BigDye® Terminator v1.1 Cycle Sequencing Kit
Contents
1. Index 3 Index 4 BigDye Terminator v1 1 Cycle Seguencing Kit Headguarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free In North America 1 800 345 5224 Fax 1 650 638 5884 Worldwide Sales and Support Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches 150 countries on six continents For sales office locations and technical support please call our local office or refer to our Web site at www appliedbiosystems com www appliedbiosystems com AB Applied Biosystems Applera Corporation is committed to providing the world s leading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses Printed in USA 08 2010 Part Number 4337036 Rev B2. Rapid thermal ramp to 95 C 95 C for 30 seconds Rapid thermal ramp to 50 55 C depending on template 50 55 C for 10 seconds Rapid thermal ramp to 60 C 60 C for 4 minutes 4 Rapid thermal ramp to 4 C and hold until ready to purify 5 Spin down the contents of the tubes in a microcentrifuge 6 Proceed to Chapter 4 Purifying Extension Products Some laboratories have found that increasing the number of cycles gives better results TRapid thermal ramp is 1 C sec BigDye Terminator v1 1 Cycle Sequencing Kit 3 7 Chapter3 Performing Cycle Seguencing 3 8 BigDye Terminator v1 1 Cycle Seguencing Kit Purifying Extension Products Chapter Summary In This Chapter The following topics are covered in this chapter Choosing a Method of Purification Ls 4 1 Ethanol EDTA Precipitation 2 0 0 0 cece cece eee 4 2 Ethanol EDTA Sodium Acetate Precipitation 4 6 Plate and Spin Column Purification lt 4 10 Plate Purification Using SDS Heat Treatment 4 13 Performing 96 Well Spin Plate Purification 4 14 Choosing a Method of Purification Purpose Purification Methods The best results are obtained when unincorporated dye terminators are completely removed prior to electrophoresis Excess dye terminators in sequencing reactions obscure data in the early part of the sequence and can interfere with basecalling
3. then remove from the centrifuge Note Start timing when the rotor starts moving 11 To continue resuspend the samples in injection buffer To store cover with aluminum foil and store at 4 C Note Make sure the wells are dry You may use a Speed Vac for 15 min to dry the plate IMPORTANT Make sure the samples are protected from light while they are drying BigDye Terminator v1 1 Cycle Sequencing Kit 4 5 Chapter4 Purifying Extension Products Ethanol EDTA Sodium Acetate Precipitation Precipitating in 96 Well Reaction Plates Note Ethano EDTA sodium acetate precipitation is recommended when good signal from base 1 is reguired However for reactions containing high concentrations of unincorporated terminators some residual terminators may be carried through the precipitation To completely remove excess terminators in these cases ethanol EDTA precipitation is recommended see page 4 2 Nome CHEMICAL HAZARD EDTA Exposure causes eye irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves WEE CHEMICAL HAZARD Ethanol is a flammable liquid and vapor Exposure causes eye skin and respiratory tract irritation and may cause central nervous system depression and liver damage Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves To precipitate 20 uL sequencing rea
4. 4 polymer 1 mL syringe P4StdSeq 1 mL E 47 cm 50 um i d capillary Ld 36 cm POP 4 polymer Rapid Sequencing P4RapidSeq 1 mL E 1 mL syringe 47 cm 50 um i d capillary Ld 36 cm POP 6 polymer 1 mL syringe 61 Seq POP6 1 mL E cm 50 um i d capillary POP 6 polymer Rapid Sequencing Seq POP6 Rapid 1 mL E 1 mL syringe 47 cm 50 um i d capillary Dye Set Primer Mobility Files Operating E Polymer System Dye Set Primer Mobility File POP 4 polymer DT310POP4 BD v2 mob e POP 6 polymer NE DT310POP6 BD LR v3 mob DT310POP6 BD mob POP 4 polymer DT310POP4 BD v2 mob Macintosh POP 6 polymer DT POP6 BD Set AnyPrimer C 6 BigDye Terminator v1 1 Cycle Sequencing Kit Electrophoresis on the ABI Prism 310 Genetic Analyzer Standards IMPORTANT The instrument matrix file for the BigDye terminators v1 1 cannot be used for the BigDye terminators v3 0 or v3 1 giri Standards for Instrument Matrix File Generation E 310 377 BigDye Terminator v1 1 Matrix Standards PN 4336805 Note Refer to the product insert for instructions on using the standards for this instrument Performing For information on how to perform sample electrophoresis on the Sample 310 instrument refer to the following manuals Electrophoresis ABI PRISM 310 Genetic Analyzer Sequencing Chemistry Guide PN 4303189 ABI PRISM 310 Genetic Ana
5. The amount of PCR product to use in sequencing also depends on the length and purity of the PCR product 2 6 BigDye Terminator v1 1 Cycle Sequencing Kit Performing Cycle Seguencing 3 Chapter Summary In This Chapter The following topics are covered in this chapter Sequencing Single and Double Stranded DNA 3 2 Sequencing Large DNA Templates as 3 5 BigDye Terminator v1 1 Cycle Sequencing Kit 3 1 Chapter3 Performing Cycle Seguencing Seguencing Single and Double Stranded DNA Overview This section describes how to prepare reactions and perform cycle seguencing on a variety of templates including M13 plasmids and PCR products Preparing the Reactions for 96 Well Reaction Plates or To prepare the reaction mixtures The type of tube reguired depends on the thermal cycler that you are using Refer to Materials for Cycle Sequencing on page 1 8 Microcentrifuge Tubes 1 For each reaction add the following reagents to a separate tube Reagent Quantity Terminator Ready Reaction 8 0 uL Mix Template See the table in Template Quantity on page 2 6 Primer 3 2 pmol Deionized water q s Total Volume 20 uL 2 Mix well and spin briefly See Using BigDye Terminator Sequencing Buffer below BigDye Terminator v1 1 Cycle Sequencing Kit Using BigDye Terminator Seguencing Buffer Sequencing Single and Double Stranded DN
6. grade Sodium acetate NaOAc 3 M pH 5 2 Sealing tape MLS Applied Biosystems PN 400320 Costar 6570 Thermowell Sealing Tape Plate Column Purification Note For 96 well reaction plates 96 Well columns for purification Sealing tape See Recommended 96 Well Spin Plates on page 4 14 Costar 6570 Thermowell Sealing Tape Spin Column Purification Centri Sep spin column 1 mL 32 columns 100 columns Sealing tape Applied Biosystems PN 401763 PN 401762 Costar 6570 Thermowell Sealing Tape You will also need a variable speed centrifuge with microtiter plate holders capable of reach a spin speed of at least 1400 x g Applied Biosystems recommends a Beckman Allegra 6A centrifuge with a GH 3 8A rotor BigDye Terminator v1 1 Cycle Sequencing Kit 1 9 Chapter 1 Introduction Materials for Electrophoresis Instrument Material Supplier ABI PRISM 3700 DNA Analyzer Hi Di Formamide 25 mL bottle Applied Biosystems PN 4311320 3700 BigDye Terminator v1 1 Matrix Standard Applied Biosystems PN 4336825 3700 3730 BigDye Terminator v1 1 Sequencing Standard Applied Biosystems PN 4336799 ABI Prism 3100 and 3100 Avant Genetic Analyzers Hi Di Formamide 25 mL bottle Applied Biosystems PN 4311320 3100 BigDye Terminator v1 1 Matrix Standard Applied Biosystems PN 4336824 BigDye Terminator
7. have been authorized for such use by Applied Biosystems or for manual DNA sequencing No license is hereby granted for use of this kit or the reagents therein in any other automated sequencing machine Such sublicense is granted solely for research or other uses that are not unlawful No other license is granted expressly impliedly or by estoppel For information concerning the availability of additional license to practice the patented methodologies contact Amersham Life Science Inc Vice President Regulatory Affairs PO Box 22400 Cleveland Ohio 44122 Patents are pending in countries outside the United States ABI PRISM Applied Biosystems BigDye GeneScan MicroAmp and Primer Island are registered trademarks of Applied Biosystems or its subsidiaries in the U S and certain other countries and ABI Avant CATALYST Hi Di POP POP 4 POP 5 and POP 6 are trademarks of Applied Biosystems or its subsidiaries in the U S and certain other countries Centricon is a trademark of W R Grace and Co Centri Sep is a trademark of Princeton Separations Inc GeneAmp are registered trademarks of Roche Molecular Systems Inc pGEM is a registered trademark of Promega Corporation All other trademarks are the sole property of their respective owners Part Number 4337036 Rev B 08 2010 Contents Chapter 1 Introduction Chapter Summary LL aks In This Ghapterss sepia aaron d i a bae About the Kit 2c iu A M a a a te New Formulatio
8. or incorrect primer sequence Redesign the primer Missing reagent Repeat the reactions carefully following the protocol Old or mishandled reagents Use fresh reagents Thermal cycler power failure Repeat the reactions Thermal cycling conditions incorrect Calibrate the thermal cycler regularly Use correct thermal cycling parameters e Use correct tube for your thermal cycler e Setramp rate to 1 C sec Extension products lost during reaction cleanup Make sure you use the correct centrifugation speeds and times for precipitation and spin column procedures Make sure the ethanol concentration is correct for the precipitation protocols BigDye Terminator v1 1 Cycle Sequencing Kit D 1 Observation Possible Causes Recommended Actions No recognizable seguence continued Extension products not resuspended Carefully resuspend the sample pellet in loading buffer Lane tracking failure 377 instrument Check the lane tracking If necessary retrack and reextract the lanes Electrokinetic injection failure capillary instruments Repeat the injections Noisy data throughout the seguence with low signal strength Insufficient DNA in the seguencing reactions Use more DNA in the sequencing reactions e Load or inject more of the resuspended sequencing reactions Degraded template Prepare fresh DNA and repeat the reactions Ol
