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DYEnamic ET Terminator Cycle Sequencing Kit

1. Make Matra EZ Name of fibe containing C bate start ot EOD E Moree oF Hee comaintg mate Start ot z009 EZ Name oF tee containing 6 ante stort ar 2000 FZ Mame or nie comaining tants startat Name af pow matrim fiio Select TP unaate raa Comment O hye Primer Matrim O tag herminatar Matrie Caster Select P gt Terminator metrin Fig 1 Make matrix window 5 Select the button T7 Terminator Matrix and then select Update File Choose the DYEnamic ET Terminator file that was copied into the ABI folder in step 1 6 The software must now be told the names of the original sequence file and the copies of the files To do this 24 Click on C then locate and open the original sequence file Click on A then locate and open one of the copies generated in step 2 above Click on G then locate and open a different copy Click on T then locate and open the remaining copy Note There must be four different copies of the same file 7 Click OK This generates the DYEnamic ET terminator matrix file After a few seconds the alert in Figure 2 is displayed Matrix already exists On you want to write over existing matrix Fig 2 8 Choose Yes to enter the new matrix data into the file Note To avoid confusion rename the matrix file to clearly identify the instrument such as ET terminator 377 serial number xxxx 9 Steps 1 8 create a
2. Do not perform this heating step after the sequencing premix has been added to the reaction because these temperatures inactivate the polymerase and result in failed or weak sequences 6 Increase the number of cycles Increasing the number of cycles to 50 100 may improve signal strengths 7 Use full strength or double volume reactions Use the recommended 20 ul reaction volume or alternatively perform 55 double scale reactions 16 pl of Sequencing reagent premix in a 40 ul total reaction volume This increases the final yield of sequencing products 7 8 Considerations for post reaction cleanup The standard cleanup protocol is designed for 20 ml reactions carried out in 0 5 ml microcentrifuge tubes or 96 well microplates This appendix provides a summary of the considerations for post reaction cleanup These recommendations are starting points for optimization The duration of precipitation length and speed of centrifugation geometry of centrifuge rotor and other parameters might need adjusting AutoSeq96 plates Dye terminator is removed efficiently using AutoSeq96 filtration plates 27 5340 10 AutoSeq 96 is a 96 well spin plate containing prehydrated G 50 Sephadex Follow the instructions that accompany the plates The purified sequencing product is recovered in approximately one reaction volume of water The sample should then be dried down in vaccuo and resuspended in loading buffer for the ABI 310 and ABI 373
3. 1989 Levedakou E N et al Biotechniques 7 438 442 1989 Lee J S DNA Cell Biol 10 67 73 1991 essing J Methods in Enzymology 101 20 78 1983 ead D A and Kemper B in Vectors A Survey of Molecular Cloning Vectors and Their Uses Butterworth Publishers assachusetts USA 1986 Dente L et al Nucleic Acids Research 11 1645 1655 1983 Holmes D S and Quigley M Anal Biochem 114 193 197 1981 Carlson A and Messing J J Biotechnology 1 253 1984 Birnboim H C and Doly J Nucleic Acids Research 24 1513 1523 1979 64 65 66 67 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 Jopan 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 Fox 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
4. 2 With the Sample Manager window active click either the Add Samples button or select File gt Add Sample s WG In the Add Samples dialog box locate and open the folder that contains the files you want to add to the Sample Manager window Click on Add Selected Samples Click OK The selected files are then loaded into the Sample Manager R a a Click on the Dye Set Primer File cell for the first sample in the Sample Manager 7 From the drop down menu select the DT3730POP7 ET 36cm mob file 20 8 Fill down the Dye Set Primer File cells so that the DT3730POP7 ET 36cm mob file is applied to all samples 9 Be sure that the Analysis check box A is checked for all samples 10 Click on the Start Analysis icon in the Toolbar 11 Click on the Save All Samples icon on the Toolbar 12 The samples are now ready for viewing and further data analysis e ABI 3700 The filenames DT3700POP 5 ET mob DT3700POP 6 ET mob and DT3700POP37 ET mob refer to the mobility files necessary to analyze runs accomplished on the ABI 3700 with POP 5 polymer POP 6 polymer and POP 37 polymer respectively 1 Quit the Data Collection and the Sequencing Analysis software 2 Copy the appropriate files from the disk to the folder Copy the mobility files from the disk to the Perkin Elmer Abi Shared Analysis Basecaller Mobility folder prior to launching the analysis program For newer version instruments the folder path is applie
5. East Europe Tel 43 1972720 Fox 43 197272 2750 Denmark Tel 45 70 25 2450 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 Italy Tel 02 26001 320 Fax 02 26001 399 Japan Tel 81 3 5331 9336 Fox 81 3 5331 9370 Korea Tel 82 2 6201 3700 Fax 82 2 6201 3803 Latin America Tel 55 11 3933 7300 Fax 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 Fox 47 815 65 666 Portugal Tel 21 417 7035 Fax 21 417 3184 imagination at work Russia amp other C LS SNALS Tel 7 495 956 5177 Fax 7 495 956 5176 South East Asia Tel 60 3 8024 2080 Fax 60 3 8024 2090 Spain Tel 902 11 72 65 Fax 935 94 49 65 Sweden Tel 018 612 1900 Fax 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 US81050PL Rev C 2006
6. acrylamide a neurotoxin and suspected carcinogen Please follow the manufacturer s Material Safety Data Sheet regarding safe handling and use of these materials 2 2 Storage Store at 15 C to 30 C 2 3 Quality control All batches of DYEnamic ET Terminator Cycle Sequencing Kit are assayed according to he recommended starting point protocol described in his booklet Reactions are analyzed on ABI Prism 377 fluorescent sequencing instrument Specifications or release are based on assessment of sequence by ength of read gt 500 bases accuracy and signal quality 3 Components of the kit Solutions included in DYEnamic ET Terminator Cycle Sequencing Kit have been carefully formulated for optimal sequencing results Each reagent has been tested extensively and its concentration adjusted to meet high standards set by GE Healthcare It is strongly recommended to use all reagents supplied in the kit exactly as described in this protocol The following components are included in the kits Kit US81050 US81060 US81070 components 100 rxns 1000 rxns 5000 rxns Sequencing 1 x 800 ul 1x8ml 1x40 ml reagent premix Control 1x50 ul 1x50 ul Not included M13mp18 DNA 0 2 pg pl Control primer 1x 100 ul 1x 100 ul Not included Universal cycle primer 0 5 pmol pl Sodium 1x 400 ul 1x4ml 1x20ml acetate EDTA buffer Formamide 1x 1200 ul 5 x 1200 ul Not included loading dye Store these kits and their com
7. that might mask software miscalls Excessive template DNA can also deplete the supply of nucleotides in the sequencing premix and lead to short sequence reads This is especially problematic with Polymerase Chain Reaction PCR products where small mass amounts of DNA are required to provide the optimal picomole amount of template Recommended buffer for dilution of DNA template Dilute the DNA template in water preferably or in a weakly buffered solution containing no more than 0 1 mM EDTA A suitable buffer is 10 mM Tris HCl pH 8 5 0 1 mM EDTA This concentration of EDTA is lower than in typical TE buffers because excess EDTA in the template or primer resuspension buffercan inhibit sequencing reactions by reducing the effective magnesium concentration Preparation of template DNA Template of suitable quality for use with the DYEnamic ET Terminator Cycle Sequencing Kit can be prepared using a variety of procedures and commercially available products Single stranded plasmid DNA Several published methods are available for preparing single stranded DNA from clones generated in M13 vectors and hybrid plasmid phage phagemid vectors 14 15 Preparation of double stranded plasmid DNA Sequencing double stranded templates with the DYEnamic ET Terminator Cycle Sequencing Kit requires no changes in the protocol Alkaline denaturation is not required for these templates For optimal results use plasmid DNA purified by cesium chlori
8. 377 instruments The eluate of the AutoSeq96 plate can be used directly for the ABI 3730 ABI 3730XL ABI 3100 ABI 3700 instrument following the instrument manufacturer s recommendations for injection from water 384 well plates and reduced volume reactions The standard ethanol precipitation protocol requires volumes that might not fit into all 384 well microplates and many researchers choose reaction volumes smaller than 20 ul In these instances the standard ethanol precipitation protocol can be scaled to match the desired reaction volume For instance if total reaction volume is one quarter the recommended volume 5 ul instead of 20 yl use 1 4 the recommended volume of sodium acetate EDTA 0 5 ul instead of 2 yl and 1 4 the recommended volume of 95 ethanol 20 pl instead of 56 80 ul Success rates may be unacceptable with such small reactions Alternately isopropanol mediated precipitation can be used lsopropanol precipitation in 384 well plates Isopropanol has two advantages over ethanol 1 Lower concentrations are required for precipitation hence smaller total volumes are involved during cleanup and 2 It is unnecessary to add salt to the reaction After cycling add 1 1 5 volumes of 80 isopropanol Spin and wash as outlined in the standard ethanol precipitation Good results are obtained using a final concentration of 40 65 isopropanol in the precipitation mix 50 60 isopropanol is optimal The disadvantage with iso
