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ABI PRISM® dGTP BigDye™ Terminator v3.0 Ready Reaction Cycle

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1. Reaction 4 6 Purifying Extension Products To precipitate in microcentrifuge tubes continued Step Action 2 Prepare the ethanol sodium acetate solution by combining the following for each sample 3 0 uL of 3 M sodium acetate NaOAc pH 4 6 62 5 uL of 95 ethanol EtOH 14 5 uL of deionized water The final volume should be 80 uL for each sample CHEMICAL HAZARD 3M sodium acetate may cause eye skin and respiratory tract irritation Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves CHEMICAL HAZARD Ethanol is a flammable liquid and vapor It may cause eye skin and upper respiratory tract irritation Prolonged or repeated contact may dry the skin Exposure may cause central nervous system depression and liver damage Keep away from heat sparks and flame Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Add 80 uL of this ethanol sodium acetate solution to 20 uL of reaction mixture Close the tubes and vortex briefly Leave the tubes at room temperature for 15 minutes to precipitate the extension products Note Precipitation times lt 15 minutes will result in the loss of very short extension products Precipitation times gt 24 hours will increase the precipitation of unincorporated dye terminators Place the tubes in a microcentr
2. 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 e g a 1 5 mL microcentrifuge tube 10 Remove the extension reaction mixture from its tube and load it carefully onto the center of the gel material 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 it was in for the first spin This is important because the surface of
3. About MSDSs 7 NY Eile CHEMICAL WASTE HAZARD Wastes 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 A site preparation and safety guide is a separate document sent to all 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 Some of the chemicals used with this instrument may be listed as hazardous by their manufacturer When hazards exist warnings are promine
4. Quantitating DNA ike RR eke ae 2 5 Template Quantity 0 00 ee eh 2 5 Template Volume sau u uh cee EA eee 2 5 3 New Cycle Sequencing Protocols Chapter Summary i is nes se eR ERU ERR RE tae eho as 3 1 In This Chapter onere Rer eee dee bab ba 3 1 Important Protocol Changes 0 0 c eee eee eee 3 1 Changes to the Cycle Sequencing Protocol 3 1 Cycle Sequencing Single and Double Stranded DNA 3 2 OVervieWo ay sd a ai hen sac AA 3 2 Preparing the Reactions 0 0 0 eee eee ee eee ee 3 2 Cycle Sequencing on the System 9700 9600 or 2400 3 3 Comparison to Original Procedure 3 3 Cycle Sequencing on the TClor480 3 4 Comparison to Original Procedure 3 4 4 Purifying Extension Products Chapter Summary lsi susya qapya E e I 4 1 In This Chapteracitn ia e A bees bE Gss 4 1 Choosing a Method of Purification 4 2 P rposSQ uc ee vU e cp Ae le EE ew ERN 4 2 Purification Methods 0 0 eee 4 2 Spin Column vs Precipitation 00 00 0000008 4 2 Ethanol Sodium Acetate Precipitation in 96 Well Reaction Plates 4 3 Recommended Protocol as eraus eee eee 4 3 Precipitating in 384 Well Reaction Plates o o oo 4 3 Precipitating in 96 Well Reaction Plates o o ooooo 4 3 Ethanol Sodi
5. S E and Boeke J D 1994 Efficient integration of artificial transposons into plasmid targets in vitro a useful tool for DNA mapping sequencing and functional analysis Nucleic Acids Res 22 3765 3772 2 Devine S E Chissoe S L Eby Y Wilson R K and Boeke J D 1997 A transposon based strategy for sequencing repetitive DNA in eukaryotic genomes Genome Res 7 551 563 2 4 Preparing the Templates DNA Quantity Quantitating DNA Template Quantity Template Volume If possible quantitate the amount of purified DNA by measuring the absorbance at 260 nm or by some other method The table below shows the amount of template to use in a cycle sequencing reaction Template Quantity PCR product 100 200 bp 13 ng 200 500 bp 3 10 ng 500 1000 bp 5 20 ng 1000 2000 bp 10 40 ng gt 2000 bp 40 100 ng Single stranded 50 100 ng Double stranded 200 500 ng Cosmid BAC 0 5 1 0 ug Bacterial genomic DNA 2 3 ug Note In general higher DNA quantities give higher signal intensities The ranges given in the table above should work for all primers You may be able to use even less DNA especially when sequencing with the 21 M13 primer The amount of PCR product to use in sequencing will also depend on the length and purity of the PCR product Cycle sequencing reactions are made up in a final volume of 20 uL The volume allows for up to 8 uL for DNA template and 4 uL for primer 0 8 pmol uL I