9. v1 1 Sequencing Standard Applied Biosystems PN 4336791 ABI PRISM 310 Genetic Analyzer Template Suppression Reagent TSR Applied Biosystems PN 401674 EDTA MLS 310 377 BigDye Terminator v1 1 Matrix Standards Applied Biosystems PN 4336805 ABI PRISM 377 DNA Sequencer Formamide MLS EDTA MLS 25 mM EDTA with 50 mg mL blue dextran Applied Biosystems PN 402055 310 377 BigDye Terminator v1 1 Matrix Standards Applied Biosystems PN 4336805 BigDye Terminator v1 1 Cycle Sequencing Kit General Safety General Safety Documentation Five user attention words appear in the text of all Applied Biosystems User Attention user documentation Each word implies a particular level of Words Observation or action as described below Note Calls attention to useful information IMPORTANT Indicates information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical N ie Ye Indicates a potentially hazardous situation which if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices N Miel Indicates a potentially hazardous situation which if not avoided could result in death or serious injury N J cidid Indicates an imminently hazardous situation which if not avoided will result in death or serious injury This signal word is to be limited to t
10. with 96 Lane Upgrade instruments IMPORTANT Spectral calibrations for the BigDye terminators v1 1 are not compatible with the v3 0 or v3 1 terminators Standards for Instrument Dye Filter Set Instrument Matrix File Generation 3700 DNA Analyzer Filter Set E 3700 BigDye Terminator v1 1 Matrix Standard PN 4336825 3100 Genetic Analyzer 3100 BigDye Filter Set E Terminator v1 1 Matrix 3100 Avant Genetic Standard Analyzer PN 4336824 The BigDye Terminator v1 1 Cycle Sequencing Kit does not require a new spectral calibration file if you currently have a BigDye Terminator v1 0 spectral file on your instrument IMPORTANT Users of the ABI Prism 3700 DNA Analyzer refer to the ABI Prism 3700 DNA Analyzer User s Manual PN 4306152 for information on instrument matrix files BigDye Terminator v1 1 Cycle Sequencing Kit Reguired Software Instructions For Generating Matrices For the 377 and 310 instruments refer to the product insert included with matrix or seguence standards for instructions on using the BigDye Matrix Standards v1 1 to generate matrices For Performing Spectral Calibrations For the 3700 instrument refer to the product insert for instructions on using the 3700 3730 BigDye Terminator v1 1 Matrix Standard to perform spectral calibration For the 3100 and 3100 Avant instruments refer to the product insert for instructions on using the 3100 BigDye Terminator v1 1 Matrix Stan
11. 1 Cycle Sequencing Kit has a new formulation that delivers ncreased robustness High guality data Improved success with difficult templates The protocols for cycle sequencing have been modified to optimize results using the new formulation Features and The BigDye Terminator v1 1 Cycle Sequencing Kit uses v1 0 Compatibilities matrix and spectral files and does not require new instrument matrix files for the ABI PRISM 310 Genetic Analyzer and ABI PRISM 377 DNA Sequencers or new spectral calibrations for the ABI PRISM 3700 DNA Analyzer ABI PRISM 3100 Genetic Analyzer and ABI PRISM 3100 Avant Genetic Analyzer BigDye Terminator v1 1 Cycle Sequencing Kit 1 1 Chapter 1 Introduction BigDye Terminator v1 1 Cycle Sequencing Kit Applied Biosystems recommends that you verify the quality of your current matrix before proceeding If it is necessary to generate a new matrix use the appropriate matrix and or sequencing standard for your instrument The 310 and 377 instruments use the 310 377 BigDye Terminator v1 1 Matrix Standards PN 4336805 for instrument matrix file generation The 3700 instrument requires the 3700 BigDye Terminator v1 1 Matrix Standard PN 4336825 for spectral calibration The 3100 and 3100 Avant instruments require the 3100 BigDye Terminator v1 1 Matrix Standard PN 4336824 for spectral calibration The alcohol precipitation methods are different from those re
12. A The BigDye Terminator v1 1 3 1 Sequencing Buffer 5X is supplied at a 5X concentration If you use it for sequencing reactions be sure the final reaction volume is at a concentration of 1X For example for a half reaction in 20 uL final volume you would use 4 UL of ready reaction premix and 2 uL of BigDye sequencing buffer as shown below Reagent Concentration Volume Ready Reaction Premix 2 5X 4 uL BigDye Sequencing 5X 2 uL Buffer Primer 3 2 pmol Template See Template Quantity on page 2 6 Water to 20 uL Final Volume 1X 20 uL Note The use of this buffer without optimization may result in deterioration of sequence quality Applied Biosystems does not support diluted reactions or guarantee the performance of BigDye chemistry when it is diluted The BigDye Terminator v1 1 3 1 Sequencing Buffer is intended for use only with BigDye Terminator v1 1 3 Cycle Sequencing Kits BigDye Terminator v1 1 Cycle Sequencing Kit 3 3 Chapter3 Performing Cycle Seguencing Preparing the Reactions for 384 Well Plates The type of tube reguired depends on the thermal cycler that you are using Refer to Materials for Cycle Sequencing on page 1 8 Note The wells in a 384 well reaction plate have a volume capacity of 35 uL Therefore Applied Biosystems recommends doing a 10 uL reaction This allows the post reaction cleanup step to be performed in the same well To prepare th
13. A Templates Overview Thermal Cyclers This section describes how to prepare reactions and perform cycle sequencing on large DNA templates such as BAC DNA Cosmid DNA Genomic DNA Only the following thermal cyclers can be used with this protocol GeneAmp PCR System 9600 GeneAmp PCR System 9700 in 9600 emulation mode BigDye Terminator v1 1 Cycle Sequencing Kit 3 5 Chapter3 Performing Cycle Seguencing Reoptimize this protocol for use on other thermal cyclers Preparing The type of tube required depends on the thermal cycler that you are Sequencing using Refer to Materials Supplied by the User on page 1 7 Reactions To prepare the sequencing reaction 1 For each reaction add the following reagents to a separate tube Reagent Quantity Terminator Ready Reaction Mix 8 0 uL DNA Template See the table in DNA Quantity on page 2 6 Primer 3 2 pmol Deionized water q s Total Volume 20 uL 2 Mix well and spin briefly Note For instructions on using BigDye sequencing buffer see Using BigDye Terminator Sequencing Buffer on page 3 3 3 6 BigDye Terminator v1 1 Cycle Sequencing Kit Seguencing Large DNA Templates Performing Cycle To perform cycle sequencing on BAC DNA Seguencing 1 Place the tubes in a thermal cycler and set the volume to 20 uL 2 Heat the tubes at 95 C for 5 minutes 3 Repeat the following for 30 cycles
14. A is desired Cesium chloride CsCl banding Commercial Kits Commercial kits are also available for BAC DNA preparation QIAGEN tip 100 QIAGEN PN 10043 25 reactions 10045 100 reactions QIAGEN tip 500 QIAGEN PN 10063 25 reactions 10065 100 reactions PCR Templates Cycle sequencing provides the most reproducible results for sequencing PCR templates Although PCR fragments can be difficult to denature with traditional sequencing methods cycle sequencing provides several chances to denature and extend the template ensuring adequate signal in the sequencing reaction Importance of Purifying Product For optimum results purify the PCR product before sequencing In general any method that removes dNTPs and primers should work We recommend Centricon 100 columns PN N930 2119 The protocol for using these columns is provided in Purifying PCR Fragments on page 2 4 Refer to the Automated DNA Sequencing Chemistry Guide for information on sequencing PCR templates Marra M Weinstock L A and Mardis E R 1966 End sequence determination from large insert cloning using energy transfer fluorescent primers Genomic Methods 6 1118 1122 BigDye Terminator v1 1 Cycle Sequencing Kit 2 3 Chapter2 Preparing the Templates Purifying PCR To purify PCR fragments by ultrafiltration Fragments 1 Assemble the Centricon 100 column according to the manufacturer s recommendations 2 Load 2 mL deionized