9. a particular capillary will be displayed as shown in Figure 5 39 12 Examine the results for each cap and verify that the profile is similar to that shown in Figure 5 Note For additional schematic illustrations see page 6 35 of the ABI 3700 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 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 Performing a spectral calibration run for POP 6 For general guidelines on performing a default calibration see page 6 25 of the ABI 3700 user s manual To create a spectral matrix for DYEnamic ET terminator chemistry follow the protocol below Left Bar Right Bar O0O000000 00000000 9 10 Fig 7 Loading the matrix standard rary Firmly place the two tubes containing the DYEnamic ET Terminator Matrix Standard into the right bar in slot positions 9 and 10 as shown in Figure 7 and on page 6 25 in the ABI 3700 user s manual Within the ABI 3700 Data Collection software open the Module Editor listed under the Instrument Utilities menu Select the Others tab under modules menu within the Module Editor Loa
10. matrix file suitable for a specific instrument This procedure must be repeated for each instrument that runs DYEnamic ET terminator sequences A matrix only needs to be created once for each instrument but this must be done before analysis Note If any error messages are generated check the raw data to confirm that the region set by the Start at and Points settings contains well defined peaks b Making a matrix from matrix standard data The analysis of matrix standards is the preferred method to 25 create a matrix DYEnamic ET Terminator Matrix Standard US80860 is specifically formulated for creating a color correction matrix file on the ABI 373 ABI 377 and ABI 310 instruments Four matrix standards consisting of purified single color reaction products in 4 ul of TE buffer are supplied in this product Important The following instructions apply to DYEnamic ET Terminator Matrix Standards US80860 with lot numbers 9614 and greater If the lot number of your matrix standard preparation is less than 9614 please contact your local GE Healthcare office for an alternate procedure Note You cannot use the DYEnamic ET Terminator Matrix Standard for the ABI 3700 US84001 to make a matrix on the ABI 310 ABI 373 or ABI 377 e ABI 373 and ABI 377 1 Add 4 ul of formamide loading dye to each matrix standard and mix well 2 For ABI 373 instruments load 2 ul of each matrix standard mixture into separate lanes of the
11. of distilled water added so that the total volume of DNA primer and water is 12 pl When combined with 8 ul of sequencing reagent premix the total volume of the reaction mix should be 20 ul For additional information concerning the amount of template and primer to use in the reaction see Appendixes 7 2 and 7 3 Note The most consistent results are obtained with 8 ul of sequencing reagent premix in a reaction volume of 20 ul However some researchers might choose to economize by diluting the premix GE Healthcare provides DYEnamic ET Terminator Dilution Buffer US84002 for this purpose Please see Appendix 7 4 for additional information concerning the use of dilution buffer 12 2 Assemble the control reaction exactly as follows M13mp18 Control template 200 ng 1 ul Primer 2 pmol 4ul Sequencing reagent premix 8 ul Water 7 ul Total volume 20 ul Note The sole purpose of the control reaction is to confirm the performance of the sequencing premix under specified and tested conditions The reaction should therefore be assembled and performed exactly as described above Customer data can then be compared with GE Healthcare quality control data should questionable performance of the sequencing premix occur 3 Mix the contents of each tube thoroughly by gentle pipetting Cap the tubes or seal the plates Centrifuge briefly to bring contents to the bottom of the tubes or wells 4 Place the tubes or plate into the therma
12. supplied in the kit 2 For the ABI 373 and ABI 377 instruments the sequencing gel was faulty Repeat the sequencing protocol using a new gel 3 Too much template DNA was used in the sequencing reaction Use less template 1 Residual terminators were not removed from the samples Carefully follow the protocol for post reaction cleanup using a final concentration of exactly 75 ethanol and the sodium acetate EDTA buffer supplied with the kit Alternatively residual terminators can be removed using spin columns If problems persist please contact GE Healthcare Technical Service for assistance 63 J Or A a 10 11 12 13 14 15 16 17 18 19 References Ju J et al Proc Nat Acad Sci USA 92 4347 4351 1995 Sanger F et al Proc Nat Acad Sci USA 74 5463 5467 1977 Prober J M et al Science 238 336 341 1987 Lee L G et al Nucleic Acids Research 20 2471 2483 1992 Tabor S and Richardson C C Proc Nat Acad Sci USA 84 4767 4771 1987 Tabor S and Richardson C C J Biol Chem 264 6447 6458 1989 Tabor S and Richardson C C Proc Nat Acad Sci USA 92 6339 6343 1995 Huibregtse J M and Engelke D R DNA and Protein Engineering Techniques 1 39 41 1988 McMahon G et al Proc Nat Acad Sci USA 84 4974 4978 1987 Carothers A M et al Biotechniques 7 494 496 498 499 1989 urray V Nucleic Acids Research 17 8889
13. the matrix used for analysis matches the run module used for collection of the sequencing data Either Run Module A or E can be used but the module used to make the matrix must match the module used for the run 4 The sequence was not correct even though the data looked good Verify that the correct mobility file is being 59 Problem 1 Sequence data have high background continued 2 Sequencing signals are weak Possible causes solutions 4 Continued used Bases are correlated with dyes via the mobility file 5 Nonspecific binding of the primer occurred Due to the formulation of the sequencing reagent premix primer annealing may be more efficient with this terminator product ncrease the annealing temperature or eliminate the annealing step in the cycling program 1 The Thermo Sequenase II DNA polymerase was denatured Do not include an initial denaturation step in the cycling program Do not incubate above 95 C 2 Samples were not centrifuged sufficiently following salt ethanol precipitation Centrifuge the samples for a longer period or at a higher speed 3 The amount of ethanol used for precipitation was insufficient Verify that the final ethanol concentration is exactly 75 during precipitation of the DNA 4 The primer or template contained excess EDTA Resuspend both primer and template in water or in dilute buffer containing lt 0 1 mM EDTA 60 Problem 2 Sequencing signa
14. 0 concentration of premix in the final reaction volume If the researcher chooses to use less sequencing premix then DYEnamic ET Terminator Dilution Buffer can be used to maintain the appropriate concentration of buffer components Use the dilution buffer according to the following formula Volume of sequencing premix dilution buffer 40 of total reaction volume For example a 20 ul reaction total volume could be assembled using 8 ul of sequencing premix and no dilution buffer according to the recommended protocol Alternatively the 20 pl reaction could be assembled with 6 ul of sequencing premix and 2 ul of dilution buffer This ratio maintains the appropriate concentration of buffer components and could be acceptable depending on the template being sequenced 7 5 Cycling conditions Of the three temperature incubation steps that comprise the cycling program denaturation annealing and extension the denaturation step is the most critical While Thermo Sequenase DNA polymerase has significant advantages over other DNA polymerases used for cycle sequencing it is not as stable against thermal inactivation The reaction buffer in the sequencing premix has been specially formulated to protect the stability of the enzyme and with proper precautions Thermo Sequenase DNA polymerase has ample stability for robust sequencing Denaturation step Important Do not use a denature at temperatures gt 95 C or for 52 longer t
15. 51 507 5 171 534 5 332 666 5 242 796 5 306 618 5 366 860 4 855 225 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 fragment analysis which has been authorized for such use by PE 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 uses 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 PE Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 GE HEALTHCARE IS LICENSED AS A VENDOR FOR AUTHORIZED SEQUENCING AND FRAGMENT ANALYSIS INSTRUMENTS NOTICE TO PURCHASER ABOUT LIMITED LICENSE The purchase of this kit reagent includes a limited non exclusive sublicense under certain patents to use the kit re
16. 6 well plate all wells for 3730XL It is essential to centrifuge the plate to position the samples at the bottom of each well Creation of Spectral Protocol 1 Expand the view in the tree pane a Click the box next to the GA Instruments icon b 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 29 a Type ET_Spectral or a similar name in the Name field b Select Spectral from the Type drop down list c 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 performed on the Dye Set selected 6 Select SeqStd Any4DyeSet par from the Params drop down list 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 Editing Spectral Calibration Section at the end of this protocol 7 Click O