6. state provincial and national environmental and health regulations Introduction 1 11 Materials for The table below lists the plates or tubes required for the recommended Cycle Sequencing Applied Biosystems thermal cyclers page 1 7 Thermal Cycler Plate or Tube Applied Biosystems Part Number GeneAmp PCR System MicroAmp 96 Well Reaction Plate N801 0560 9700 MicroAmp Reaction Tubes 0 2 mL 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 mL N801 0533 MicroAmp Caps 12 or 8 strip N801 0534 N801 0535 ABI Prism Optical Adhesive Cover Starter 4313663 or 4311971 Pack or ABI PRISM Optical Adhesive Covers GeneAmp PCR System MicroAmp Reaction Tubes 0 2 mL N801 0533 2400 MicroAmp Caps 12 or 8 strip N801 0534 N801 0535 DNA Thermal Cycler 4808 GeneAmp Thin Walled Reaction Tubes 0 5 mL N801 0537 GeneAmp Thin Walled Reaction Tubes N801 0737 with Flat Cap DNA Thermal Cycler TC1 GeneAmp Thin Walled Reaction Tubes N801 0537 0 5 mL a The DNA Thermal Cycler 480 and the DNA Thermal Cycler TC1 thermal cyclers require mineral oil that can be obtained from Applied Biosystems P N 0186 2302 1 12 Introduction Materials for Purif
7. 4 3 Plates Ethanol Sodium Acetate Precipitation in Microcentrifuge Tubes 4 6 Ethanol Precipitation in 96 Well Reaction Plates 4 9 Ethanol Precipitation in Microcentrifuge Tubes 4 11 Spin Column Purification 4 13 Spin Column vs Use the method that works best for your particular application Precipitation 4 Precipitation methods are cheaper and faster but they may remove less of the unincorporated dye labeled terminators that can obscure data at the beginning of the sequence 4 The spin column procedure removes more terminators but is more costly and may take additional time to perform 4 2 Purifying Extension Products Ethanol Sodium Acetate Precipitation in 96 Well Reaction Plates Recommended Protocol Precipitating in 384 Well Reaction Plates Precipitating in 96 Well Reaction Plates With the dGTP BigDye terminators v3 0 the ethanol sodium acetate precipitation method for 96 well reaction plates produces consistent signal while minimizing unincorporated dyes A final 70 ethanol wash is required Note While this method produces the cleanest signal it may cause loss of small molecular weight fragments To use the ethanol sodium acetate precipitation method for 384 well reaction plates refer to AB Prism BigDye Terminator v3 0 Ready Reaction Cycle Sequencing Kit Protocol P N 4390037 IMPORTANT Use non denatured 95 ethanol rather than absolute 100 ethanol Absolute ethanol absorb
8. AAA 1 11 Materials for Cycle Sequencing 1 12 Materials for Purifying Extension Products 1 13 Safety ias EI IN A ir 1 14 Documentation User Attention Words 1 14 Chemical Hazard Warning 1 14 Chemical Waste Hazard Warning 1 15 Site Preparation and Safety Guide 1 15 About MSDSS sui bee lala lisa 1 15 Ordering MSDS So 4 5 a Iu en ERR RR ERES 1 16 2 Preparing the Templates ChapterSumiaty iisiieieiered rene AA sere 2 1 In This Chapter cs de eR Rose Re eR EUER ROG ER IUe 2 1 Control DNA Templates 0 00 eee ee 2 2 Using Control DNA ium esse eased A Ro RES 2 2 Control DNA Sequence 0 0 eee eee 2 2 An Additional Control Sold Separately 2 2 Template Preparation Methods 0 0 cece ee eee 2 3 Single and Double Stranded Templates 2 3 PER Templates lt a a lt AE 2 3 Importance of Purifying Product 2 3 Purifying PCR Fragments 0 00 0 0 eee ee eee 2 3 Use of the Primer Island Transposition Kit 2 4 OVERVIEW cid Sede ay ae vec eae eee es EE 2 4 About Transposons 2 4 Inserting Artificial Transposons 2 4 Techniques el p Rx E ds ee a oes 2 4 DNA Quantity iis 6 ri a Ree REA EA Hor 2 5
9. BigDye primers original Dye Filter Set Standards for Instrument Matrix File Generation A ABI Prism BigDye Matrix Standards v3 0 For use with P N 4390421 the BigDye Filter Wheel Note For instructions on using the matrix standards P N 4390421 for the 373 instruments contact Technical Support 2 Includes the ABI PRISM 373 and ABI PRISM 373 with XL Upgrade instruments 5 6 Sample Electrophoresis Resuspending and To resuspend and load the samples Loading the Samples Step Action 1 Prepare a loading buffer by combining the following in a 5 1 ratio 5 parts deionized formamide to 1 part EDTA with blue dextran Deionized formamide 25 mM EDTA pH 8 0 with blue dextran 50 mg mL CHEMICAL HAZARD Formamide causes eye skin and respiratory tract irritation It is a possible reproductive and birth defect hazard Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves CHEMICAL HAZARD EDTA may cause eye skin and respiratory tract irritation Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Resuspend each sample pellet in loading buffer as follows Volume pL 180r24 32or36 Template well well 48 well 64 well PCR product 3 4 3 4 2 4 2 4 plasmid M13 Vortex and spin the samples Heat the samples
10. U S Patent Nos 5 498 523 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 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 P O Box 22400 Cleveland Ohio 44122 Patents are pending in countries outside the United States Notice to Purchaser Limited License The purchase price of the ABI PRISM dGTP BigDye Terminator v3 0 Ready Reaction Cycle Sequencing Kit includes a limited nontransferable license under U S patents or its foreign counterparts to use only this amount of the product for DNA Sequencing and related processes described in said patent solely for the research and development activities of