15. BigDye Terminator v1 1 Cycle Sequencing Kit Protocol Applied AB Biosystems O Copyright 2002 2010 Applied Biosystems All rights reserved For Research Use Only Not for use in diagnostic procedures Notice To Purchaser Limited License A license under the process claims of U S patents or their foreign counterpart claims has an up front fee component and a running royalty component The purchase price of BigDye Terminator v1 1 Cycle Seguencing Kit includes limited non transferable rights under the running royalty component to use only this amount of the product to practice the DNA sequence and fragment analysis processes described in said patents when this product is used in conjunction with an Authorized DNA sequence analysis instrument whose use is covered under the up front fee component of these patents No other rights are granted expressly by implication or by estoppel or under any other patent rights owned or licensable by Applied Biosystems Further information re lating to the purchase of licenses for DNA sequence and fragment analysis and other applications may be obtained by contacting the Director of Licensing at Applied Biosystems 850 Lincoln Centre Drive Foster City CA 94404 U S A Notice to Purchaser Limited License The purchase of the BigDye Terminator v1 1 Cycle Sequencing Kit includes a limited nontransferable non exclusive license without the right to resell repackage or sublicense under the process
16. T CCGCTTCCTC 360 GCTCACTGAC TCGCTGCGCT CGGTCGTTCG GCTGCGGCGA 400 GCGGTATCAG CTCACTCAAA GGCGGTAATA CGGTTATCCA 440 CAGAATCAGG GGATAACGCA GGAAAGAACA TGTGAGCAAA 480 AGGCCAGCAA AAGGCCAGGA ACCGTAAAAA GGCCGCGTTG 520 CTGGCGTTTT TCCATAGGCT CCGCCCCCCT GACGAGCATC 560 ACAAAAATCG ACGCTCAAGT CAGAGGTGGC GAAACCCGAC 600 AGGACTATAA AGATACCAGG CGTTTCCCCC TGGAAGCTCC 640 BigDye Terminator v1 1 Cycle Seguencing Kit Appendix B Control DNA Seguence CTCGTGCGCT CTCCTGTTCC GACCCTGCCG CTTACCGGAT 680 ACCTGTCCGC CTITCTCCCT TCGGGAAGCG TGGCGCTTTC 720 TCATAGCTCA CGCTGTAGGT ATCTCAGTTC GGTGTAGGTC 760 GTTCGCTCCA AGCTGGGCTG TGTGCACGAA CCCCCCGTTC 800 AGCCCGACCG CTGCGCCTTA TCCGGTAACT ATCGTCTTGA 840 GTCCAACCCG GTAAGACACG ACTTATCGCC ACTGGCAGCA 880 GCCACTGGTA ACAGGATTAG CAGAGCGAGG TATGTAGGCG 920 GTGCTACAGA GTTCTTGAAG TGGTGGCCTA ACTACGGCTA 960 CACTAGAAGG ACAGTATTTG GTATCTGCGC TCTGCTGAAG 1000 B 2 BigDye Terminator v1 1 Cycle Seguencing Kit Sample Electrophoresis Some Important Reminders Dye set primer mobility files for the BigDye terminators v1 1 are the same as those for BigDye terminators original but different than those for the dRhodamine terminators and BigDye terminators v3 1 Ifa mobility file for the wrong sequencing chemistry is used some bases may be miscalled due to different dye labeling for the different chemistries Note See Required Software on page 1 4 for information on obtaining
17. The methods recommended below have produced clean sequencing data for BigDye Terminator v1 1 Cycle Sequencing Kit products Ethanol EDTA precipitation Ethanol EDTA sodium acetate precipitation Plate and Spin column purification Earlier methods developed for other sequencing kits may not remove unincorporated dyes efficiently Use the method that works best for your particular application BigDye Terminator v1 1 Cycle Sequencing Kit 4 1 Chapter4 Purifying Extension Products Note These precipitation protocols have been optimized for use with the v1 1 formulation at the specified seguencing reaction volumes 20 uL in 96 well format and 10 uL in 384 well format and are not recommended for other versions IMPORTANT To clean up sequencing reactions at volumes less than those specified reduce each component of the precipitation protocol proportionately Ethanol EDTA Precipitation Recommended Protocol Precipitating in 96 Well Reaction Plates With the BigDye terminators v1 1 the ethanol EDTA precipitation method produces consistent signal while minimizing unincorporated dyes It is particularly good at getting rid of unincorporated dye labelled terminators that obscure data in the early part of the sequence Note While this method produces the cleanest signal it may cause loss of small molecular weight fragments IMPORTANT Absolute ethanol absorbs water from the atmosphere gradually decreasing its conce
18. and denaturation before use BigDye Terminator v1 1 Cycle Sequencing Kit 2 1 Chapter2 Preparing the Templates 2 2 There are two forms of the v1 0 and v1 1 seguencing standard as shown in the table below Please use the correct sequencing standard for your instrument Refer to the product inserts for instructions on using each sequencing standard Instrument Kit PN ABI PRISM 3700 DNA Analyzer 3700 3730 BigDye Terminator v1 1 4336799 Sequencing Standard ABI PRISM 3100 Genetic Analyzer ABI PRISM 3100 Avant Genetic PAN Analyzer BigDye Terminator v1 1 Sequencing 4336791 Standard ABI PRISM 310 Genetic Analyzer ABI PRISM 377 DNA Sequencers Includes the ABI PRISM 377 ABI Prism 377 18 ABI PRISM 377 with XL Upgrade and the ABI PRISM 377 with 96 Lane Upgrade instruments BigDye Terminator v1 1 Cycle Seguencing Kit Template Preparation Methods Template Preparation Methods Single and Refer to the Automated DNA Sequencing Chemistry Guide Double Stranded PN 4305080 for information on preparing single and double Templates stranded templates BAC DNA With larger DNA targets such as bacterial artificial chromosomes Templates BACS the quality of DNA template is important to the success of the sequencing reaction Two methods have given good sequencing results Alkaline lysis with extra phenol extraction and isopropanol precipitation if very clean DN
19. anufacturer BigDye Terminator v1 1 Cycle Sequencing Kit 1 13 Chapter1 Introduction 1 14 BigDye Terminator v1 1 Cycle Seguencing Kit Preparing the Templates Chapter Summary In This Chapter The following topics are covered in this chapter Control DNA Templates o oooooooooooorrrnmom 2 1 Template Preparation Methods lt kaka kas 2 3 DNA Quality vei Habe L O ee Pie B ee 2 4 DNA Quantity ii A RA itr 2 6 Control DNA Templates Using Control DNA Control DNA Sequence An Additional Control Sold Separately Include a control DNA template as one of the templates in a set of sequencing reactions The results from the control can help determine whether failed reactions are the result of poor template quality or sequencing reaction failure Applied Biosystems recommends M13mp18 as a single stranded control and pGEM 3Zf as a double stranded control All Applied Biosystems DNA sequencing kits provide pGEM control DNA All dye terminator cycle sequencing kits include a 21 M13 forward primer for use in performing control reactions The partial sequence of pGEM 3Zf from the 21 M13 forward primer followed by the ensuing 1000 bases is shown in Appendix A Control DNA Sequence The BigDye Terminator v1 1 Sequencing Standard provides an additional control to help in troubleshooting electrophoresis runs This standard contains lyophilized sequencing reactions that require only resuspension
20. ator v1 1 Cycle Sequencing Kit 4 11 Chapter4 Purifying Extension Products To perform spin column purification continued 11 Spin the column in a microcentrifuge at 730 x g for 2 minutes Note If using a centrifuge with a fixed angle rotor place the column in the same orientation as 1t was in for the first spin This is important because the surface of the gel will be at an angle in the column after the first spin 12 Discard the column The sample is in the sample collection tube 13 Dry the sample in a vacuum centrifuge for 10 15 minutes or until dry Do not over dry 4 12 BigDye Terminator v1 1 Cycle Sequencing Kit Plate Purification Using SDS Heat Treatment Plate Purification Using SDS Heat Treatment Overview This section provides instructions for adding a sodium dodecyl sulfate SDS heat treatment to the spin plate purification method This SDS heat treatment effectively eliminates high concentrations of unincorporated dye terminators from cycle seguencing reactions Preparing Use this procedure to prepare extension products for 96 well spin Extension plate purification Products Nene PAN CHEMICAL HAZARD Sodium dodecyl sulfate SDS Exposure causes eye skin and respiratory tract irritation Exposure may cause severe allergic respiratory and skin reaction Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves To pr
21. claims of U S patents and corresponding claims in foreign counterpart patents and patent applications to use this product solely with an Applied Biosystems commercial automated DNA sequencing machine or other authorized automated DNA sequencing machines that have been authorized under these patents by Applied Biosystems No license is hereby granted for the use of this kit or the reagents therein in any other automated sequencing machine Such license is granted solely for research and other uses that are not unlawful No other license is granted expressly impliedly or by estoppel For information concerning the availability of additional licenses to practice the patented methodologies contact Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA Patents are pending in countries outside the United States Notice to Purchaser About Limited License This kit reagent is sold pursuant to a limited sublicense from Amersham International plc under one or more U S Patent Nos 5 498 523 and 5 614 365 and corresponding foreign patents and patent applications The purchase of this kit reagent includes a limited non exclusive sublicense without the right to resell repackage or further sublicense under such patent rights to use this reagent for DNA sequencing or fragment length analysis solely with an Applied Biosystems commercial automated sequencing machine or other authorized DNA sequencing machines that