17. ABI Prism 310 ABI 3100 ABI Prism 3700 and ABI 3730 ABI 3730XL DNA Analyzer instruments Thermal cycler For thermally cycled incubations between 50 C and 95 C 1 100 cycles 5 Introduction DYEnamic ET Terminator Cycle Sequencing Kit is designed for sensitive and robust sequencing using novel energy transfer dye labelled terminators 1 and Thermo Sequenase I DNA polymerase For sequencing with this product a sequencing reagent premix is combined with template DNA and primer and thermally cycled The reaction products are then precipitated with ethanol to remove unincorporated dye labelled terminators Samples are finally dissolved in an appropriate loading solution for separation and detection using the ABI 373 ABI 377 ABI 310 ABI 3100 ABI 3700 or the ABI 3730 ABI 3730XL sequencing instruments Dye terminator based sequencing DYEnamic ET Terminator Cycle Sequencing Kits are based on a modification of traditional dideoxynucleotide chain termination chemistry 2 in which terminators are labelled with fluorescent dyes for automated detection 3 4 In this case however each of the four dideoxy terminators ddG ddA ddT and ddC is labelled with two dyes fluorescein and one of four different rhodamine dyes rather than a single dye Fluorescein has a large extinction coefficient at the wavelength 488 nm of the argon ion laser used in the sequencing instrument Acting as the donor dye fluorescein absorbs energy fr
18. DYEnamic ET matrix standards but the DYEnamic ET terminators can also be used DYEnamic ET terminators can be used on either the run module A setting or the run module E setting with equivalent performance However the module used to generate the matrix file must match the module used for sample files If both types of runs are routinely performed it is convenient to make a matrix for each run module A and E and use the corresponding matrix to analyze files a Making a matrix from sequence data A matrix can be created after DYEnamic ET terminator sequences have been electrophoresed but it must be done prior to data analysis 23 Ww Copy the matrix file DYEnamic ET Terminator from the mobility file disk into the ABI folder found in the system folder This is a template must be modified because it is not optimal for the instrument it is simpler to modify an existing file than to create a new one Select a sequence run that appears successful based on the raw data view i e good signal well resolved peaks throughout the run Locate this file on the computer s hard drive Make three copies of the file using the Duplicate command on the computer s file menu This will produce four copies of the file with four distinct names Open the DataUtility program that is located within the ABI software folder Choose the menu function Make Matrix under the Utilities menu This function will display the window in Figure 1
19. GE Healthcare DYEnamic ET Terminator Cycle Sequencing Kit Product Booklet Codes US81050 US81060 US81070 Page finder 1 2 Or ON E Legal Handling 2 1 Safety warnings and precautions 2 2 Storage 2 3 Quality control Components of the kit Materials not supplied Introduction Protocols Oo AN DAAAAHD W 11 6 1 Preliminary preperation and general handling instructions 11 6 2 Preparation of sequencing reactions 6 3 Resuspension of samples and electrophoresis Appendixes 7 1 Setting up the instrument to run and analyze DYEnamic ET Terminators 7 2 Template DNA general considerations 7 3 Primers general considerations 7 4 Dilution and reduced reaction volumes 7 5 Cycling conditions 7 6 Optimal solutions for sample resuspension and injection for the ABI 3730 ABI 3730XL ABI 3700 and ABI 3100 7 7 Sequencing BACs and other large templates 7 8 Considerations for post reaction cleanup 8 Troubleshooting all instruments 9 References 12 15 19 19 46 49 50 52 54 54 56 59 64 1 Legal GE and GE monogram are trademarks of General Electric Company DYEnamic MegaBACE Sequenase AutoSeq Sephadex and Thermo Sequenase are trademarks of GE Healthcare companies NOTICE TO PURCHASER LIMITED LICENSE This kit is sold pursuant to Authorization from PE Applied Biosystems under one or more of the following US Patent numbers 4 849 513 5 015 733 5 118 800 5 118 802 5 1
20. K The module is saved and displayed in the Instrument Protocols section of the Protocol Manager view Creating a Plate Record 1 Expand the view in the tree pane a Click the box next to the GA Instruments icon b Click the box next to the ga3730 icon 2 Click the Plate Manager icon The Plate Manager view opens Terminator Matrix Standard to bring the contents 40 ul to the bottom of the tube 30 3 Click NEW The New Plate Dialog opens 4 Complete the plate information a Type a name for the plate ID in the ID barcode field b Type a name for the plate in the Name field c Select Spectral Calibration from the application drop down list d Select 96 Well from the Plate Type drop down list e Select Heat Sealing or Septa from the Plate Sealing drop down list f Type a name for the owner and operator in the appropriate fields 5 Click OK A blank plate record opens 6 Complete the plate record a In the sample name column type a name b In the Instrument Protocol column select the protocol created in the Creation of Spectral Protocol section c Select the Sample Name and Instrument Protocol columns and fill down 7 Click OK 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 a plate record a Select th
21. 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 This allows the selection of a value from any capillary Choose any acceptable capillary 36 Spectral Calibration Profile for F x AE es el 6 8 0 1 2 9 4 95 w y e o 0 Intensity vs Bin Number 000 Intensity vs Soan Number Capillary Number 52 4 Condition Number 7 41 Q Value 0 966 Data Source From capillary 5t Override matrix from another source From capiltary From data fle J cancer Fig 5 Spectral calibration profile for F using POP 5 Performing a spectral calibration run for POP 37 For general guidelines on performing a default calibration see page 6 25 of the ABI 3700 user s manual To create a spectral matrix for DYEnamic ET terminator chemistry follow the protocol on page 38 Left Bar Right Bar O0O000000 OO000000 Fig 6 9 10 37 a Firmly place the two tubes containing the DYEnamic ET Terminator Matrix Standard into the right bar in slot positions 9 and 10 as shown in Figure 6 below and on page 6 25 in the ABI 3700 user s manual A 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 w
22. agent to perform one or more patented DNA sequencing methods in those patents solely for use with Thermo Sequenase I DNA polymerase purchased from GE Healthcare for research activities No other license is granted expressly impliedly or by estoppel For information concerning availability of additional licenses to practice the patented methodologies contact GE Healthcare Bio Sciences Corp Director Business Development 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA US Patent numbers 4 962 020 5 173 411 5 409 811 5 498 523 5 614 365 and 5 674 716 Patents pending tThis product is sold under licensing arrangements with Roche Molecular Systems F Hoffmann La Roche Ltd and the Perkin Elmer Corporation Purchase of this product is accompanied by a limited license to use it in the Polymerase Chain Reaction PCR process for research in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up front license fee either by payment to Perkin Elmer or as purchased i e an authorized thermal cycler Energy Transfer dyes and primers US Patent numbers 5 654 419 5 688 648 and 5 707 804 Thermo Sequenase is a heat stable DNA polymerase producing even band intensities in DNA sequencing ABI Prism Hi Di and POP are trademarks of Applied Biosystems Inc a Division of the Perkin Elmer Corporation Windows NT is a trademark of Microsoft Corp 2006 Gene
23. al 9 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 page 6 34 of the ABI 3700 user s manual 10 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 Select the dye set to display will appear 11 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
24. aration of DYEnamic ET Terminator Matrix Standard 1 Briefly centrifuge the tube containing the DYEnamic ET Terminator 41 Matrix Standard to bring the contents 40 pl to the bottom of the tube 2 Transfer 5 ml of the DYEnamic ET terminator matrix standard to a tube containing 195 ml of distilled water 3 Mix the contents of the tube thoroughly by vortexing vigorously 4 Briefly centrifuge the tube and dispense 10 ml of the DYEnamic ET Terminator Matrix Standard into a 96 well plate as shown in Figure 8 It is essential to centrifuge the plate to position the samples at the bottom of each well 5 Assemble the plate for loading on the ABI 3100 See diagram on page 3 9 of the ABI 3100 user s manual gt m _ 12 O OO OOOO O GOCCs O OOO0O000 OO 0000 O O Fig 8 Performing a spectral calibration For general guidelines on performing a default calibration see page 4 17 of the ABI 3100 user s manual To create a spectral matrix for DYEnamic ET terminator chemistry follow the protocol below 42 1 Place the plate on the autosampler Note For a schematic of the procedure see page 4 24 in the ABI 3100 user s manual 2 Ww aw Open the ABI 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 Cl
25. background or double superimposed sequences Excess primer also can contribute to an artifact known as the cliff effect that typically appears as a short region 50 200 bases of strong sequence abruptly followed by weak sequence The likely cause of this artifact is the inadvertent generation of PCR products during cycle sequencing that accumulate rapidly and deplete the nucleotide supply in the sequencing premix Determine the concentration of primer and use 5 pmol for each sequencing reaction 2 pmol for the control primer in the kit 49 The following method is useful for measuring the concentration of sequencing primer Dissolve the primer in water preferably or in buffer containing no more than 0 1 mM EDTA and determine its optical density at 260 nm OD 69 For primers containing N bases measured in a cuvette with a 1 cm path lingth the approximate concentration pmol ul is given by the formula Concentration pmol ul OD gt 0 0 01 x N where N is the number of bases Designing a sequencing primer The length and sequence of a primer determines its melting temperature and specificity For cycling temperatures recommended in this protocol the primer should be 18 25 bases in length Check the sequence of the primer for potential self annealing or hairpin formation especially at its 3 end Possible sites of false priming in the vector or other known sequences should also be identified again stressing match