11. antibiotics and transposons 2 4 C chemical hazards 1 14 color coding dye base 1 4 gelimage 1 4 compressions and secondary structures 1 6 bands 1 6 control DNA pGEM sequence B 1 sequences used 2 2 troubleshooting 2 2 using 2 2 customer support See technical support C 1 cycle sequencing and PCR templates 2 3 reaction preparation 3 2 required materials 1 12 template quantities 2 5 template volumes 2 5 D DNA quantitating 2 5 dye set primer downloading files 1 9 filenames 5 2 files where to get 1 9 installing files 1 9 required files 5 3 dye filter sets required 1 8 E early signal loss avoiding 1 2 M matrix standards compatibilities 1 2 required 1 8 5 3 5 6 mobility files See dye set primer MSDSs about 1 15 ordering 1 16 P primers sequence selection A 1 purification methods for 4 2 PCR fragments 2 3 required materials 1 13 Q quantitating DNA 2 5 R ramping time speed 1 7 requirements cycle sequencing 1 12 dye set primer files 5 3 5 6 matrix standards 5 3 5 6 purifying extension products 1 13 run modules 5 3 S safety 1 14 to 1 16 samples 373loading 5 7 377 loading 5 4 secondary structure and compression 1 6 and migration 1 6 sequencing primers selecting A 1 Index 1 sequencing kit choice C rich 1 5 GC rich 1 4 G rich 1 4 GT rich 1 4 secondary structure 1 5 unapparent characteristic 1 5 T technical support C 1 to C 1 to thermal cycler settings 3 3 to 3 4 tran
12. at 95 C for 2 minutes to denature Place on ice until ready to load Load each sample into a separate lane of the gel as follows Volume pL 180r24 32or36 Template well well 48 well 64 well PCR product 3 4 3 4 2 2 5 2 plasmid M13 Sample Electrophoresis 5 7 Selecting Sequencing Primers Selecting Sequencing Primers Overview The choice of sequencing primer sequence method of primer synthesis and approach to primer purification can have a significant effect on the quality of the sequencing data obtained in dye terminator cycle sequencing reactions with this kit Recommendations These decisions are particularly important when sequencing 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 This is especially true for 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 Tm above 45 C produce better results than primers with lower Tm For primers with a G C content less than 50 it may b
13. of reaction mixture Seal the tubes with strip caps or by applying a piece of 3M Scotch Tape 431 or 439 adhesive backed aluminum foil tape Press the foil onto the tubes to prevent any leakage Invert the plate a few times to mix Leave the plate at room temperature for 15 minutes to precipitate the extension products Note Precipitation times 15 minutes will result in the loss of very short extension products Precipitation times gt 24 hours will increase the precipitation of unincorporated dye terminators 4 4 Purifying Extension Products To precipitate in 96 well reaction plates continued Step Action 7 Place the plate in a table top centrifuge with a tube tray adaptor and spin it at the maximum speed which must be 21400 x g but 3000 x g 1400 2000 x g 45 minutes 2000 3000 x g 30 minutes Note A MicroAmp tube in a MicroAmp plate can withstand 3000 x g for 30 minutes IMPORTANT Proceed to the next step immediately If this is not possible then spin the tubes for 2 minutes more immediately before performing the next step 8 Without disturbing the precipitates remove the adhesive tape and discard the supernatant by inverting the plate onto a paper towel folded to the size of the plate 9 Place the inverted plate with the paper towel into the table top centrifuge and spin at 50 x g for 1 minute 10 Add 150 uL of 70 ethanol to each pellet 11 Cap or seal the t
14. 77 and 373 instruments Color of Bands on ABI Prism 377 Base Terminator or 373 Instrument Gel Image A V3 Dye 2 Green C V3 Dye 4 Red G V3 Dye 1 Blue T V3 Dye 3 Yellow When problems are encountered with a particular template use the table below to choose the best approach This table shows which types of difficult templates should be sequenced with the dGTP BigDye Terminator v3 0 Kit and which are best sequenced with the ABI Prism BigDye Terminator v3 0 Ready Reaction Cycle Sequencing Kit using altered reaction conditions Note Sequence contexts refer to the extension strand i e the sequence that is seen in the electropherogram Template Characteristic at or Near the Stop Region Chemistry to Use GT rich dGTP BigDye terminator v3 0 G rich dGTP BigDye terminator v3 0 GC rich BigDye terminator v3 0 Alter reaction conditions to improve template denaturation refer to pages 7 32 and 7 33 of the Automated DNA Sequencing Chemistry Guide for methods Ifthe sequence data does not improve with the BigDye Terminator v3 0 Kit using altered reaction conditions use the dGTP BigDye Terminator v3 0 Kit Ifthe sequence data still does not improve using altered reaction conditions with the dGTP BigDye Terminator v3 0 Kit can be helpful in some cases Template Characteristic at or Near the Stop Region Chemistry to Use C rich BigDye terminator