22. commended for the original and other versions of BigDye terminators The existing mobility files can be used with their respective platforms New mobility files are not necessary The BigDye Terminator v1 1 Cycle Sequencing Kit provides the required reagent components for the sequencing reaction in a ready reaction pre mixed format You need only provide your template and template specific primer These reagents are suitable for performing fluorescence based cycle sequencing reactions on single stranded or double stranded DNA templates on polymerase chain reaction PCR fragments and on large templates for example BAC clones Note This kit includes BigDye Terminator v1 1 3 1 Sequencing Buffer 5X which has been specifically optimized for use with the new BigDye ready reaction mixes This buffer should be used for any reaction optimization you undertake BigDye Terminator v1 1 Cycle Sequencing Kit Instruments Instruments Instrument The BigDye Terminator v1 1 Cycle Sequencing Kit is for use with Platforms the following instruments ABI PRISM 3700 DNA Analyzer ABI PRISM 3100 Genetic Analyzer ABI PRISM 3100 Avant Genetic Analyzer ABI PRISM 310 Genetic Analyzer ABI PRISM 377 DNA Sequencer all models General instructions are given for using the kit reagents to generate samples for these instruments For more detailed instructions refer to the appropriate instrument user s manual or c
23. ctions in 96 well reaction plates 1 Remove the 96 well reaction plate from the thermal cycler and briefly spin 2 Add 2 uL of 125 mM EDTA to each well Note Make sure the EDTA reaches the bottom of the wells 3 Add 2 uL of 3 M sodium acetate to each well Note Make sure the sodium acetate reaches the bottom of the wells 4 Add 50 uL of 100 ethanol to each well 5 Seal the plate with aluminum tape and mix by inverting 4 times 6 Incubate at room temperature for 15 min BigDye Terminator v1 1 Cycle Sequencing Kit Ethanol EDTA Sodium Acetate Precipitation To precipitate 20 uL sequencing reactions in 96 well reaction plates continued VA If you are using Then a Beckman Allegra set it at 4 C and spin the plate at 6A centrifuge with a 1650 x g for 45 min GH 3 8A rotor any other centrifuge use a plate adapter and spin the plate at the maximum speed as follows 1400 2000 x g for 45 min or e 2000 3000 x g for 30 min IMPORTANT Proceed to the next step immediately If this is not possible then spin the plate for an additional 2 min before performing the next step 8 Invert the plate and spin up to 185 x g then remove from the centrifuge 9 Add 70 uL of 70 ethanol to each well 10 With the centrifuge set to 4 C spin at 1650 x g for 15 min 11 Invert the plate and spin up to 185 x g for 1 min then r
24. d as follows 1400 2000 x g for 45 min or e 2000 3000 x g for 30 min IMPORTANT Proceed to the next step immediately If this is not possible then spin the plate for an additional 2 min before performing the next step Invert the plate and spin up to 185 x g then remove from the centrifuge Add 60 uL of 70 ethanol to each well With the centrifuge set to 4 C spin at 1650 x g for 15 min Invert the plate and spin up to 185 x g for 1 min then remove from the centrifuge Note Start timing when the rotor starts moving BigDye Terminator v1 1 Cycle Sequencing Kit 4 3 Chapter4 Purifying Extension Products Precipitating in 384 Well Reaction Plates To precipitate 20 uL sequencing reactions in 96 well reaction plates continued 11 To continue resuspend the samples in injection buffer To store cover with aluminum foil and store at 4 C Note Make sure the wells are dry You may use a Speed Vac for 15 min to dry the plate IMPORTANT Make sure the samples are protected from light while they are drying WEE CHEMICAL HAZARD EDTA Exposure causes eye irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves NE CHEMICAL HAZARD Ethanol is a flammable liquid and vapor Exposure causes eye skin and respiratory tract irritation and may cause central nervous system depression and l
25. d or mishandled reagents Use fresh reagents Thermal cycling conditions incorrect Calibrate the thermal cycler regularly Use correct thermal cycling parameters Use correct tube for your thermal cycler Setramp rate to 1 C sec Lane tracking failure 377 instrument Check the lane tracking If necessary retrack and reextract the lanes Electrokinetic injection failure capillary instruments Repeat the injections Noisy data throughout the sequence with good signal strength Inhibitory contaminant in the template Clean up the template Multiple templates in the sequencing reaction Examine template on an agarose gel to be sure only one template is present Multiple priming sites Make sure primer has only one priming site e f necessary redesign primer Multiple primers when sequencing PCR products Purify your PCR template to remove excess primers Observation Possible Causes Recommended Actions Noisy data throughout the seguence with good signal strength continued Primer with N 1 contamination Use HPLC purified primer High signal saturating the detector Use less DNA in the sequencing reactions e Load or inject less of the resuspended sequencing reactions Incorrect run module Use correct run module Incorrect instrument matrix file Use correct instrument file for terminator chemistry Noise up to or a
26. dard to perform spectral calibration BigDye Terminator v1 1 Cycle Sequencing Kit 1 5 Chapter 1 Introduction Dye Set Primer To analyze sequencing data generated with BigDye chemistries Mobility Files v1 1 you need dye set primer mobility files that were created for v1 0 chemistries The dye set primer mobility files can be downloaded from the Internet Dye set primer mobility files can be downloaded from the Applied Biosystems website http www appliedbiosystems com support software If you do not have access to the Internet contact Applied Biosystems Technical Support or your local field applications specialist call your local sales office for more information Reagents and Storage 1 6 Available Kits The following kits are available Number of Kit Reactions Part Number BigDye Terminator v1 1 Cycle 100 4337450 Sequencing Kit 1000 4337451 5000 4337452 25 000 4337453 The BigDye Terminator v1 1 Cycle Sequencing Kit Protocol PN 4337036 is available separately and can be ordered at no charge Kit Reagents A listing of the kit components is given below Ready Reaction Mix pGEM 3Zf double stranded DNA Control Template 21 MI3 Control Primer forward BigDye Terminator v1 1 3 1 Sequencing Buffer 5X BigDye Terminator v1 1 Cycle Sequencing Kit Materials Supplied by the User Storage and Use Store the kit at 15 to 25 C of the Kit
27. e Terminator v1 1 Matrix Standards PN 4336805 Note Refer to the product insert for instructions on using the standards for this instrument If you are using the BigDye chemistries v1 1 on the 377 instrument in conjunction with the ABI PRISM Lane Guide M Lane Identification Kit refer to that kit s protocol PN 4313804 for instructions on resuspending and loading samples For longer sequencing read lengths follow the gel and buffer formulations described in the user bulletin entitled Achieving Longer High Accuracy Reads on the 377 Sequencer PN 4315153 For information on how to perform sample electrophoresis on the 377 instrument refer to the following manuals Automated DNA Sequencing Chemistry Guide PN 4305080 ABI PRISM 377 DNA Sequencer User s Manual PN 4307164 BigDye Terminator v1 1 Cycle Sequencing Kit C 9 Appendix C Sample Electrophoresis C 10 BigDye Terminator v1 1 Cycle Seguencing Kit Troubleshooting Observation Possible Causes Recommended Actions No recognizable seguence Insufficient template Quantitate DNA template e Increase amount of DNA in the seguencing reactions Inhibitory contaminant in the template Clean up the template Insufficient primer Quantitate the primer e Increase amount of primer in the sequencing reactions Primer has no annealing site Use a primer that is complementary to the template Poor primer design
28. e reaction mixtures l For each reaction add the following reagents to a separate tube Reagent Quantity Terminator Ready Reaction 4 0 uL Mix Sample Block Module Template See the table in Template Quantity on page 2 6 Primer 3 2 pmol Deionized water q s Total Volume 10 pL 2 Mix well and spin briefly 3 Use on a GeneAmp PCR System 9700 Dual 384 Well Note For instructions on using BigDye sequencing buffer see Using BigDye Terminator Sequencing Buffer on page 3 3 BigDye Terminator v1 1 Cycle Sequencing Kit Cycle Seguencing on the System 9700 9600 2700 or 2400 Seguencing Large DNA Templates To sequence single and double stranded DNA on the GeneAmp PCR System 9700 in 9600 emulation mode 9600 2700 or 2400 1 Place the tubes in a thermal cycler and set to the correct volume Perform an initial denaturation a Rapid thermal ramp to 96 C b 96 C for 1 min Repeat the following for 25 cycles Rapid thermal ramp to 96 C 96 C for 10 seconds Rapid thermal ramp to 50 C 50 C for 5 seconds Rapid thermal ramp to 60 C 60 C for 4 minutes Rapid thermal ramp to 4 C and hold until ready to purify 5 Spin down the contents of the tubes in a microcentrifuge 6 Proceed to Chapter 4 Purifying Extension Products Rapid thermal ramp is 1 C second Sequencing Large DN
