of deionized formamide! 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.! WARNING! Please dispose of hazardous waste in accordance with all local, state, and federal health and environmental laws and regulations. 3 Heat the cocktails at 95 C for 2 minutes. Place on ice until ready to load. IMPORTANT DNA samples should not be stored in formamide for more than a few hours. Select filter set A. 5 Perform the following steps to prepare the sample sheet: a. List sample names exactly as they are written in the Matrix Standard column of step 8 below. b. Select any dye set/primer. c. Select any instrument (matrix) file. d. De-select Auto Analysis (refer to your user s manual for instructions). User Bulletin: ABI PRISM 373 DNA Sequencer Page 9 of 28

10 To run matrix standards on the instrument: (continued) Step Action 6 Load each of the four matrix standard cocktails into a separate lane of the gels. Refer to the table below for the load amount that corresponds to the comb you are using. It is a good idea to load duplicates of each standard in case one of the lanes is unusable due to an unforeseen gel artifact. Instrument Loading Volume (µl) ABI 373A-18 comb 3.0 ABI 373A-2 comb 3.0 ABI 373A-36 comb ABI 373AXL-8 comb 2.0 ABI 373AXL-6 comb Perform electrophoresis according to the user s manual for your instrument. 8 After electrophoresis is complete, check the lane tracking in the gel image for the matrix standards before making the matrix. The matrix standards should display the following colors: Matrix Standard Color in Gel Image Color in Electropherogram dr110 Blue Blue dr6g Green Green dtamra Yellow Black drox Red Red Page 10 of 28 User Bulletin: ABI PRISM 373 DNA Sequencer

11 Making the Matrix Put the correct data file for each matrix standard into the correct box in the Data Utility application. You will be directed to do this at the appropriate step. Table. Placement of Standards in the Data Utility Application Box Dye Primer Matrix Taq Terminator Matrix T7 Terminator Matrix C dr110 drox dr6g A dr6g dr6g dtamra G dtamra dr110 drox T 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 Taq terminator matrix in the file. An error message will appear saying, Multicomponent matrix error, Bad Data. The T7 Terminator Matrix file is needed to analyze BigDye terminator and drhodamine terminator chemistry data. To make the Dye Primer Matrix: Step Action 1 Launch the Data Utility software (located in the Utilities folder). 2 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. User Bulletin: ABI PRISM 373 DNA Sequencer Page 11 of 28

12 To make the Dye Primer Matrix: (continued) Step Action 3 Click on the box for each nucleotide base and select the sample file that corresponds to the correct matrix standard as shown in the table below. Box C A G T Dye Primer Matrix dr110 dr6g dtamra drox For each matrix standard sample, start with the default value of 2000 for the start point. Start with the default value of 1500 for the number of data points to analyze. Note If the default values do not work, follow the instructions for using other values in steps 8 and 9 below. 5 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. Page 12 of 28 User Bulletin: ABI PRISM 373 DNA Sequencer

13 To make the Dye Primer Matrix: (continued) Step Action 6 The Make Matrix dialog box should look like that shown below. a. Click OK. The computer makes the matrix. When finished, a dialog window appears with the message Make matrix successfully completed. b. Click OK. 7 If the computer is unable to make a matrix, examine the raw data again in the Sequencing Analysis software. If you used the default values, then select new start points as directed in steps 8 and 9 below. If many peaks are off-scale, dilute the matrix standards and rerun them. User Bulletin: ABI PRISM 373 DNA Sequencer Page 13 of 28

14 To make the Dye Primer Matrix: (continued) Step Action 8 If the matrix cannot be made with the default values, proceed with steps a, b, and c below. a. In the Sequencing Analysis software, open a matrix standard sample and examine the raw data. An example is 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 000 relative fluorescence units (RFU), and that the baseline at the end of the range is flat. A typical number of data points is Repeat step 8 for each matrix standard sample. Record the results for later 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 000 RFU and that both the starting and ending points have flat baselines and no peaks. Page 1 of 28 User Bulletin: ABI PRISM 373 DNA Sequencer

15 To make the Taq 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 Taq 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 below. Box C A G T Taq Terminator Matrix drox dr6g dr110 dtamra IMPORTANT The order of matrix standard data files is different from that in the Dye Primer Matrix (see Table on page 11). Enter the same numbers for each matrix standard sample in the Start at and Points boxes as were used for the Dye Primer Matrix. 5 Click Update File A dialog window appears. User Bulletin: ABI PRISM 373 DNA Sequencer Page 15 of 28

16 To make the Taq Terminator Matrix: (continued) Step Action 6 Choose drhod from the ABI folder within the System folder and click Save. The Make Matrix dialog box is displayed. Note The numbers in the Start at and Points boxes are default values. Your numbers may vary. 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. Page 16 of 28 User Bulletin: ABI PRISM 373 DNA Sequencer