26. buffer DYEnamic ET Terminator Dilution Buffer is specially formulated for use with this product Dilution buffers from other manufacturers are not optimized for use with DYEnamic ET terminators 14 For capillary instruments the sample contained too much salt Salt can interfere with electrokinetic injection used on capillary instruments Perform a 70 ethanol wash of the pellet after precipitation or use a gel filtration medium to remove salt 1 Excess template DNA was present in the reaction In some cases the use of too much template especially PCR product DNA can exhaust the supply of ddNTPs in the reaction If this occurs the sequence will suddenly fade before reaching 350 bases This problem is especially prevalent if excess primer is also used Use lt 1 pmol of template DNA and 5 pmol of primer for each reaction By using less template 62 Problem 3 Extensions appear short with read length limited to lt 350 bases Continued Peaks in the sequence are broad and poorly resolved Localized broad peaks terminator blobs are prevalent in the sequences Possible causes solutions 1 Continued the concentration of any potential contaminant is also reduced 2 The extension step incubation was too brief Increase the duration of the extension step in the cycling program to 2 4 minutes 1 The DNA preparation was contaminated Repeat the sequencing protocol using the control DNA
27. by aspiration from each microcentrifuge tube For plates a brief inverted spin lt 1 minute at 300 x g is sufficient for supernatant removal Remove as much liquid as possible at this step to prevent dye blobs 9 Wash the DNA pellets with 70 ethanol Use as large a volume of 70 ethanol as the tube or well can safely accommodate Centrifuge briefly Note Scientists at GE Healthcare routinely use 250 500 ul for 0 5 ml microcentrifuge tubes 100 ul for 96 well plates and 50 ul for 384 well plates 10 Remove the supernatants by aspiration or by an inverted spin Air dry preferably or vacuum dry in a vacuum centrifuge the pellets for 2 5 minutes Do not overdry 14 6 3 Resuspension of samples and electrophoresis Protocols from this point on vary with the instrument being used Be sure to follow the specific recommendations described below for the instrument that will be used to analyze the sequencing reactions General recommendations for resuspension of samples a The DNA pellet must be completely dissolved at this step for optimal sequencing results If a fixed angle rotor was used for centrifugation the DNA pellet will be on the side of the microcentrifuge tube This material must be washed to the bottom of the tube to ensure that the entire reaction product is loaded onto the gel It is not necessary to heat samples prior to injection In fact heating samples may cause excessive evaporation of the resusp
28. cessarily before the run A compact disk containing the DYEnamic ET terminator mobility files for the ABI 373 ABI 377 ABI 310 ABI 3100 ABI 3700 ABI 3730 and ABI 3730XL instruments can be ordered separately US84003 Mobility files are also available on the GE Healthcare web site The DYEnamic ET terminator mobility files for the ABI 373 ABI 377 and ABI 310 are unchanged from those previously provided with DYEnamic ET Terminator Mobility Disk US80872 Note The sequencing analysis software must be restarted after the mobility files are installed to properly recognize them e ABI 3730 The filename DT3730POP 7 ET mob refer to the mobility files 19 necessary to analyze runs accomplished on the ABI 3730 with POP 7 polymer 1 Quit the Sequencing Analysis software 2 Copy the file from the disk to the folder Copy the mobility files from the disk to the E Applied Biosystems SegA 5 0 Shared Analysis Basecaller Mobility folder prior to launching the analysis program ET Terminator Data Analysis Automatic data analysis is not yet available for DYEnamic ET Terminators on the ABI 3730 Therefore one must load the completed sequencing run into the ABI Sequence Analysis software and select the correct DYEnamic ET Terminator mobility file prior to sequence analysis The following protocol describes the necessary steps 1 Select Start gt Programs gt Applied Biosystems gt Sequencing Analysis 5 0 gt Sequencing Analysis 5 0
29. d N 40 the SpectSQ1_1POP 6DefaultModule parameters into the Module Editor by clicking on its listing in the Others tab Ww Scroll down to the Run Time parameter number 15 listed in the Module Parameters window Edit the Run Time by clicking on the default value of 2 700 seconds and replace that with 4 000 seconds Click the Save As button and enter SpecSQ1_1POP 6Extended as the new module file name Click OK to save the new parameters Click Done to exit the Module Editor 5 In the Data Collection software initiate a spectral calibration run by selecting the Spectral Run command within the Run Setup page This opens up the Calibration Module and Dye Set dialog window From here the appropriate type of calibration module dye set and parameter files are selected Note For a schematic of the procedure see page 6 26 in the ABI 3700 user s manual 6 Using the pull down menu under Calibration Module choose the SpecSQ1_1POP 6Extended File for spectral calibration for POP 6 Note For a schematic of the procedure see page 6 26 in the user s manual 7 Continue the spectral calibration run by following steps 4 12 in the previous section Performing a spectral calibration run for POP 5 page 34 e ABI 3100 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 3100 sequencing instrument Prep
30. dbio Shared Analysis Basecaller Mobility 3 Restart the Data Collection and the Sequencing Analysis software e ABI 3100 The filenames DT3100POP 6 ET 36cm mob DT3100POP 6 ET 50cm mob and DT3100POP4 ET 80cm mob refer to the mobility files necessary for analysis of runs accomplished on the ABI 3100 with POP 6 polymer and POP 4 polymer 1 Quit the Data Collection and the Sequencing Analysis software A Windows NT version of this mobility file can be downloaded from www gehealthcare com sequencing_downloads 21 2 Copy the appropriate files from the disk to the folder Copy the mobility files from the disk to the Perkin Elmer Abi Shared Analysis Basecaller Mobility folder prior to launching the analysis program For newer version instruments the folder path is appliedbio Shared Analysis Basecaller Mobility 3 Restart the Data Collection and the Sequencing Analysis software e ABI 310 The filename DYEnamic ET Term POP 6 refers to the mobility file necessary for analysis of runs accomplished on the ABI 310 with POP 6 polymer 1 Quit the Sequencing Analysis software 2 Copy the appropriate file from the disk to the ABI folder in the system folder 3 Restart the Sequencing Analysis software e ABI 377 and ABI 373 The filename DYEnamic ET Term US80872 refers to the mobility file necessary to analyze runs accomplished on the ABI 373 or ABI 377 1 Quit the Sequencing Analysis software 2 Copy the appropriat
31. de gradients polyethylene glycol PEG precipitation adsorption to glass columns and other common DNA purification methods However because 48 such small quantities of DNA are used in the reactions even impure DNA samples can yield acceptable sequence data Although there are many popular protocols for purifying plasmid DNA from 2 ml to 10 ml cultures scientists at GE Healthcare achieve consistent success using boiling 16 17 and alkaline 18 miniprep methods Templates for sequencing can also be prepared using TempliPhi DNA sequencing template amplification kits manufactured by GE Healthcare for more details contact your local GE Healthcare office or visit us at www gehealthcare com lifesciences and search with the keyword TempliPhi A variety of templates can be amplified by rolling circle amplification using Phi29 DNA polymerase and sequencing quality DNA can be prepared within 4 6 hours directly from bacterial colonies 19 Microgram quantities of template DNA can be prepared at isothermal conditions from picogram amounts of starting material Amplified DNA can be used directly for cycle sequencing without purification 7 3 Primers general considerations Primer amount The optimal amount of primer for sequencing with these protocols is 5 pmol If not enough primer is used signals may be weak If too much primer is present nonspecific priming can occur resulting in noisy sequences characterized by high
32. e F from the pull down menu 5 Important Under Parameter File choose SeqStd AnyDyeSet par from the pull down menu 9 Click OK to accept these chosen fields Sad 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 8 After electrophoresis is completed lt 3 hours a dialog window will display Spectral Calibration Result as shown on page 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 page 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 successful caps The capillary status bar will indicate the 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 9 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 decreases so does the condition number A condition number of 1 0 would indicate no spectral overlap for a particular dye set The expected condit