29. ed DNA should run as a single band on an agarose gel Note Uncut plasmid DNA can run as three bands supercoiled nicked and linear Spectrophotometry The A5 amp A5go ratio should be 1 7 to 1 9 Smaller ratios usually indicate contamination by protein or organic chemicals Agarose gels reveal the presence of contaminating DNAs and RNAs but not proteins Spectrophotometry can indicate the presence of protein contamination but not DNA and RNA contamination Use these methods together to get the most information about your DNA before sequencing BigDye Terminator v1 1 Cycle Sequencing Kit 2 5 Chapter2 Preparing the Templates DNA Quantity Quantitating DNA If possible quantitate the amount of purified DNA by measuring the absorbance at 260 nm or by some other method Template The table below shows the amount of template to use in a cycle Quantity sequencing reaction Template Quantity PCR product 100 200 bp 1 8 ng 200 500 bp 3 10 ng 500 1000 bp 5 20 ng 1000 2000 bp 10 40 ng 22000 bp 20 50 ng Single stranded 25 50 ng Double stranded 150 300 ng Cosmid BAC 0 5 1 0 ug Bacterial genomic DNA 2 3 ug Note In general higher DNA quantities give higher signal intensities Higher DNA quantities may also give shorter read lengths and top heavy data The template quantities given above should work with all primers You may be able to use even less DNA when using capillary instruments for detection
30. emove from the centrifuge Note Start timing when the rotor starts moving 12 To continue resuspend the samples in injection buffer To store cover with aluminum foil and store at 4 C Note Make sure the wells are dry You may use a Speed Vac for 15 min to dry the plate IMPORTANT Make sure the samples are protected from light while they are drying BigDye Terminator v1 1 Cycle Sequencing Kit 4 7 Chapter4 Purifying Extension Products Precipitating in 384 Well Reaction Plates WEE CHEMICAL HAZARD EDTA Exposure causes eye irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves NE CHEMICAL HAZARD Ethanol is a flammable liquid and vapor Exposure causes eye skin and respiratory tract irritation and may cause central nervous system depression and liver damage Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves To precipitate 10 uL sequencing reactions in 384 well reaction plates and briefly spin 1 Remove the 384 well reaction plate from the thermal cycler 2 Add 1 uL of 125 mM EDTA to each well Note Make sure the EDTA reaches the bottom of the wells 3 Add 1 uL of 3 M sodium acetate to each well Note Make sure the sodium acetate reaches the bottom of the wells 4 Add 25 uL of 100 ethanol to each well 5 Seal the pla
31. epare extension products 1 Prepare a solution of 2 2 SDS in deionized water This SDS solution is stable at room temperature 2 Add an appropriate amount of the 2 2 SDS solution to each sample to bring the final SDS concentration to 0 2 For example Add 2 uL of 2 2 SDS solution to each 20 uL completed cycle sequencing reaction 3 Seal the tubes and mix thoroughly 4 Heat the tubes to 98 C for 5 min then allow the tubes to cool to ambient temperature before proceeding to the next step Note A convenient way to perform this heating cooling cycle is to place the tubes in a thermal cycler and set it as follows 98 C for 5 min 25 C for 10 min 5 Spin down the contents of the tubes briefly 6 Continue with 96 well plate purification BigDye Terminator v1 1 Cycle Sequencing Kit 4 13 Chapter4 Purifying Extension Products Performing 96 Well Spin Plate Purification Recommended For large scale procedures you can use 96 well spin plates such as 96 Well Spin the Gel Filtration Kit from Edge Biosystems Plates Note Other spin plate systems may be used to successfully remove unincorporated dye terminators However due to the large number of variables associated with using spin plate systems you will need to optimize the performance of your system in your own laboratory Purifying with the To perform purification with the spin plate 96 Well Spin Plate 1 Prepare the exten
32. equencing Kit 4 9 Chapter4 Purifying Extension Products Plate and Spin Column Purification Overview Recommended Spin Columns Optimizing Spin 4 10 Column Purification This section describes the recommended plate and spin columns for purifying extension products IMPORTANT Extra caution is required when dispensing samples onto the column bed Residual dye peaks will result if samples flow through the sides of the column We recommend Centri Sep spin columns Applied Biosystems PN 401763 for 32 columns and PN 401762 for 100 columns IMPORTANT When using the BigDye terminators v1 1 hydrate the column for 2 hours see below Tips for optimizing spin column purification when using individual columns Do not process more columns than you can handle conveniently at one time Loadthe sample in the center ofthe column bed slowly Make sure that the sample does not touch the sides of the column and that the pipet tip does not touch the gel surface Note If samples are not properly loaded peaks from unincorporated dye terminators can result Spin the column at 325 730 x g for best results Use the following formula to calculate the best speed for your centrifuge g 711 18x r x rpm 1000 where g relative centrifugal force r radius of the rotor in cm rpm revolutions per minute Do not spin for more than 2 minutes Perform the entire procedure without interruption to ensure optimal res
33. etter results than primers with lower T m For primers with a G C content less than 50 it may be necessary to extend the primer sequence beyond 18 bases to keep the T gt 45 C Use of primers longer than 18 bases also minimizes the chance of having a secondary hybridization site on the target DNA Avoid primers that have secondary structure or that can hybridize to form dimers Several computer programs for primer selection are available They can be useful in identifying potential secondary structure problems and determining if a secondary hybridization site exists on the target DNA BigDye Terminator v1 1 Cycle Sequencing Kit A 1 Appendix A Selecting Seguencing Primers A 2 BigDye Terminator v1 1 Cycle Seguencing Kit Control DNA Seguence Control Seguence Partial Sequence The pGEM 3Zf sequence below is the sequence of the 21 M13 forward primer followed by the ensuing 1000 bases of pGEM 3Zf TGTAAAACGACGGCCAGT 21 M13 primer GAATTGTAAT ACGACTCACT ATAGGGCGAA TTCGAGCTCG 40 GTACCCGGGG ATCCTCTAGA GTCGACCTGC AGGCATGCAA 80 GCTTGAGTAT TCTATAGTGT CACCTAAATA GCTTGGCGTA 120 ATCATGGTCA TAGCTGTTTC CTGTGTGAAA TTGTTATCCG 160 CTCACAATTC CACACAACAT ACGAGCCGGA AGCATAAAGT 200 GTAAAGCCTG GGGTGCCTAA TGAGTGAGCT AACTCACATT 240 AATTGCGTTG CGCTCACTGC CCGCTTTCCA GTCGGGAAAC 280 CTGTCGTGCC AGCTGCATTA ATGAATCGGC CAACGCGCGG 320 GGAGAGGCGG TTTGCGTATT GGGCGCTCT
34. fter a specific point in the sequence Mixed plasmid separation Make sure you have only one template Multiple PCR products Make sure you have only one template Primer dimer contamination in PCR sequencing e Optimize your PCR amplification Make sure there is no sequence complementarity between the two PCR primers Make sure your sequencing primer does not overlap the sequences of the PCR primers e Use a Hot Start technique such as with Amplitag Gold polymerase Slippage after repeat region in template Try an alternate sequencing chemistry Use an anchored primer Poor mobility correction Incorrect dye set primer mobility file Use correct dye set primer file Incorrect Peak 1 location for data analysis Select a new Peak 1 location Gel with very different separation properties than the gel matrices used to construct the dye set primer mobility file Use correct dye set primer file for your gel type BigDye Terminator v1 1 Cycle Sequencing Kit D 3 Appendix D Troubleshooting Observation Possible Causes Recommended Actions Excess dye peaks at beginning of seguence Poor removal of unincorporated dye terminators Use ethanol EDTA precipitation protocol to remove unincorporated dye terminators With Centri Sep spin columns take care to load sample on the center of the gel surface Note Do not touch the gel surface with the pipe
35. he most extreme situations Site Preparation A site preparation and safety guide is a separate document sent to all and Safety Guide customers who have purchased an Applied Biosystems instrument Refer to the guide written for your instrument for information on site preparation instrument safety chemical safety and waste profiles Chemical Safety Chemical Hazard NTT CHEMICAL HAZARD Some of the chemicals Warning used with Applied Biosystems instruments and protocols are potentially hazardous and can cause injury illness or death Read and understand the material safety data sheets MSDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials Minimize contact with chemicals Wear appropriate personal protective eguipment when handling chemicals e g safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS BigDye Terminator v1 1 Cycle Seguencing Kit 1 11 Chapter 1 Introduction Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation e g fume hood For additional safety guidelines consult the MSDS Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended on the MSDS Comply with all local state provincial or national laws and regulations related to chemical storage handling a