15. ABI PRISM dGTP BigDye Terminator v3 0 Ready Reaction Cycle Sequencing Kit Protocol KS Beams Copyright 2002 2010 Applied Biosystems All rights reserved For Research Use Only Not for use in diagnostic procedures Information in this document is subject to change without notice Applied Biosystems assumes no responsibility for any errors that may appear in this document This document is believed to be complete and accurate at the time of publication In no event shall Applied Biosystems be liable for incidental special multiple or consequential damages in connection with or arising from the use of this document 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 run ning royalty component The purchase price of ABI PRISM dGTP BigDye Terminator v3 0 Ready Reaction Cycle Sequencing Kit includes limited non transferable rights under 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 inform
16. AGTGAGCT CCGCTTTCCA ATGAATCGGC GGGCGCTCTT CGGTCGTTCG GGCGGTAATA GGAAAGAACA ACCGTAAAAA CCGCCCCCCT CAGAGGTGGC CGTTTCCCCC GACCCTGCCG TCGGGAAGCG ATCTCAGTTC TGTGCACGAA TCCGGTAACT ACTTATCGCC CAGAGCGAGG TGGTGGCCTA GTATCTGCGC TTCGAGCTCG AGGCATGCAA GCTTGGCGTA TTGTTATCCG AGCATAAAGT AACTCACATT GTCGGGAAAC CAACGCGCGG CCGCTTCCTC GCTGCGGCGA CGGTTATCCA TGTGAGCAAA GGCCGCGTTG GACGAGCATC GAAACCCGAC TGGAAGCTCC CTTACCGGAT TGGCGCTTTC GGTGTAGGTC CCCCCCGTTC ATCGTCTTGA ACTGGCAGCA TATGTAGGCG ACTACGGCTA TCTGCTGAAG Control DNA Sequence B 1 40 80 120 160 200 240 280 320 360 400 440 480 520 560 600 640 680 720 760 800 840 880 920 960 1000 Technical Support Services amp Support Applied To access the Applied Biosystems Web site go to Biosystems Web Site http www appliedbiosystems com At the Applied Biosystems Web site you can 4 4 4 4 4 4 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 Applied Biosystems Web site provides a list of telephone and fax numbers that can be used to contact Technical Support Technical Support C 1 Index A AmpliTaq DNA Polymerase FS advantages 1 3 and cycle sequencing 1 3
17. Recommended Spin Columns Optimizing Spin Column Purification This section describes the recommended 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 CentriSep spin columns Applied Biosystems P N 401763 for 32 columns and P N 401762 for 100 columns IMPORTANT When using the dGTP BigDye terminators v3 0 hydrate the column for 2 hours Tips for optimizing spin column purification Donot process more columns than you can handle conveniently at one time Load the sample in the center of the column bed slowly Make sure that the sample does not touch the sides of the column and that the pipette tip does not touch the gel surface If samples are not loaded properly 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 11 18 x rx 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 results Do not allow the column to dry out Purifying Extension Products 4 13 Performing Spin To perform spin column purification Column Purification Step Action
18. al ramp to 96 C No change 96 C for 30 seconds No change Rapid thermal ramp to 50 C Rapid thermal ramp to 68 C 50 C for 15 sec Eliminated Rapid thermal ramp to 60 C Eliminated 60 C for 4 minutes 68 C for 2 minutes Note Two steps have been modified and two steps have been eliminated This is a faster procedure because of the change from a 3 step to a 2 step protocol and the use of a higher extension temperature 3 4 New Cycle Sequencing Protocols Purifying Extension Products Chapter Summary In This Chapter The following topics are covered in this chapter Topic See Page Choosing a Method of Purification 4 2 Ethanol Sodium Acetate Precipitation in 96 Well Reaction 4 3 Plates Ethanol Sodium Acetate Precipitation in Microcentrifuge Tubes 4 6 Ethanol Precipitation in 96 Well Reaction Plates 4 9 Ethanol Precipitation in Microcentrifuge Tubes 4 11 Spin Column Purification 4 13 Purifying Extension Products 4 1 Choosing a Method of Purification Purpose Unincorporated dye terminators must be removed before the samples can be analyzed by electrophoresis Excess dye terminators in sequencing reactions obscure data in the early part of the sequence and can interfere with basecalling Purification There are several methods you can use to purify extension products Methods Purification Method See page Ethanol Sodium Acetate Precipitation in 96 Well Reaction