17 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 the table below (note the order of the matrix standard files). Box C A G T T7 Terminator Matrix dr6g dtamra drox dr110 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. 5 Click Update File A dialog window appears. User Bulletin: ABI PRISM 373 DNA Sequencer Page 17 of 28

18 To make the T7 Terminator Matrix: (continued) Step Action 6 Choose drhod from the ABI folder within the System folder and click Save. The Make Matrix dialog box should look like that shown below. Note The numbers in the Start at and Points boxes are default values. Your numbers may vary. 7 a. Click OK. The computer makes the matrix. When finished, a dialog window appears with the message Make matrix successfully completed. Click OK. Page 18 of 28 User Bulletin: ABI PRISM 373 DNA Sequencer

19 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. 3 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 11. Note The corresponding numbers for all three matrix files will be the same. Click Cancel. 5 Open or restart the Sequencing Analysis software and use drhod as the instrument file to analyze your sequencing data. User Bulletin: ABI PRISM 373 DNA Sequencer Page 19 of 28

20 Making a Matrix from a Sample File An instrument file can be made from matrix standards as explained above, or it can be made from a sample file that was run with one of the three drhodamine-based chemistries. This procedure requires fewer steps than running matrix standards, however, the matrix made from a sample file may not be as good as one made from matrix standards. The quality of a matrix file made from a sample file depends on the quality of the sample file used. The best samples to choose for making a matrix have approximately 25% each of A, C, G, and T. A good example of this is the pgem DNA with the -21M13 primer that is included as a control in every Ready Reaction Sequencing Kit. To create a matrix from a sample file, follow the steps below. To create a matrix from a sample file: Step Action 1 Prepare a sequencing reaction according to the protocol booklet for the chemistry you choose. 2 Select filter set A. 3 Perform the following steps to prepare the sample sheet: a. List sample names. b. Select any dye set/primer. c. Select any instrument (matrix) file. d. De-select Auto Analysis (refer to your user s manual for instructions). Load the sample according to the load chart for your comb size. See Table 5 on page Perform electrophoresis according to the user s manual for your instrument. 6 Before making the matrix, verify that lane tracking is accurate. Adjust if necessary. 7 Duplicate the unanalyzed sample file three times. Use the Duplicate command from the File menu in the Finder. You will have a total of four copies of the same sample file with the following names: Sample name Sample name Copy 1 Sample name Copy 2 Sample name Copy 3 Page 20 of 28 User Bulletin: ABI PRISM 373 DNA Sequencer

21 To create a matrix from a sample file: (continued) Step Action 8 These four sample files can now be used in the same way as the four matrix standard samples. The same instructions can be used with these four samples as with the four matrix standard samples. Follow the directions in To make the Dye Primer Matrix: on page 11; begin at step. Whenever the protocol indicates a specific matrix standard to be used, follow the table below: Matrix Standard Standard File dr110 Sample name dr6g Sample name Copy 1 dtamra Sample name Copy 2 drox Sample name Copy 3 Sequencing Reaction Setup Follow the relevant protocol booklet for all steps in the sequencing reaction preparation except for the loading recommendations. For the loading recommendations, see Table 5 on page 23 or the same table in Appendix C: Load Chart. The following kits are compatible with the 373 Sequencer BigDye filter wheel: ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase, FS ABI PRISM drhodamine Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase, FS ABI PRISM BigDye Primer Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase, FS, -21M13 ABI PRISM BigDye Primer Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase, FS, M13Rev IMPORTANT When the BigDye filter wheel is installed, the FS rhodamine terminators and FS dye primers will no longer be compatible. In addition, you cannot perform GeneScan software analysis with the BigDye filter wheel because the emissions from the GeneScan software dyes are not collected efficiently. The protocol booklets for these kits were written specifically for the ABI PRISM 310 Genetic Analyzer and ABI PRISM 377 DNA User Bulletin: ABI PRISM 373 DNA Sequencer Page 21 of 28

22 Sequencer before the ABI PRISM 373 BigDye filter wheel was available. Therefore, disregard comments in the protocol booklet saying This kit is not designed for use with the ABI 373 DNA Sequencer or the ABI 377 DNA Sequencer with XL Upgrade. Sample Loading Recommendations Table 5 on page 23 below gives the recommended loading amounts for all the comb configurations and drhodamine-based sequencing chemistries, as well as the drhodamine terminator sequencing standard. The amounts given are for full-volume reactions. If you are doing reduced-volume reactions or diluting the Ready Reaction Mix, the load amounts will have to be increased. The load amount depends on the amount of signal, which is directly related to the quality of the DNA template and primer performance. A very clean template with a perfectly matched primer of good cycle sequencing characteristics will produce ample signal. The optimal range of total signal for drhodamine-based chemistries is between 300 and 000 total signal strength. The total signal is calculated by adding the signal for each of the four bases. These numbers are located in the annotation view of the analyzed sample file or at the top of the printed electropherogram. Acceptable sequence can be obtained from total signal below 300 total signal strength. However, the background may be more noticeable and may interfere with basecalling, especially at the end of the run. If the total signal is below 300 total signal strength and the quality of the data is unacceptable, load more of the reaction. If 100% of the reaction is already being loaded, try any of the following: Check the quantitation of the template and adjust the amount of template. Sometimes adding 25 50% more template can raise signals. Repurify the template to ensure salt and ethanol contaminants are removed. Ensure the primer is suitable for the template and has good melting characteristics (ideally having a T m of approximately C). If you are running these chemistries for the first time, use the upper limit of the load amount. It is better to have more signal than you need rather than risk having an insufficient amount of signal to analyze the data. Page 22 of 28 User Bulletin: ABI PRISM 373 DNA Sequencer