36. hemistry guide Thermal Cyclers The protocols provided in this document were optimized using Applied Biosystems thermal cyclers including the GeneAmp PCR Systems 9700 9600 2700 and 2400 Note If you use a thermal cycler not manufactured by Applied Biosystems you may need to optimize thermal cycling conditions Ramping time is very important If the thermal ramping time is too fast gt 1 sec poor noisy data may result Includes the ABI PRISM 377 ABI PRISM 377 18 ABI PRISM 377 with XL Upgrade and the ABI PRISM 377 with 96 Lane Upgrade instruments BigDye Terminator v1 1 Cycle Sequencing Kit 1 3 Chapter 1 Introduction Required Software Dye Filter Sets and Matrix Standards for the 310 and 377 Instruments Dye Sets and Spectral Standards for the 3700 3100 and 3100 Avant Instruments 1 4 The dye filter sets and matrix standards required for the 310 and 377 instruments are listed in the table below Standards for Instrument Dye Filter Set Instrument Matrix File Generation 310 Genetic Analyzer Filter Set E 310 377 BigDye Terminator v1 1 Matrix 377 DNA Sequencer Filter Set E Standards PN 4336805 The BigDye Terminator v1 1 Cycle Sequencing Kit does not require a new spectral calibration file if you currently have a BigDye Terminator v1 0 spectral file on your instrument TIncludes the ABI PRISM 377 ABI PRISM 377 18 ABI PRISM 377 with XL Upgrade and the ABI PRISM 377
37. iver damage Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves To precipitate in 384 well reaction plates 1 Remove the 384 well reaction plates from the thermal cycler Remove the seal from each plate and briefly spin the plates 2 Add 2 5 uL of 125 mM EDTA to each well Note Make sure the EDTA reaches the bottom of the wells 3 Add 25 uL of 100 ethanol to each well 4 Seal the plates with aluminum tape and mix by inverting 4 times 5 Incubate at room temperature for 15 min BigDye Terminator v1 1 Cycle Sequencing Kit Ethanol EDTA Precipitation To precipitate in 384 well reaction plates continued 6 If you are using Then a Beckman Allegra set it at 4 C and spin the plate at 6A centrifuge with a 1650 x g for 45 min GH 3 8A rotor any other centrifuge use a plate adapter and spin the plate at the maximum speed as follows 1400 2000 x g for 45 min or e 2000 3000 x g for 30 min IMPORTANT Proceed to the next step immediately If this is not possible then spin the plate for an additional 2 min before performing the next step 7 Invert the plate and spin up to 185 x g then remove from the centrifuge 8 Add 30 UL of 70 ethanol to each well 9 With the centrifuge set to 4 C spin at 1650 x g for 15 min 10 Invert the plate and spin up to 185 x g for 1 min
38. lyzer User s Manual PN 4317588 BigDye Terminator v1 1 Cycle Sequencing Kit C 7 Appendix C Sample Electrophoresis Electrophoresis on the ABI PRISM 377 DNA Sequencers Requirements Electrophoresis and data analysis of samples on the ABI PRISM 377 DNA Sequencers all models require the following Filter Set E Run Modules Configuration Run Module 36 cm wtr 1200 scans hr any comb Seg Run 36E 1200 36 cm wtr 2400 scans hr any comb Seq Run 36E 2400 48 cm wtr 1200 scans hr any comb Seq Run 48E 1200 Any plate check and prerun module can be used on the ABI Prism 377 DNA Sequencers Dye Set Primer Mobility Files Ranger gel 3 Operating a Gel Formulation System Dye Set Primer Mobility File 4 5 acrylamide NT DT377 BD mob 29 1 or 5 Long Macintosh DT BD Set Any Primer Includes the ABI PRISM 377 ABI PRISM 377 18 ABI PRISM 377 with XL Upgrade and the ABI PRISM 377 with 96 Lane Upgrade instruments C 8 BigDye Terminator v1 1 Cycle Seguencing Kit Using the Lane Guide Kit Using Long Read Gel and Buffer Formulations Performing Sample Electrophoresis Electrophoresis on the ABI Prism 377 DNA Sequencers Standards IMPORTANT The instrument matrix file for the BigDye terminators v1 1 cannot be used for the BigDye terminators v3 0 or v3 1 giri Standards for Instrument Matrix File Generation E 310 377 BigDy
39. n 0 0 00 eee Features and Compatibilities llli BigDye Terminator v1 1 Cycle Sequencing Kit Instruments aaa idad cei n a eoe ee tied Instrument Platforms sellers Thermal Cyclers o oocoocccccccooo een Required Software LL Pakaks Dye Filter Sets and Matrix Standards for the 310 and 377 Instruments ooo Dye Sets and Spectral Standards for the 3700 3100 and 3100 Avant Instruments 2000 0c eee eens Instr ctlons baaa Seen ya So ce e gaan Pt Dye Set Primer Mobility Files Ls Reagents and Storage 0oooccooccco tte Available Kits sia a a EX es Kit Reagents cos obere exui DET er Ree x Rn Storage and Use of the Kits 0000 e eee Materials Supplied by the User NI iii A A eae Gen ed Materials for Cycle Sequencing ooooccccocccon ee eee Materials for Purifying Extension Products Other Equipment 000 cee rere Materials for Electrophoresis 00000e eee eee General Safety cui cose sie bx raptada BigDye Terminator v1 1 Cycle Sequencing Kit lii Chapter 2 Chapter 3 Preparing the Templates Chapter SUMME y iia ALI u Shae OE ee 2 1 In ThiS Ghaptet cert kote a 2 1 Control DNA Templates o ooocccccocoocc 2 1 Using Control DNA ooocccoccccococo eh 2 1 Control DNA Sequence ccococcccccccc eee 2 1 An Additional Control Sold Separately o o oo o 2 1 Template Preparatio
40. n Methods sells 2 3 Single and Double Stranded Templates 2 3 BAC DNA Templates ceneni trenetan benp eee 2 3 PCR Templates iniri me oira eaa y aea eee 2 3 Purifying PCR Fragments LPL kaka 2 4 DNA Q alily 22 eini ad iR id 2 4 Poor Template Quality Ls 2 4 Contamination a o 2 oe tea rre RS E EE RE 2 5 Determining DNA Quality Ls 2 5 DNA Quantity c nere ye Pay ud WAR RO da EG 2 6 Quantitating DNA 2 6 Template Quantity Likas 2 6 Performing Cycle Sequencing Chapter Summary suelen 3 1 In This Chapter xem i ask ap RARO rn 3 1 Sequencing Single and Double Stranded DNA 3 2 S Sado aka aa Drogues E a eet a s een 3 2 Preparing the Reactions for 96 Well Reaction Plates or Microcentrifuge Tubes LL aks 3 2 Using BigDye Terminator Sequencing Buffer 3 3 Preparing the Reactions for 384 Well Plates 3 4 Cycle Sequencing on the System 9700 9600 2700 or 2400 3 5 Sequencing Large DNA Templates Lk ks 3 5 OVOIVIOW 3 5 Thermal Cyclets 32e cc pee awn gay pep RR READ MEUS 3 5 Preparing Sequencing Reactions o ooccooccoooomo ooo 3 6 Performing Cycle Sequencing lt Lk 3 7 BigDye Terminator v1 1 Cycle Sequencing Kit Chapter 4 Appendix A Appendix B Purifying Extension Products Chapter Summary ika 4 1 In This Chapter cota ac E e a it 4 1 Choosing a Method of Purification Ls 4 1 Bier g
41. nd disposal Chemical Waste NTT CHEMICAL WASTE HAZARD Wastes Hazard Warning 1 12 About MSDSs produced by Applied Biosystems instruments are potentially hazardous and can cause injury illness or death Read and understand the material safety data sheets MSDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste Handle chemical wastes in a fume hood Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals e g safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation e g fume hood For additional safety guidelines consult the MSDS After emptying the waste container seal it with the cap provided Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or national environmental and health regulations Some of the chemicals used with this instrument may be listed as hazardous by their manufacturer When hazards exist warnings are prominently displayed on the labels of all chemicals Chemical manufacturers supply a current material safety data sheet MSDS before or with shipments of hazardous chemicals to new customers and with the first shipment of a haza
42. nt Genetic Analyzers require the following Run Modules Configuration Run Module POP 4 polymer 36 cm UltraSeg_POP4Default Module POP 4 polymer 80 cm LongSeg80 POP4DefaultModule POP 6 polymer 36 cm RapidSeq36 POP6DefaultModule POP 6 polymer 50 cm StdSeq50 POP6DefaultModule Dye Set Primer Mobility Files Polymer Dye Set Primer Mobility File POP 4 polymer DT3100POP4LR BD v1 mob POP 6 polymer DT3100POP6 BD v1 mob Standards IMPORTANT Use Dye Set E Dye Set Standards E 3100 BigDye Terminator v1 1 Matrix Standard PN 4336824 Note Refer to the product insert for instructions on using the standards for this instrument C 4 BigDye Terminator v1 1 Cycle Sequencing Kit Electrophoresis on the ABI Prism 3100 and 3100 Avant Genetic Analyzers Performing For information on how to perform sample electrophoresis on the Sample 3100 instrument refer to the following manuals Electrophoresis ABI PRISM 3100 Genetic Analyzer Sequencing Chemistry Guide PN 4315831 ABI PRISM 3100 Genetic Analyzer User s Manual PN 4315834 BigDye Terminator v1 1 Cycle Sequencing Kit C 5 Appendix C Sample Electrophoresis Electrophoresis on the ABI PRISM 310 Genetic Analyzer Requirements Electrophoresis and data analysis of samples on the ABI PRISM 310 Genetic Analyzer reguires the following Filter Set E Run Modules Configuration Run Module POP