19. ation relating 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 ABI PRISM dGTP BigDye Terminator v3 0 Ready Reaction Cycle Sequencing Kit includes a limited nontransferable non exclusive license without the right to resell repackage or sublicense under U S patents corresponding foreign counterparts 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 Patents are pending in countries outside the United States Notice to Purchaser About Limited License This kit is sold pursuant to a limited sublicense from Amersham International plc under one or more
20. ded in precipitation protocols purchase non denatured ethanol at this concentration rather than absolute 100 ethanol Absolute ethanol absorbs water from the atmosphere gradually decreasing its concentration This can lead to inaccurate final concentrations of ethanol which can affect some protocols To precipitate in 96 well reaction plates Step Action 1 Remove the 96 well MicroAmp plate from the thermal cycler Remove the caps from each tube Add the following for each sample 16 uL of deionized water 64 uL of non denatured 95 ethanol The final ethanol concentration should be 60 3 MENNA CHEMICAL HAZARD Ethanol is a flammable liquid and vapor It may cause eye skin and upper respiratory tract irritation Prolonged or repeated contact may dry the skin Exposure may cause central nervous system depression and liver damage Keep away from heat sparks and flame Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Seal the tubes with strip caps or by applying a piece of 3M Scotch Tape 431 or 439 adhesive backed aluminum foil tape Press the foil onto the tubes to prevent any leakage Invert the plate a few times to mix Leave the plate at room temperature for 15 minutes to precipitate the extension products Note Precipitation times 15 minutes will result in the loss of very short extension products Precipitation t
21. e Stranded DNA Overview This section describes how to prepare reactions and perform cycle sequencing on a variety of templates including M13 plasmids and PCR products Preparing the The type of tube required depends on the thermal cycler that you are Reactions using Refer to Materials for Cycle Sequencing on page 1 12 To prepare the reaction mixtures Step Action 1 For each reaction add the following reagents to a separate tube Reagent Quantity Terminator Ready Reaction Mix 8 0 uL Template See the table under Template Quantity on page 2 5 Primer 3 2 pmol Deionized water q s Total Volume 20 uL Mix well and spin briefly If using the DNA Thermal Cycler TC1 or DNA Thermal Cycler 480 Overlay the reaction mixture with 40 uL of light mineral oil 3 2 New Cycle Sequencing Protocols Cycle Sequencing To sequence single and double stranded DNA on the GeneAmp PCR on the System System 9700 in 9600 emulation mode 9600 or 2400 9700 9600 or 2400 Step Action 1 Place the tubes in a thermal cycler and set the volume to 20 pL 2 Repeat the following for 25 cycles Rapid thermal ramp to 96 C 96 C for 10 seconds Rapid thermal ramp to 68 C 68 C for 2 minutes 3 Rapid thermal ramp to 4 C and hold until ready to purify 4 Spin down the contents of the tubes in a microcentrifuge 5 Proceed to Chapter 4 Purifyi
22. e 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 Selecting Sequencing Primers A 1 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 A 2 Selecting Sequencing Primers Control DNA Sequence Control Sequence Partial Sequence The pGEM 3Zf sequence below is the sequence of the 21 M13 of pGEM 3Zf forward primer followed by the ensuing 1000 bases TGTAAAACGACGGCCAGT 21 M13 primer GAATTGTAAT GTACCCGGGG GCTTGAGTAT ATCATGGTCA CTCACAATTC GTAAAGCCTG AATTGCGTTG CTGTCGTGCC GGAGAGGCGG GCTCACTGAC GCGGTATCAG CAGAATCAGG AGGCCAGCAA CTGGCGTTTT ACAAAAATCG AGGACTATAA CTCGTGCGCT ACCTGTCCGC TCATAGCTCA GTTCGCTCCA AGCCCGACCG GTCCAACCCG GCCACTGGTA GTGCTACAGA CACTAGAAGG ACGACTCACT ATCCTCTAGA TCTATAGTGT TAGCTGTTTC CACACAACAT GGGTGCCTAA CGCTCACTGC AGCTGCATTA TTTGCGTATT TCGCTGCGCT CTCACTCAAA GGATAACGCA AAGGCCAGGA TCCATAGGCT ACGCTCAAGT AGATACCAGG CTCCTGTTCC CTTTCTCCCT CGCTGTAGGT AGCTGGGCTG CTGCGCCTTA GTAAGACACG ACAGGATTAG GTTCTTGAAG ACAGTATTTG ATAGGGCGAA GTCGACCTGC CACCTAAATA CTGTGTGAAA ACGAGCCGGA TG
23. ee hae pai 5 6 Resuspending and Loading the Samples 5 7 A Selecting Sequencing Primers Selecting Sequencing Primers 0 0 00 0c eee eee eee eee ee A 1 OVerVIG WA da a sae Maes Bag A 1 Recommendations A 1 B Control DNA Sequence Control Sequence ene ae Kee o ld a e te B 1 Partial Sequence of pGEM 3Zf B 1 C Technical Support Services amp Support s 0 cs neces s a toa eae te ae C 1 Applied Biosystems Web Site 0 00 0 02 eee eee C 1 Index Index I Introduction Chapter Summary In This Chapter The following topics are covered in this chapter Topic See Page About the Kit 1 2 Instruments 1 7 Required Software 1 8 Reagents and Storage 1 10 Materials Supplied by the User 1 11 Safety 1 14 Introduction 1 1 About the Kit Reagent The ABI PRism dGTP BigDye Terminator v3 0 and the Requirements dGTP BigDye Terminator v3 0 1 2 Introduction Kit ABI Prism BigDye v3 0 Ready Reaction Cycle Sequencing Kit with AmpliTaq DNA Polymerase FS requires unique instrument matrix files for the ABI PRisM 377 DNA Sequencer and ABI Prism9 373 DNA Sequencers with the ABI PRISM BigDye Filter Wheel installed The instrument file created for the BigDye chemistry v1 0 v2 0 and dRhodamine chemi