33. e file from the disk to the ABI folder in the System folder 3 Restart the Sequencing Analysis software Making a color correction file e General comments for all instruments The color correction file allows the computer to identify a unique spectral signature for each of the four different dye terminators A unique color correction file must be generated on each sequencing instrument that analyzes DYEnamic ET 22 terminator sequences The color correction file is specific to the performance of each individual instrument so these files should not be copied from one instrument to another A color correction matrix only need to be created once for the ABI 377 or 373 slab gel instruments For the ABI 3100 ABI 3700 ABI 3730 and ABI 3730XL capillary instruments preform a spectral calibration whenever a new capillary array is installed or when switching from one separation medium to another GE Healthcare supplies matrix standards to produce color correction files for each type of ABI instrument as described below If a matrix file has been generated previously with any DYEnamic ET terminator kit it is not necessary to generate a new one Otherwise prepare a new matrix specific for DYEnamic ET terminator chemistry for each instrument that will use the kit for each instrument e ABI 310 ABI 377 and ABI 373 Two methods are available to create a matrix on ABI 310 ABI 377 and ABI 373 instruments The preferred method is to use the
34. e plate you want to use in the name column b Click Add 31 c Click Done The plate is added to the Run Scheduler view 5 Click on the Run Instrument button Evaluating Spectral Calibration results To view pass fail status of each capillary Locate the log file at the following location E AppliedBiosystems UDC Data Collection Data ga3730 instrument name SpectralCalMclFiles E BigDyeV1 Open the file in Notepad 3 View the results The condition number c is a measure of the spectral overlap of the dyes As the spectral overlap of the dye 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 spectralcalibration fits the 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 m Editing Spectral Calibration Files If the SeqStd Any4DyeSet par is not available when setting up the ET terminator spectral calibration protocol on the insrument an existing parameter file must be edited to be compatible with the DYEnamic ET teminators 1 Locate the Spectral Calibration ParamFiles folder in the following file path E Appliedbiosystems UDC ga3730 Calib
35. ection time and voltage parameters may require some adjustment for optimal results according to the instrument manufacturer s recommendations ABI 3700 1 Dissolve each pellet in 10 20 ul of MegaBACE loading solution and vortex vigorously for 10 20 seconds to ensure complete resuspension Centrifuge briefly to collect the sample at the bottom of the tube well Note For additional information on the use of MegaBACE loading solution for samples analyzed on the ABI 3700 see Appendix 7 6 2 Inject and electrophorese the samples according to the instructions provided with the ABI 3700 instrument Use the appropriate run settings for DYEnamic ET terminators Note For information on setting up the instrument to run and analyze DYEnamic ET terminators see Appendix 7 1 Note Recommended injection parameters for MegaBACE loading solution with the control DNA is 1 kV for 30 seconds Injection time and voltage parameters may require some adjustment 16 for optimal results according to the instrument manufacturer s recommendations ABI 3100 1 Dissolve each pellet in 20 30 ul of MegaBACE loading solution and vortex vigorously for 10 20 seconds to ensure complete resuspension Briefly centrifuge to collect the sample at the bottom of the tube well 2 Inject and electrophorese the samples according to the instructions provided with the ABI 3100 instrument Use the appropriate run settings for DYEnamic ET terminators Note For i
36. ension buffer and speed the breakdown of the dye abelled sequencing products Two buffers are recommended for resuspension of the samples prior to electrophoresis The pink formamide loading dye US79448 supplied with the kit is recommended only for use on slab gel instruments ABI 373 ABI 377 MegaBACE loading solution US79916 is recommended for capillary instruments ABI 310 ABI 3100 ABI 3700 ABI 3730 ABI 3730XL If the same samples are to be analyzed on both slab gel and capillary instruments dissolve the samples in a small volume 4 ul of MegaBACE loading solution Remove enough volume of the samples for slab gel analysis then add MegaBACE loading solution up to a final volume of 10 ul to the remainder of the sample and mix well prior to injection on the capillary instrument 15 ABI 3730 3730XL 1 Dissolve each pellet in 10 20 ul of MegaBACE loading solution and vortex vigorously for 10 20 seconds to ensure complete resuspension Centrifuge briefly to collect the sample at the bottom of the tube well 2 Inject and electrophorese the samples according to the instructions provided with the ABI 3730 3730XL instrument Use the appropriate run settings for DYEnamic ET terminators Note For information on setting up the instrument to run and analyze DYEnamic ET terminators see Appendix 7 1 Note Recommended injection parameters for MegaBACE loading solution with the control DNA is 1 kV for 20 seconds Inj
37. es involving the 3 end of the primer 7 4 Dilution and reduced reaction volumes DYEnamic ET terminator sequencing products are designed to provide robust performance over a wide range of conditions The DYEnamic ET terminator sequencing reagent premix is carefully formulated for use with Thermo Sequenase II DNA polymerase and the conditions that yield the best sequencing results have been determined empirically during product development Optimal results are achieved when the following conditions are observed 1 the concentration of the buffer components is maintained to protect the stability and activity of the DNA polymerase 2 the enzyme cofactors and substrates are present in excess such that the reaction is efficient and 3 the volume of the sequencing reaction is sufficient to withstand evaporation For the DYEnamic ET terminator 50 sequencing products the most consistent results are obtained using 8 ul of sequencing reagent premix in a reaction volume of 20 ul as described previously in the protocols These are the recommended conditions for routine use and should be the starting point for any optimization experiments Some researchers might prefer to dilute the sequencing reaction mix to achieve greater economy albeit at the expense of data consistency The DYEnamic ET Terminator Dilution Buffer US84002 provides the appropriate buffer conditions salts metal cofactors and stabilizing agents necessary for optima
38. et 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 9 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 45 At the bottom left of the dialog box under Override matrix from another source click on the button From capillary This will allow you to choose values from any capillary Choose any acceptable capillary 13 Examine the results for each cap and verify that the profile is similar to that shown in Figure 9 Note For additional schematic illustrations see page 4 29 of the ABI 3100 user s manual 7 2 Template DNA general considerations Template amount This protocol usually produces optimal results using 0 1 0 2 pmol of template DNA but these numbers should be considered as guidelines In some cases more or less template can be used due to the sensitivity and robustness of DYEnamic ET terminators For example scientists at GE Healthcare have obtained good resu
39. fragments was inefficient and the trace was weak early in the sequence Final ethanol concentration Salt added 85 1 5M NaOAc 250mM EDTA Blobs 7 5M ammonium acetate Blobs 7 5M NH40Ac 60mM EDTA Blobs 1 5M NaOAc Blobs No Salt Blobs Table 1 Precipitation efficiency 80 Good Blobs Blobs Good Blobs 58 75 Good Blobs Good Good Blobs 70 Good Good Good 65 60 Delayed Delayed Delayed Delayed Delayed Delayed Delayed Delayed Delayed Blobs Blobs Blobs 8 Troubleshooting all instruments Under ideal conditions the strengths of the G A and T signal values reported by the analysis software should be roughly equal and between 200 and 1 000 The C signal will be lower but should be at least 50 of the G A and T values Signal strengths lt 50 or gt 1500 for ABI 373 ABI 377 and ABI 310 or lt 10 or gt 1500 for ABI 3700 3100 3730 3730XL may indicate problems Note Always run the kit control reagents in parallel with test samples during optimization Problem Possible causes solutions 1 Sequence data 1 The matrix was bad have high Confirm that a matrix file has been made background from the standards on each machine on which the kit will be used 2 The wrong matrix was used Confirm that the matrix file being used was generated with the same run module as the samples on the specific instrument being used 3 Sequences were run with the wrong run module Confirm that
40. gel and run the gel For ABI 377 instruments load 1 ul of each mixture into separate lanes and run the gel These matrix standards can analyzed on the same gel as the sequencing reations Note Be sure to record the lane order and file name of the standards because the colors displayed in the gel file will not be those commonly observed for these bases 3 Follow steps 1 5 in the previous section Making a matrix from sequence data 4 The software must now be told the names of the four sample files created by the four standard lanes that were run in steps 1 and 2 above Because the association of the dyes with bases 26 for the DYEnamic ET terminators is different from that for T7 terminators different standard sample lanes from those called for in the software must be used As is illustrated in Figure 3 Click on C then locate and open the file containing the T matrix standard data Click on A then locate and open the file containing the A matrix standard data Click on G then locate and open the file containing the C matrix standard data Click on T then locate and open the file containing the G matrix standard data Make Matrix T standard c Name of file containing X data Start at A standard a Name of file containing X data Start at C standard 6 Name of file containing X data Start at G standard A Name of file containing Xdata Start at Fig 3 5 Continue with steps 7 9 i