43. ntration and leading to inaccurate final concentrations of ethanol which can affect some sequencing results IMPORTANT 9594 ethanol is usable but you must make sure the final ethanol concentration for precipitation remains the same 67 71 WEE CHEMICAL HAZARD EDTA Exposure causes eye irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves NE CHEMICAL HAZARD Ethanol is a flammable liguid and vapor Exposure causes eye skin and respiratory tract irritation and may cause central nervous system depression and liver damage Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves BigDye Terminator v1 1 Cycle Seguencing Kit Ethanol EDTA Precipitation To precipitate 20 uL sequencing reactions in 96 well reaction plates 1 Remove the 96 well reaction plate from the thermal cycler and briefly spin Add 5 uL of 125 mM EDTA to each well Note Make sure the EDTA reaches the bottom of the wells Add 60 uL of 100 ethanol to each well Seal the plate with aluminum tape and mix by inverting 4 times Incubate at room temperature for 15 min If you are using Then a Beckman Allegra set it at 4 C and spin the plate at 6A centrifuge with a 1650 x g for 45 min GH 3 8A rotor any other centrifuge use a plate adapter and spin the plate at the maximum spee
44. occccoconnocoooo B 1 BigDye Terminator v1 1 Cycle Sequencing Kit V Appendix C Sample Electrophoresis Some Important Reminders LLP Lakis Electrophoresis on the ABI Prism 3700 DNA Analyzer Requirements llle eee Performing Sample Electrophoresis Electrophoresis on the ABI PRISM 3100 and 3100 Avant Genetic Analyzers LPL Pakaks Requirements ia iaa ag dks Performing Sample Electrophoresis Electrophoresis on the ABI Prism 310 Genetic Analyzer Requirements 00 00 cece eee eee eee Performing Sample Electrophoresis Electrophoresis on the ABI PRISM 377 DNA Sequencers Requirements oos sel il land Using the Lane Guide Kit o Using Long Read Gel and Buffer Formulations Performing Sample Electrophoresis Appendix D Troubleshooting Appendix E Obtaining Technical Support Services and Support oooocccccc cee eee Applied Biosystems Web Site Index vi BigDye Terminator v1 1 Cycle Sequencing Kit Introduction Chapter Summary In This Chapter The following topics are covered in this chapter Aboutithe Kit eem ita tha hs 1 1 A aa sias A a a aa o dieu 1 3 Required Software 0 cect eens 1 4 Reagents and Storage 0 ccc eens 1 6 Materials Supplied by the USer o ooooooomomo o 1 7 General Safety i co oves ee ee A se ep ee 1 11 About the Kit New Formulation The BigDye Terminator v1
45. rdous chemical after an MSDS update MSDSs provide you with the safety information you need to store handle transport and dispose of the chemicals safely BigDye Terminator v1 1 Cycle Sequencing Kit Chemical Safety We strongly recommend that you replace the appropriate MSDS in your files each time you receive a new MSDS packaged with a hazardous chemical erem CHEMICAL HAZARD Be sure to familiarize yourself with the MSDSs before using reagents or solvents Ordering MSDSs You can order free additional copies of MSDSs for chemicals manufactured or distributed by Applied Biosystems using the contact information below To order documents by automated telephone service 1 From the U S or Canada dial 1 800 487 6809 2 Follow the voice instructions to order documents for delivery by fax Note There is a limit of five documents per fax request To obtain documents through the Applied Biosystems Web site 1 Goto http docs appliedbiosystems com msdssearch html 2 In the SEARCH field type in the chemical name part number or other information that will appear in the MSDS and click SEARCH Note You may also select the language of your choice from the drop down list 3 When the Search Results page opens find the document you want and click on it to open a PDF of the document For chemicals not manufactured or distributed by Applied Biosystems call the chemical m
46. s C 9 M matrix standards 310 1 4 377 1 4 general requirements 1 2 mobility files 1 2 about C 1 downloading 1 6 MSDSs ordering 1 13 P pGEM 3Zf sequence B 1 primers importance of selection A 1 optimizing selection A 1 purification SDS and 96 well 4 13 spin plate 4 14 purifying methods 2 3 4 1 methods for PCR 2 3 PCR by ultrafiltration 2 4 Q quality problems template 2 5 quality examining DNA 2 5 quantitating DNA 2 6 Index 2 R reaction mixtures BAC DNA 3 7 for 384 well 3 4 for 96 well 3 2 for microcentrifuge tubes 3 2 reagents electrophoresis 1 10 purifying 1 9 required files matrix 1 1 spectral 1 1 S safety chemical 1 11 to 1 13 guide 1 11 to 1 13 SDS and purification 4 13 sequencing buffer optimizing 1 2 sequencing kits ordering 1 6 sequencing problems 2 4 sequencing standards for each instrument 2 2 general requirements 1 2 shorter read lengths and DNA quantity 2 6 signal intensities and DNA quantity 2 6 site preparation and safety guide 1 11 spectral standards 3100 1 4 3700 1 4 spin column purification optimizing 4 10 performing 4 11 storage temperatures 1 7 BigDye Terminator v1 1 Cycle Sequencing Kit T technical support E 1 templates BAC DNA 2 3 PCR 2 3 poor quality characteristics 2 4 preparing 2 3 top heavy data and DNA quantity 2 6 U user attention words 1 11 W warning symbols 1 11 BigDye Terminator v1 1 Cycle Seguencing Kit
47. s Note The BigDye sequencing buffer can be stored at 4 C Avoid excess that is no more than 5 to 10 freeze thaw cycles Aliquot reagents in smaller amounts if necessary Before each use of the kit allow the frozen stocks to thaw at room temperature do not heat IMPORTANT Mix each stock thoroughly and then centrifuge briefly to collect all the liquid at the bottom of each tube Whenever possible keep thawed materials on ice during use Do not leave reagents at room temperature for extended periods Materials Supplied by the User Overview In addition to the reagents supplied in this kit other items are required This section lists general materials needed for Cycle sequencing Purifying extension products Note Many of the items listed in this section are available from major laboratory suppliers MLS unless otherwise noted Equivalent sources may be acceptable where noted Refer to the individual instrument protocols for the specific items needed for each instrument NITE CHEMICAL HAZARD Before handling the chemical reagents needed for cycle sequencing read the safety warnings on the reagent bottles and in the manufacturers Material Safety Data Sheets MSDSs and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Dispose of waste in accordance with all local state provincial and national environmental and health regulations BigDye Terminator v1 1 C
48. sion products according to Preparing Extension Products on page 4 13 if desired 2 Prepare the 96 well spin plate per the manufacturer s instructions 3 Perform the purification per the manufacturer s instructions IMPORTANT When using the Edge Biosystems gel filtration kit only centrifuge at 850 x g for 2 min 4 14 BigDye Terminator v1 1 Cycle Sequencing Kit Selecting Seguencing Primers Selecting Seguencing Primers Overview Optimizing Primer Selection The choice of seguencing primer seguence method of primer synthesis and approach to primer purification can have a significant effect on the guality of the seguencing data obtained in dye terminator cycle seguencing reactions with this kit These decisions are particularly important when seguencing is done on real time detection systems where signal strength is critical Some of the recommendations given here are based on information that is general knowledge while others are based on practical experience gained by Applied Biosystems scientists The following recommendations are provided to help optimize primer selection Primers should be at least 18 bases long to ensure good hybridization Avoid runs of an identical nucleotide especially guanine where runs of four or more Gs should be avoided Keep the G C content in the range 30 80 For cycle sequencing primers with melting temperatures T above 45 C produce b
49. t PIE 4 1 Purification MethodS ooccccooccco eee 4 1 Ethanol EDTA Precipitation 000 000 eee eee 4 2 Recommended Protocol cece eee eee 4 2 Precipitating in 96 Well Reaction Plates 4 2 Precipitating in 384 Well Reaction Plates 4 4 Ethanol EDTA Sodium Acetate Precipitation 4 6 Precipitating in 96 Well Reaction Plates 4 6 Precipitating in 384 Well Reaction Plates 4 8 Plate and Spin Column Purification LL is 4 10 OVerVIGW A uita sad dem 4 8 2 as se pistas Ma op dU s aea 4 10 Recommended Spin Columns eee nena 4 10 Optimizing Spin Column Purification LL 4 10 Performing Spin Column Purification oo o 4 11 Plate Purification Using SDS Heat Treatment 4 13 OVERVIEW Cisse E uer ala c deni EC BAN Ba pia Ee ERR mA 4 13 Preparing Extension Products 000 e eee eee eee 4 13 Performing 96 Well Spin Plate Purification 4 14 Recommended 96 Well Spin Plates oooooooo 4 14 Purifying with the 96 Well Spin Plate 4 14 Selecting Sequencing Primers Selecting Sequencing Primers LL aka as A 1 Overview u cce ege bebe duos er arvo i E dee eae eee A 1 Optimizing Primer Selection ooooocooooocorooooo A 1 Control DNA Sequence Control Sequence o ooccococccc ere B 1 Partial Sequence of pGEM 3Zf o o oo