23 Table 5. Suggested Load Amounts for Different Comb Sizes and Chemistry Choices Chemistry Volume (µl) 18-well 2-well 32/36-well 8-well 6-well drhodamine Resuspend 2 2 Load BigDye Resuspend terminator Load BigDye primer Resuspend Load drhodamine Resuspend 2 2 dye terminator Load sequencing 2 2 standard Quick Start Guide for the ABI PRISM 373 BigDye Filter Wheel This Quick Start Guide provides general steps for running the ABI PRISM 373 or 373XL DNA Sequencer with the BigDye filter wheel. For more detailed instructions, please refer to the user s manual for your ABI PRISM 373 DNA Sequencer model. Step Action 1 Prepare the sequencing reactions according to the chemistry protocol. 2 Prepare any of the following gels: 5.75% Long Ranger, 3 cm WTR.75% 19:1 acrylamide:bis acrylamide, 3 cm WTR 5% Long Ranger, 8 cm WTR % 19:1 acrylamide:bis acrylamide, 8 cm WTR 5% 29:1 acrylamide:bis acrylamide, 3-cm WTR 5.75% PagePlus, 3-cm WTR 5.25% PagePlus, 8-cm WTR 3 Select filter set A. User Bulletin: ABI PRISM 373 DNA Sequencer Page 23 of 28

24 Step Action Prepare a sample sheet: a. Input the sample name. b. Select the correct dye set/primer (mobility) file: 373BDP Rev Use with ABI PRISM BigDye Primer Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase, FS, M13Rev. 373BDP -21M13 Use with ABI PRISM BigDye Primer Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase, FS, -21M dRhod Use with ABI PRISM drhodamine Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase, FS. 373BDT Use with ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase, FS. IMPORTANT Dye set/primer (mobility) 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. In addition, if a mobility file for the wrong sequencing chemistry is used, bases will be miscalled. c. Select the drhodamine instrument (matrix) file. Note If you are using the ABI PRISM 373 BigDye filter wheel for the first time, refer to the instructions in Creating a drhodamine Instrument (Matrix) File on page 8. 5 Load samples according to the loading recommendations for your chemistry and comb size. See Table 5 on page 23. Note If you are running these chemistries for the first time, use the upper limit of the load amount. It is better to have more signal than you need rather than risk having an insufficient amount of signal to analyze the data. Page 2 of 28 User Bulletin: ABI PRISM 373 DNA Sequencer

25 Appendix A: Part Numbers Kits Chemistry ABI PRISM drhodamine Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase, FS ABI PRISM BigDye Primer Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase, FS, -21M13 ABI PRISM BigDye Primer Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase, FS, M13Rev ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase, FS Reactions Part Numbers Long Read drhodamine Terminator Cycle Sequencing Standard with AmpliTaq FS drhodamine Matrix Standards 0307 User Bulletin: ABI PRISM 373 DNA Sequencer Page 25 of 28

26 Appendix B: Dye/Base Relationships Table 6. Dye/Base Relationships for all ABI PRISM Sequencing Chemistries Dye primers 5-FAM JOE TAMRA ROX C A G T FS dye terminators R110 R6G TAMRA ROX G A T C dr110 dr6g dtamra drox BigDye terminators G A U C drhod terminators G A C T BigDye primers C A G T Page 26 of 28 User Bulletin: ABI PRISM 373 DNA Sequencer

27 Appendix C: Load Chart Suggested Load Amounts Chemistry drhodamine BigDye terminator BigDye primer drhodamine dye terminator sequencing standard Volume (µl) Resuspend Load Resuspend Load Resuspend Load Resuspend Load 18-well well /36-well well well User Bulletin: ABI PRISM 373 DNA Sequencer Page 27 of 28

28 Copyright 2001 Applied Biosystems. All rights reserved. ABI, and BigDye are trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries. ABI PRISM, Applied Biosystems, and GeneScan are registered 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. Macintosh is a registered trademark of Apple Computer Systems, Inc. P/N Rev. B, Stock No