41. han 30 seconds The inclusion of a long denaturation step prior to cycling is a common PCR strategy but is unnecessary and not recommended for cycle sequencing reactions Extended denaturing can prematurely in activate Thermo Sequenase II DNA polymeraseand ultimately produce weak signals Annealing step The appropriate annealing temperature varies with the length and sequence of the primer In general temperatures from 45 55 C work well An annealing step is usually required only with primers lt 20 bases in length Optimal annealing temperatures are up to 5 C higher in DYEnamic ET terminator reactions than with other dye terminator sequencing products For primers with sufficiently high melting temperatures the annealing step can be omitted and a two step cycling program alternating between denaturation and extension temperatures can be used Extension step Extension at 60 C for 60 seconds is optimal Because Thermo Sequenase II DNA polymerase incorporates dITP more rapidly than other DNA sequencing enzymes there is no apparent advantage to increase the time or temperature of the extension step Number of cycles Twenty five to thirty cycles are sufficient to sequence the recommended amounts of plasmids or PCR products More cycles are usually not necessary and may lead to artifacts Increasing the number of cycles might be appropriate when sequencing extremely large DNAs such as BACs See appendix 7 7 for more details and rec
42. hich the appropriate calibration module dye set and parameter files are selected Note For a schematic of the procedure see page 6 26 in the ABI 3700 user s manual 3 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 page 6 26 in the ABI 3700 user s manual 4 Important Under Dye Set choose F from the pull down menu 5 Important Under Parameter File choose SeqStd AnyDyeSet par from the pull down menu 6 Click OK to accept these chosen fields 7 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 8 After electrophoresis is complete lt 3 hours a dialog window Spectral Calibration Result appears as shown on page 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 page 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 successful caps The capillary 38 status bar will indicate 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 manu
43. ick Finish This opens the Plate Editor spreadsheet Within the Plate Editor spreadsheet complete the following for the 16 samples in the sample plate e Name the samples e Select Dye Set F Important e Select the run module appropriate for you capillary array size 36 cm Spect36_POP 6DefaultModule 50 cm Spect50_POP 6DefaultModule 80 cm Spect80_POP 4DefaultModule Select the spectral calibration parameters SeqStd AnyDyeSet par Important Click OK 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 plate you just created from the Pending Plate Records table 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 page 3 39 in the ABI 3100 user s manual Click the Run Instrument button on the toolbar to begin the run After the run is completed 40 150 minutes a dialog window displays Spectral Calibration Result as shown on page 4 23 43 10 11 in ABI 3100 User s Manual indicating the number of caps that passed spectral calibration Accept the result by clicking OK The software then assigns proper calibration values to failed caps automatically from the adjoining successful caps Upon completion of the spectral run and data processing the quality of the spectra
44. ion 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 35 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 page 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 10 Select F from the pull down menu Important Click OK This action opens a dialog window title Spectral Calibration Profile for F The fluorescence emission spectra for all four dyes in a particular capillary will be displayed as shown in Figure 1 11 Examine the results for each cap and verify that the profile is similar to that shown in Figure 5 Note For additional schematic illustrations see page 6 35 of the 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
45. ity and excellent performance on GC rich templates make it an efficient and robust sequencing enzyme Cycle sequencing Thermostable DNA polymerases allow sequencing reactions to be cycled through alternating periods of thermal denaturation primer annealing and extension termination which increases the signal levels generated from template DNA 8 13 This amplification process employs a single primer so the amount of product increases linearly with the number of cycles By using a thermostable enzyme such as Thermo Sequenase II DNA polymerase programmed cycling can proceed without multiple additions of enzyme A cycling protocol is especially useful when the amount of template is limiting or the sensitivity of the detection system is low 10 6 Protocols 6 1 Preliminary preparation and general handling instructions Important Researchers who work with DYEnamic ET terminators for the first time should carefully read Appendix 7 1 Setting up the instrument to run and analyze DYEnamic ET terminators page 19 A mobility file must be installed and a color correction matrix must to be created prior to their use on ABI sequencing instruments Thaw and maintain all kit reagents on ice prior to use Whenever possible cap the tubes to minimize evaporation of the small volumes of reagents used Dispense reagents using disposable tip micropipettes and exercise caution to avoid contamination of stock solutions Thoroughly mix reac
46. l cycler Run the following cycling program for 25 cycles 95 C 20 seconds 50 C 15 seconds 60 C 60 seconds Cycling is completed in approximatley 1 hour Note For additional information concerning cycling conditions see Appendix 7 5 5 When cucling is complete add 2 ul 1 10 volume of sodium acetate EDTA buffer to each tube or well 6 Add 80 ul of 95 ethanol to each reaction and mix well using a vortex mixer Note When working with 384 well plates scientists at GE Healthcare routinely use 30 ul of 95 ethanol Please refer to 13 Appendix 7 8 for additional information regarding 384 well plates and small volume reactions Note Post reaction cleanup is most easily and inexpensively achieved by ethanol precipitation using the sodium acetate EDTA buffer provided with the kit This buffer contains 1 5 M sodium acetate pH gt 8 0 and 250 mM EDTA EDTA dramatically reduces the amount of unincorporated terminators that precipitate with the DNA virtually eliminating dye blobs that distort sequence data near the primer and in adjacent lanes Note Do not combine the sodium acetate EDTA buffer and ethanol prior to addition to the sequencing reaction EDTA has very low solubility in ethanol and will precipitate 7 Centrifuge the tubes at room temperature in a microcentrifuge for 15 minutes at 12 000 rpm Centrifuge 96 well or 384 well plates for at least 30 minutes at 2 500 x g or greater 8 Remove the supernatant
47. l enzyme activity In any case the level of dilution that produces acceptable data must be determined empirically by the researcher DYEnamic ET Terminator Dilution Buffer is not included with this kit but can be ordered separately US84002 Dilution buffers provided by other manufacturers should not be used with DYEnamic ET terminator products Note GE Healthcare makes every effort to develop products that are consistent in performance over a broad range of conditions Dilution of this kit s components is not recommended or supported and no guarantee is provided concerning the extent to which a product can be diluted DYEnamic ET Terminator Dilution Buffer is provided as a courtesy for those researchers who choose to economize with the understanding that the researcher must accept the risk of reduced performance How to use the dilution buffer For optimal sequencing results it is important to maintain the appropriate concentration of buffer components in the sequencing reaction To achieve this use 8 ul of DYEnamic ET Terminator sequencing reagent premix in a 20 ul reaction volume such that 40 of the final reaction mixture is sequencing premix If the total 51 volume of the sequencing reaction mixture changes then scale all of the components of the reaction appropriately For example if 10 ul reactions are assembled in a 384 well microplate then 4 ul of sequencing premix would be required in each reaction to achieve a 4
48. l profile the emission spectra for all 4 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 matrix replacement are provided in the ABI 3100 user s manual on page 4 27 Overriding a Spectral Calibration Profile The condition number is a measure of the spectral overlap of the dyes As the spectral overlap of 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 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 capability 44 Matrices for dye set F x AA a na Sa a Ty E E TRG On TR ne Intensity vs Bin Number Intensity vs Sean Number Capillary Number 16 aj at Condition Number 7 43 Q Value 0 957 Data Source _x Fig 9 Spectral calibration profile for F using POP 6 with the ABI 3100 12 Review the 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 s