24. entrifuge Tubes With ethanol precipitation residual terminator peaks may be seen However the recovery of small molecular weight fragments will be improved using this precipitation method IMPORTANT Where 95 ethanol is recommended in precipitation protocols purchase non denatured ethanol at this concentration rather than absolute 100 ethanol Absolute ethanol absorbs water from the atmosphere gradually decreasing its concentration This can lead to inaccurate final concentrations of ethanol which can affect some protocols To precipitate in microcentrifuge tubes Step Action 1 pipette the entire contents of each extension reaction into a 1 5 mL microcentrifuge tube Note Ifthe TC1 or DNA Thermal Cycler 480 was used for thermal cycling remove the reactions from the tubes as shown in step 1 on page 4 6 2 Add the following for each sample 16 uL of deionized water 64 uL of non denatured 95 ethanol The final ethanol concentration should be 60 3 CHEMICAL HAZARD Ethanol is a flammable liquid and vapor It may cause eye skin and upper respiratory tract irritation Prolonged or repeated contact may dry the skin Exposure may cause central nervous system depression and liver damage keep away from heat sparks and flame Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves 3 Close the tubes and vortex briefly Leave the tubes at
25. equencing methods cycle sequencing provides several chances to denature and extend the template which ensures adequate signal in the sequencing reaction 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 P N N930 2119 The protocol for using these columns is provided in Purifying PCR Fragments below To purify PCR fragments by ultrafiltration Step Action 1 Assemble the Centricon 100 column according to the manufacturer s recommendations Load 2 mL deionized water onto the column Add the entire sample to the column 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 the sample This should yield approximately 40 60 pL of sample 7 Add deionized water to bring the purified PCR fragments to the original volume Preparing the Templates 2 3 Use of the Primer Island Transposition Kit Overview About Transposons Inserting Artificial Transposons Technique The dGTP BigDye terminators v3 0 are a
26. er Wheel Note We are currently investigating methods to run the dGTP BigDye terminators v3 0 on capillary instruments including the ABI PRISM9 310 Genetic Analyzer ABI PRISM9 3700 DNA Analyzer and the ABI PRISM 3100 Genetic Analyzer However the use of this chemistry on capillary instruments is not recommended or supported by this protocol The protocols provided in this document were optimized using Applied Biosystems thermal cyclers including GeneAmp PCR Systems 9700 9600 and 2400 DNA Thermal Cycler 480 DNA Thermal Cycler TC1 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 1 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 2 Includes the ABI Prism 373 and ABI PRISM 373 with XL Upgrade instruments Introduction 1 7 Required Software Dye Filter Sets and The dye filter sets and matrix standards required for the 377 and 373 Matrix Standards instruments are listed in the table below for the 377 and 373 IMPORTANT Instrument matrix file for the dGTP BigDye terminators v3 0 Instruments cannot be used for the BigDye terminators original BigDye terminators v2 0 dRhodamine terminators or BigDye primers original Standards f
27. f your DNA is not concentrated enough and you need to add more than 8 uL of DNA template then you can compensate for the additional volume by using a more concentrated solution of primer For example if your concentration of primers is increased from 0 8 pmol uL to 3 2 pmol uL then the volume of primers can be reduced from 4 uL to 1 uL Because less volume is used for the primers more volume can then be added for the template In this example the volume of DNA template could be increased from 8 pL to 11 uL Preparing the Templates 2 5 New Cycle Sequencing Protocols Chapter Summary In This Chapter The following topics are covered in this chapter Topic See Page Important Protocol Changes 3 1 Cycle Sequencing Single and Double Stranded DNA 3 2 Important Protocol Changes Changes to the The cycle sequencing protocols used for the ABI PRisM dGTP Cycle Sequencing BigDye Terminator v3 0 Ready Reaction Cycle Sequencing Kit with Protocol ABI PRism DNA Polymerase FS are changed from those used for the ABI PRisM BigDye Terminator v3 0 Ready Reaction Cycle Sequencing Kit These protocols are also different than those used in the previous version of the dGTP BigDye Terminator v3 0 kit The cycling conditions protocols have been improved to reduce cycling time by half They have been optimized for Applied Biosystems thermal cyclers New Cycle Sequencing Protocols 3 1 Cycle Sequencing Single and Doubl