50. t tip IMPORTANT Be sure you hydrate the column for at least 2 hours Spin samples for recommended times spinning too long precipitates more dyes with the sample With microfuge tubes aspirate the supernatant rather than decanting decanting leaves excess ethanol on the sides of the tube Select the start point for data analysis to exclude excess dye peaks Pull up peaks or Total signal strength over Quantitate DNA template bleedthrough 4000 Decrease amount of DNA in the seguencing reactions Load or inject less of the resuspended seguencing reactions D 4 BigDye Terminator v1 1 Cycle Seguencing Kit Obtaining Technical Support Services and Support Applied A services and support page is available on the Applied Biosystems Biosystems Web site To access this go to Web Site http www appliedbiosystems com and click the link for services and support At the services and support page you can e e e Search through frequently asked questions FAQs Submit a question directly to Technical Support Order Applied Biosystems user documents MSDSs certificates of analysis and other related documents Download PDF documents Obtain information about customer training Download software updates and patches In addition the services and support page provides worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities BigD
51. te with aluminum tape and mix by inverting 4 times 6 Incubate at room temperature for 15 min BigDye Terminator v1 1 Cycle Sequencing Kit Ethanol EDTA Sodium Acetate Precipitation To precipitate 10 uL sequencing reactions in 384 well reaction plates continued VA If you are using Then a Beckman Allegra set it at 4 C and spin the plate at 6A centrifuge with a 1650 x g for 45 min GH 3 8A rotor any other centrifuge use a plate adapter and spin the plate at the maximum speed as follows 1400 2000 x g for 45 min or e 2000 3000 x g for 30 min IMPORTANT Proceed to the next step immediately If this is not possible then spin the plate for an additional 2 min before performing the next step 8 Invert the plate and spin up to 185 x g then remove from the centrifuge 9 Add 35 uL of 70 ethanol to each well 10 With the centrifuge set to 4 C spin at 1650 x g for 15 min 11 Invert the plate and spin up to 185 x g for 1 min then remove from the centrifuge Note Start timing when the rotor starts moving 12 To continue resuspend the samples in injection buffer To store cover with aluminum foil and store at 4 C Note Make sure the wells are dry You may use a Speed Vac for 15 min to dry the plate IMPORTANT Make sure the samples are protected from light while they are drying BigDye Terminator v1 1 Cycle S
52. the v1 1 dye set primer mobility files BigDye Terminator v1 1 Cycle Sequencing Kit C 1 Appendix C Sample Electrophoresis Electrophoresis on the ABI PRISM 3700 DNA Analyzer Reguirements Run Modules Configuration Run Module POP 5 polymer 50 cm Seg1 1POP5DefaultModule Seg1 2POP5DefaultModule POP 6 polymer 50 cm Seg1 1POP6DefaultModule Seg1 2POP6DefaultModule Dye Set Primer Mobility Files Polymer Dye Set Primer Mobility File POP 5 polymer DT3700POP5 BD v3 mob POP 6 polymer DT3700POP6 BD v5 mob Standards IMPORTANT Use Dye Set E Dye Set Standards E 3700 BigDye Terminator v1 1 Matrix Standard PN 4336825 Note Refer to the product insert for instructions on using the standards for this instrument C 2 BigDye Terminator v1 1 Cycle Sequencing Kit Electrophoresis on the ABI Prism 3700 DNA Analyzer Performing For information on how to perform sample electrophoresis on the Sample 3700 instrument refer to the following manuals Electrophoresis ABI PRISM 3700 DNA Analyzer Sequencing Chemistry Guide PN 4309125 ABI PRISM 3700 DNA Analyzer User s Manual PN 4306152 BigDye Terminator v1 1 Cycle Seguencing Kit C 3 Appendix C Sample Electrophoresis Electrophoresis on the ABI PRISM 3100 and 3100 Avant Genetic Analyzers Requirements Electrophoresis and data analysis of samples on the ABI PRISM 3100 and 3100 Ava
53. ults Do not allow the column to dry out BigDye Terminator v1 1 Cycle Sequencing Kit Plate and Spin Column Purification Performing Spin To perform spin column purification Column Purification 1 Gently tap the column to cause the gel material to settle to the bottom of the column Remove the upper end cap and add 0 8 mL of deionized water Replace the upper end cap and vortex or invert the column a few times to mix the water and gel material Allow the gel to hydrate at room temperature for at least 2 hours Note Hydrated columns can be stored for a few days at 2 6 C Longer storage in water is not recommended Allow columns stored at 2 6 C to warm to room temperature before use Remove any air bubbles by inverting or tapping the column and allowing the gel to settle Remove the upper end cap first then remove the bottom cap Allow the column to drain completely by gravity Note If flow does not begin immediately apply gentle pressure to the column with a pipette bulb Insert the column into the wash tube provided Spin the column in a microcentrifuge at 730 x g for 2 minutes to remove the interstitial fluid Remove the column from the wash tube and insert it into a sample collection tube for example a 1 5 mL microcentrifuge tube Remove the extension reaction mixture from its tube and load it carefully onto the center of the gel material BigDye Termin
54. water onto the column 3 Add the entire sample to the column 4 Spin the column at 3000 x g in a fixed angle centrifuge for 10 minutes Note The manufacturer recommends a maximum speed of 1000 x g but 3000 x g has worked well in Applied Biosystems laboratories If you are following the manufacturer s guidelines increase the time to compensate 5 Remove the waste receptacle and attach the collection vial 6 Invert the column and spin it at 270 x g for 2 minutes to collect approximately 40 60 uL of sample 7 Add deionized water to bring the purified PCR fragments to the original volume DNA Quality Poor Template Poor template quality is the most common cause of sequencing Quality problems The following are characteristics of poor quality templates Noisy data or peaks under peaks No usable sequence data Weak signal Always follow recommended procedures to prepare templates 2 4 BigDye Terminator v1 1 Cycle Sequencing Kit DNA Guality Contamination Potential contaminants include Proteins RNA Chromosomal DNA Excess PCR primers dNTPs enzyme and buffer components from a PCR amplification used to generate the sequencing template Residual salts Residual organic chemicals such as phenol chloroform and ethanol Residual detergents Determining DNA The following methods can be used to examine DNA quality Quality Agarose gel electrophoresis Purifi
55. ycle Sequencing Kit 1 7 Chapter 1 Introduction Materials for Cycle Sequencing The table below lists the plates or tubes required for the recommended Applied Biosystems thermal cyclers Thermal Cycler Plate or Tube Applied Biosystems Part Number GeneAmp PCR System MicroAmp 384 Well Reaction Plate 4305505 9700 MicroAmp 96 Well Reaction Plate N801 0560 MicroAmp Reaction Tubes 0 2 uL N801 0533 MicroAmp Caps 12 or 8 strip N801 0534 or N801 0535 ABI PRISM Optical Adhesive Cover 4313663 or 4311971 Starter Pack or ABI PRISM Optical Adhesive Covers GeneAmp PCR System MicroAmp 96 Well Reaction Plate N801 0560 9600 MicroAmp Reaction Tubes 0 2 uL N801 0533 MicroAmp Caps 12 or 8 strip N801 0534 N801 0535 ABI PRISM Optical Adhesive Cover 4313663 or 4311971 Starter Pack or ABI PRISM Optical Adhesive Covers GeneAmp PCR System MicroAmp Reaction Tubes 0 2 mL N801 0533 2400 and 2700 MicroAmp Caps 12 or 8 strip N801 0534 N801 0535 1 8 BigDye Terminator v1 1 Cycle Sequencing Kit Materials for Purifying Extension Products Other Eguipment Materials Supplied by the User Sealing taped Method Material Supplier Ethanol EDTA Ethanol EtOH 200 MLS Precipitation proof Molecular Biology grade MLS Ebi TES MNI Costar 6570 Thermowell Sealing Tape Ethanol Sodium Acetate Precipitation Ethanol EtOH 200 proof Molecular Biology
56. ye Terminator v1 1 Cycle Sequencing Kit E 1 Appendix E Obtaining Technical Support E 2 BigDye Terminator v1 1 Cycle Seguencing Kit Index Numerics 310 reguirements mobility files C 6 run modules C 6 sequencing standards C 7 3100 requirements mobility files C 4 run modules C 4 spectral calibration standards C 4 3700 requirements mobility files C 2 run modules C 2 spectral calibration standards C 2 377 requirements matrix file standards C 9 mobility files C 8 run modules C 8 A attention words 1 11 B bacterial artificial chromosomes 2 3 C centrifuge recommended 1 9 components sequencing kits 1 6 contaminants of templates 2 5 control double stranded 2 1 electrophoresis 2 1 single stranded 2 1 control DNA reason for 2 1 customer support See technical support E 1 BigDye Terminator v1 1 Cycle Sequencing Kit cycle sequencing BAC DNA 3 7 protocol forGeneAmp 3 5 D disposables cycle sequencing 1 8 purifying 1 9 DNA quantity characteristics 2 6 quantity touse 2 6 dye set primer files downloading 1 6 dye filter set 3100 1 4 3700 1 4 377 1 4 E ethanol EDTA precipitation 384 well 4 4 96 well 4 3 ethanol EDTA sodium acetate precipitation in 384 well 4 8 precipitation in 96 well 4 6 F formulation new 1 1 freeze thaw limits 1 7 H hazards chemical 1 11 waste 1 12 Index 1 instruments sequencing 1 3 thermal cyclers 1 3 L Lane Guide C 9 long read length
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