49. ls are weak continued Possible causes solutions 5 10 11 The DNA preparation was faulty Repeat the protocol using the control DNA supplied in the kit Either the quantity of template DNA or the number of cycles used for amplification was insufficient Increase either the amount of DNA used in the reaction or the number of cycles The annealing temperature was too high for the primer being used Use a lower annealing temperature for cycling Insufficient primer was used The recommended amount of primer is 5 pmol per reaction The primer was faulty Some primers form dimers or hairpins that can interfere with annealing Use a different preparation of the primer or a primer of different sequence The wrong volume of sequencing reagent premix was used The reagents are carefully formulated to work optimally with 8 ul of premix in a 20 ul reaction volume No other configuration is recommended or supported Check the volumes of reagents added Primer and template DNA were used in excess Check the quantities added to the sequencing reaction mix 61 Problem 2 Sequencing signals are weak continued 3 Extensions appear short with read length limited to lt 350 bases Possible causes solutions 12 The sequencing reagent premix was diluted Repeat the experiment using the recommended amounts of reaction components 13 The sequencing reagent premix was diluted in the wrong dilution
50. lts on an ABI 377 using 10 10 000 ng 4 fmol 4 pmol of pure M13mp18 DNA For routine sequencing however follow the guidelines described above The following formula is useful for calculating the optimal mass 0 15 pmol of double stranded template to include in the sequencing reaction Mass of template ng Total length of DNA in base pairs x 0 1 For example a typical plasmid that is 3 800 base pairs in length vector plus insert should produce optimal data using 380 ng in these protocols The recommended range for template amount is 250 500 ng 0 1 0 2 pmol These relationships are shown graphically in the figures 10 and 11 46 These relationships are shown graphically in the figures 10 and 11 Short PCR products Mass of DNA in ng 0 200 400 600 800 1000 Length of template in base pairs Fig 10 The recommended amount of template DNA to use in a sequencing reaction PCR products Plasmids and large PCR products 1400 1200 an Q Q fo Mass of DNA in ng fon 8 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 Length of template in base pairs Fig 11 The recommended amount of template DNA to use in a sequencing reaction Plasmids 47 Insufficient template DNA in the sequencing reaction can produce low signal strengths lt 50 that can cause poor basecalling and short reads In contrast too much template can yield very high signal strengths gt 1500
51. lysis System 1 Resuspend each pellet in 10 20 ul of MegaBACE loading solution Vortex vigorously 10 20 seconds to ensure complete resuspension Briefly centrifuge to collect the sample at the bottom of the tube or plate and to remove bubbles 2 The analysis of DYEnamic ET terminator sequences on MegaBACE DNA Analysis Systems does not require the installation of a mobility file or the creation of a color correction file The MegaBACE analysis software automatically performs both of these functions 18 7 Appendixes 7 1 Setting up the instrument to run and analyze DYEnamic ET Terminators The analysis of DYEnamic ET terminator sequences on ABI instruments requires a mobility file and a color correction file also known as a spectral separation file matrix file or instrument file The procedures are different for each ABI instrument Please proceed to the section below that pertains to the instrument being used Mobility file installation e General comments for all instruments Two functions are performed by the mobility file 1 to correlate the specific dye with its corresponding base call designation and 2 to correct for differences in the electrophoretic mobility of sequencing products terminated with the four different terminators The correct mobility file is supplied by GE Healthcare and need only be installed once on the computer used for analysis Note The mobility file must be installed before analysis but not ne
52. n the previous section Making a matrix from sequence data page 23 Note If any error messages are generated check the raw data to be sure that the region set by the Start at and Points settings contains well defined peaks More importantly make absolutely sure that the order of the matrix standard data files is correct as shown above Do not use the order suggested by the DataUtility software because errors will result and no matrix will be created 27 e ABI 310 1 Add 6 ul of MegaBACE loading solution to each of the four matrix standards and mix well 2 For each of the four matrix standards inject the entire 10 ul volume using standard conditions for injection Note Each standard will require a separate run for the ABI 310 instrument Note Be sure to record the lane order and file name of the standards because the colors displayed in the gel file will not be those commonly observed for these bases 3 Follow steps 1 5 in the previous section Making a matrix from sequence data page 23 4 The software must now be told the names of the four sample files created by the four standard lanes that were run in steps 1 and 2 immediately above Because the association of the dyes with bases for the DYEnamic ET terminators is different from that for T7 terminators standard sample lanes different from those called for in the software must be selected Click on C then locate and open the file containing the T matri
53. nformation on setting up the instrument to run and analyze DYEnamic ET terminators see Appendix 7 1 Note Recommended injection parameters for MegaBACE loading solution with the control DNA is 1 5 kV for 20 seconds Injection time and voltage parameters may require some adjustment for optimal results according to the instrument manufacturer s recommendations ABI 310 1 Dissolve each pellet in 10 pl of MegaBACE loading solution preferably or in 10 ul of formamide loading dye and vortex vigorously for 10 20 seconds to ensure complete resuspension Briefly centrifuge to collect the sample at the bottom of the tube well 2 Carry out injection and electrophoresis according to the instructions provided with the ABI 310 instrument Note For information on setting up the instrument to run and analyze DYEnamic ET terminators see Appendix 7 1 17 ABI 377 373 1 Dissolve each pellet in 4 pl of formamide loading dye and vortex vigorously for 10 20 seconds to ensure complete resuspension Briefly centrifuge to collect the sample at the bottom of the tube well 2 For the ABI 373 load as much sample as feasible into a single well For the ABI 377 load half of the sample 2 ul into a single well 3 Perform electrophoresis according to the instructions provided with the sequencing instrument Note For information on setting up the instruments to run and analyze DYEnamic ET terminators see Appendix 7 1 MegaBACE DNA Ana
54. om incident laser light and transfers it to the rhodamine acceptor dye on the same terminator molecule Each acceptor dye then emits light at its characteristic wavelength for detection that identifies the nucleotide that terminates extension of the DNA chain This energy transfer format 1 is more efficient than direct excitation of the acceptor dye by the laser and produces a sequencing method that is very sensitive and robust The acceptor dyes used in the kits are the same standard rhodamine dyes rhodamine 110 rhodamine 6 G tetramethyl rhodamine and rhodamine X used in earlier Taq DNA polymerase dye terminator 9 methodologies so the DYEnamic ET reaction products can be detected on any instrument that can monitor the original Taq dye terminator chemistry The kit also features dITP as well as Thermo Sequenase II DNA polymerase a thermostable enzyme that efficiently incorporates dITP By replacing dGTP with dITP even very strong compression artifacts common to high GC content DNA are resolved for more accurate data interpretation Thermo Sequenase II DNA polymerase Thermo Sequenase DNA polymerase is a thermostable DNA polymerase engineered by GE Healthcare specifically for cycle sequencing The enzyme readily accepts dideoxynucleotide terminators 5 and generates bands of uniform intensity much like T7 Sequenase DNA polymerase 6 7 Its tolerance to high salt conditions efficient utilization of dITP high processiv
55. ommendations 53 7 6 Optimal solutions for sample resuspension and injection for the ABI 3730 ABI 3730XL ABI 3700 and ABI 3100 Several solutions were tested for their ability to protect sequenced samples from breakdown prior to injection These solutions included water the ABI HiDi formamide and several concentrations of formamide deionized and nondeionized in aqueous solutions at different pH in the presence and absence of EDTA In a comparison experiment the purified sequencing reaction products were dissolved in various solutions placed onto the ABI 3700 loading deck and incubated at ambient temperature for 24 hours During this period samples were loaded and run every 3 hours to simulate unattended operation Each run was analyzed for the appearance of breakdown products G blobs appear first with a concurrent decrease in G signal strength and for the loss of resolution in the sequence ladder C peaks show this first The MegaBACE loading solution low conductivity 70 formamide in water 1 mM EDTA pH 8 protected the sequenced samples from decomposition better than formamide solutions without EDTA and much better than water Samples resuspended in MegaBACE loading solution generated high quality sequence data with minimal breakdown after 24 hours Sequencing products resuspended in MegaBACE loading solution do not have to be covered with foil Other loading solutions might also be appropriate but must be tested by