28. h to analyze your data using a If your data was collected on a 377 instrument computer with the Windows NT platform Windows NT platform 377 or 373 instrument Macintosh computer with a CD ROM drive PN 4326479 For Macintosh platform 377 or 373 instrument Macintosh computer with a floppy drive PN 4326480 For Macintosh platform Downloading Files from the Internet Dye set primer mobility files can be downloaded from our Web site http www appliedbiosystems com techsupp swpps SAsw html If you do not have access to the Internet you can get the files from Applied Biosystems Technical Support or from your local field applications specialist call your local sales office for more information Introduction 1 9 Reagents and Storage Available Kit The following kit is available Number of Kit Reactions Part Number The ABI Prism dGTP BigDye 100 4390229 Terminator v3 0 Ready Reaction Cycle Sequencing Kit with AmpliTag DNA Polymerase FS Description of A description of the kit reagents is listed below Reagents 1 10 Introduction Terminator Ready Reaction Mix A Dye Terminator C Dye Terminator G Dye Terminator TDye Terminator Deoxynucleoside triphosphates dATP dCTP dGTP dUTP AmpliTaq DNA Polymerase FS with thermally stable pyrophosphatase MgCl Tris HCl buffer pH 9 0 pGEM 3Zf double
29. han one fragment can migrate at the same position Due to the replacement of dITP by dGTP compressions can be a problem for the dGTP BigDye Terminator v3 0 Kit IMPORTANT Because of band compressions we do not recommend using the dGTP BigDye Terminator v3 0 Kit for routine sequencing It should be used only if the standard terminator kits do not give good data IMPORTANT When using the ABI PRISM 373 DNA Sequencers compressions can be severe Use the dGTP BigDye Terminator v3 0 Kit to sequence through the difficult regions Then sequence the opposite strand using the ABI Prism BigDye Terminator v3 0 Ready Reaction Cycle Kit with a primer that anneals to the opposite strand beyond the stop region Instruments Instrument Platforms Thermal Cyclers The ABI PRISM dGTP BigDye Terminator v3 0 Ready Reaction Cycle Sequencing Kit is for use with the following instruments ABI Prism 377 DNA Sequencer all models ABI PRISM 373 DNA Sequencers with the ABI Prism BigDye Filter Wheel installed Refer to the ABI Prism BigDye Filter Wheel User Bulletin P N 4304367 for more information 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 IMPORTANT This kit is not designed for use with ABI PRISM 373 DNA Sequencers and ABI PRISM9 373 DNA Sequencers with XL Upgrade that do not have the ABI PRISM BigDye Filt
30. ifuge and mark their orientations Spin the tubes for 20 minutes at maximum speed IMPORTANT Proceed to the next step immediately If this is not possible then spin the tubes for 2 minutes more immediately before performing the next step Carefully aspirate the supernatants with a separate pipette tip for each sample then discard Pellets may or may not be visible IMPORTANT The supernatants must be removed completely as unincorporated dye terminators are dissolved in them The more residual supernatant left in the tubes the more unincorporated dye terminators will remain in the samples Purifying Extension Products 4 7 To precipitate in microcentrifuge tubes continued Step Action 8 Add 250 uL of 70 ethanol to the tubes and mix briefly 9 Place the tubes in the microcentrifuge in the same orientation as step 5 and spin for 5 minutes at maximum speed 10 Aspirate the supernatants carefully as in step 6 11 Dry the samples in a vacuum centrifuge for 10 15 minutes or to dryness Do not over dry 4 8 Purifying Extension Products Ethanol Precipitation in 96 Well Reaction Plates Unincorporated Terminators Precipitating in 96 Well Reaction Plates With ethanol precipitation residual terminator peaks may be seen However the recovery of small molecular weight fragments will be improved using this precipitation method IMPORTANT Where 95 ethanol is recommen