56. ponents at 15 C to 30 C not in a frost free freezer When the reagent is not in a freezer keep it on ice prior to use For convenience the kit can be stored at 2 4 C for up to three months with no loss of performance however avoid this if the reagents will not be consumed completely within three months 7 4 Materials not supplied Reagents Water Use only deionized distilled water for the sequencing reactions Sequencing primers Use primers appropriate for the template being sequenced For most applications 5 pmol of primer is sufficient Dissolve each primer in water and determine its optical density at 260 nm ODz 9 For primers containing N bases measured in a cuvette with a 1 cm path length the approximate concentration pmol l is given by the formula Concentration pmol l ODz 9 0 01 x N where N is the number of bases Ethanol 95 and 70 For post reaction cleanup Note Do not use denatured alcohol Gel reagents Prepare sequencing gels according to instructions provided by the manufacturer of the sequencing instrument Use electrophoresisgrade reagents Equipment Liquid handling supplies Vials pipettes microcentrifuge and vacuum centrifuge Perform all sequencing reactions in plastic microcentrifuge tubes typically 0 5 ml or in 96 well or 384 well plates suitable for thermal cycling Instrument This kit is designed for use with the ABI 373 DNA Sequencer and the ABI Prism 377
57. propanol precipitation is that the pelleted DNA is less firm than with ethanol precipitation and may be lost during wash and inverted spins It is important to add an isopropanol solution that is less than 100 isopropanol because addition of pure isopropanol even to the same final concentration causes dye blobs These blobs are caused by very high local concentrations of isopropanol before and during mixing Alternate salts for ethanol precipitation The sodium acetate EDTA buffer included with the kit produces the most efficient precipitation over the widest range of ethanol terminators The disadvantage to this salt is that it cannot be mixed with ethanol prior to addition to the sequencing reaction Alternate salt solutions have been investigated and the results are presented in Table 1 To generate this table control reactions were purified by ethanol precipitation with various salts and ethanol concentrations To each 20 ul reaction 1 10 volume 2 ul of the given salt was added the solution was mixed and 95 ethanol was added to the tabulated final ethanol concentration The mixture was centrifuged and washed as described in the standard protocol Blobs denotes that unincorporated terminators were precipitated and not removed by the 70 ethanol wash Good denotes no terminator blobs observed 57 and the sequence was strong and started at the expected position Delayed denotes that the precipitation of sequencing
58. ral 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 and precautions 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 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 data sheet s and or safety statement s for specific advice Warning This kit contains formamide The protocol also requires the use of ethanol a flammable liquid Gel reagents may contain
59. rationData SupportFiles SpectralCalibration ParamFiles 32 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 callibration is 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 m Edit the copied parameter file to include the following minQ 0 92 conditionBounds 1 0 10 0 maxScansAnalyzed 5000 6 Save the file Rename the edited file SeqStd E par or SeqStd Z par as appropriate N 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 e ABI 3700 DYEnamic ET Terminator Matrix Standard for the ABI 3700 US84001 is specifically formulated for performing a spectral calibration and creating a spectral matrix for the ABI 3700 sequencing instrument Preparing the matrix standard 1 Briefly centrifuge the tube containing the DYEnamic ET 33 2 Add 360 ul of distilled wa
60. ter to the tube containing the matrix standard Mix the contents of the tube thoroughly by vigorous vortexing Briefly centrifuge the tube 3 Transfer 200 ul of the matrix solution to each of two 200 ul microtubes as recommended by the instrument manufacturer Performing a spectral calibration run for POP 5 For general guidelines on performing a default calibration see page 6 25 of the ABI 3700 user s manual To create a spectral matrix for DYEnamic ET terminator chemistry follow the protocol below 1 Firmly place the two tubes containing the DYEnamic ET Terminator Matrix Standard into the right bar in slot positions 9 and 10 as shown in the Figure 4 below and on page 6 25 in the ABI 3700 user s manual N Open the ABI 3700 Data Collection software Initiate a spectral calibration run by selecting the Spectral Run command under the Run Setup page This opens up the Calibration Module and Dye Left Bar Right Bar ooo0oo0oo0oo0o00 0 00000000 9 10 Fig 4 Set dialog window From here the appropriate type of calibration module dye set and parameter files are selected Note For a schematic of the procedure see page 6 26 in the ABI 3700 user s manual 3 Using the pull down menu under Calibration Module choose the SpecSQ1_1POP 5DefaultModule file for spectral calibration for POP 5 34 Note For a schematic of the procedure see page 6 26 in the ABI 3700 user s manual 4 Important Under Dye Set choos
61. the investigator 7 7 Sequencing BACs and other large templates Bacterial Artificial Chromosomes BACs and other large templates gt 10 kb in length can be difficult to sequence reliably A large mass 54 of template DNA is required to provide enough sequencing targets picomoles for efficient sequencing For example 10 ug of a BAC 100 kb long would be required to supply the 0 15 pmol recommended for a sequencing reaction Additionally with a large mass of DNA in the sequencing reaction specific and efficient priming becomes difficult The following suggestions are recommended for sequencing large templates 1 Start with the purest template possible Clean BAC preparations yield much more consistent sequencing results than quick and dirty preparations 2 Include at least 1 ug of template DNA in the reaction Although purified BACs are typically difficult to acquire in large amounts ap proximately 1 ug is a good general rule for minimum template mass 3 Increase the amount of primer Greater primer amounts 20 50 pmol per reaction can improve priming efficiency 4 Linearize the template Linearizing or fragmenting the template by digestion with restriction enzymes mechanical shearing or sonication can improve sequencing results 5 Denature the template Before adding the sequencing premix denature the template by heating for 5 minutes at 95 98 C This denatures the template more completely and helps priming
62. tion mixtures after each addition typically by pumping the solution two or three times with a micropipettor without creating air bubbles Centrifuge briefly tubes plates to collect the reaction mixtures at the bottoms of the vessel With care and experience reactions can be completed in 15 20 min The protocol described below provides high quality sequencing results using the control DNA and primer provided in the kit However regard this protocol only as a starting point Optimization of protocols might be necessary to obtain best sequencing results for specific templates For additional information on optimization and some important precautions please refer to Appendixes 7 2 7 7 and the troubleshooting section For general information concerning templates primers and cycling conditions see Appendixes 7 2 7 5 and 7 7 11 6 2 Preparation of sequencing reactions This section describes the procedure and important parameters for performing the sequencing reaction The protocols in this section are the same for all current ABI and MegaBACE sequencing instruments 1 Assemble each sequencing reaction as follows Template DNA 0 1 0 2 pmol ul Primer 5 pmol pl Water yl Sequencing reagent premix 8 ul Total volume 20 ul Note 0 1 0 2 pmol of DNA and 5 pmol of primer are recommended for routine sequencing The volumes of DNA and primer added to each reaction will depend on their concentrations Adjust the amount
63. x standard data Click on A then locate and open the file containing the A matrix standard data Click on G then locate and open the file containing the C matrix standard data Click on T then locate and open the file containing the G matrix standard data 5 Continue with steps 7 9 in the previous section Making a matrix from sequence data page 23 Note If any error messages are generated check the raw data to be sure that the region set by the Start at and Points settings contains 28 well defined peaks More importantly make certain that the order of the matrix standard data files is correct as shown above Do not use the order suggested by the DataUtility software because errors will result and no matrix will be created e ABI 3730 DYEnamic ET Terminator Matrix Standard for the ABI 3730 US84001 is formulated for performing a spectral calibration and creating a spectral matrix for the ABI 3730 sequencing instrument Preparing the 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 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 Note Combine 2 tubes for 3730XL 3 Dispense 10 ul of the DYEnamic ET terminator matrix standard into every other column of a 9

DYEnamic ET Terminator Cycle Sequencing Kit

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