31. imes gt 24 hours will increase the precipitation of unincorporated dye terminators Purifying Extension Products 4 9 To precipitate in 96 well reaction plates continued Step Action 6 Place the plate in a table top centrifuge with a tube tray adaptor and spin it at the maximum speed which must be 21400 x g but 3000 x g 1400 2000 x g 45 minutes 2000 3000 x g 30 minutes Note A MicroAmp tube in a MicroAmp plate can withstand 3000 x g for 30 minutes IMPORTANT Proceed to the next step immediately If this is not possible then spin the tubes for 2 minutes more immediately before performing the next step Without disturbing the precipitates remove the adhesive tape and discard the supernatant by inverting the plate onto a paper towel folded to the size of the plate Place the inverted plate with the paper towel into the table top centrifuge and spin at 50 x g for 1 minute Add 150 uL of 70 ethanol to each pellet 10 Cap or seal the tubes then invert the plate a few times to mix 11 Spin the plate for 10 minutes at maximum speed See step 6 above 12 Repeat steps 7 and 8 13 Remove the plate and discard the paper towel Note Pellets may or may not be visible Vacuum drying of the samples is not necessary 4 10 Purifying Extension Products Ethanol Precipitation in Microcentrifuge Tubes Unincorporated Terminators Precipitating in Microc
32. ion for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses Printed in the USA 06 2010 Part Number 4390038 Rev D Contents 1 Introduction Chapter Summary ci e eee es eso ss rac eedem 1 1 In This Chapter oo eee aula RETE 1 1 About the Kit ose fete ats wie tee eG Heh ev uei RU 1 2 Reagent Requirements o ooocoocococcococ o 1 2 dGTP BigDye Terminator v3 0 Kit oooooooocoocoocoooooo 1 2 Kit Formats nico g a td eatin Paes 1 3 Cycle Sequencing with AmpliTaq DNA Polymerase FS 1 3 dGTP BigDye Terminator v3 0 Appearance on the 377 or 373 Instrument Gel Image 1 4 Difficult Templates a GA ie Nae a kausa 1 4 COMPreSSIONS iii Bae ee dae dae E RM SEDET 1 6 InstrumentS sr A Sees RAE Heer ok nes 1 7 Instrument Platforms 1 7 Thermal Cyclers ic Rl b eU MER 1 7 Required Softwaren eiu lla ker eR RR eR E ER 1 8 Dye Filter Sets and Matrix Standards for the 377 and 373 Instruments taria Ri eS he te ee eee eas 1 8 Instructions for Generating Matrices 1 8 Dye Set Primer Mobility Files 1 9 Reagents add Storage cin mia ao ihe am eee hae eee ede eed 1 10 Available Kit iii IS Ge ude eeu e Ue ss 1 10 Description of Reagents 0 0 0 0 eee eee eee 1 10 Storage and Use of the Kit 1 11 Materials Supplied by the User 1 11 QVerVIe Wi vec pU O AA
33. is 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 Seq 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 a Any plate check and prerun module can be used on the ABI Prism 377 DNA Sequencers Dye Set Primer Mobility Files Gel Formulation Dye Set Primer Mobility File 4 5 acrylamide 29 1 or DT377 BDv3 v1 mob 5 Long Ranger gel Matrix Standards IMPORTANT Instrument matrix file for the dGTP BigDye terminators v3 0 and BigDye terminators v3 0 cannot be used for the BigDye terminators original BigDye terminators v2 0 dRhodamine terminators or BigDye primers original Dye Filter Set Standards for Instrument Matrix File Generation E ABI Prism BigDye Matrix Standards v3 0 P N 4390421 Note Refer to the product insert for instructions on using the standards for this instrument 1 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 Sample Electrophoresis 5 3 Using the Lane Guide Kit Using Long Read Gel and Buffer Formulations Resuspending and Loading the Samples If you are using the BigDye chemistries v3 0 on the 377 instrument in conjunctio
34. lso suitable for sequencing plasmid templates generated using the Primer Island Transposition Kit P N 402984 This kit uses transposons to insert primer binding sites into cloned DNA Transposons are mobile genetic elements regions of DNA capable of inserting themselves or copies of themselves into the genome Transposons encode the proteins that facilitate their insertion into the target DNA This property of transposons can be exploited to place unique primer binding sites randomly throughout any large segment of DNA These primer sites may be used subsequently as templates for PCR and or sequencing reactions Transposon insertion is an alternative to subcloning or primer walking when sequencing a large cloned DNA region 1 2 The Primer Island Transposition Kit provides reagents for generating artificial transposon insertions into target DNA in vitro The artificial transposon contains the Pl and Pl priming sites The Primer Island reagents are combined with a target DNA of choice and used to transform Escherichia coli To identify the E coli carrying the transposon the transformed bacteria are plated on Luria Bertani LB agar plates containing carbenicillin and trimethoprim antibiotics Each carbenicillin and trimethoprim resistant colony has integrated a copy of the transposon into the target DNA Follow Primer Island Transposition Kit Protocol P N 402920 for transposon insertion and template preparation 1 Devine
35. n with the ABI PRISM Lane Guide Lane Identification Kit refer to that kit s protocol P N 4313804 for instructions on resuspending and loading samples For longer sequencing read lengths follow the gel and buffer formulations described in user bulletin Achieving Longer High Accuracy Reads on the 377 Sequencer P N 4315153 Note You can use any plate check and prerun modules To resuspend and load the samples Step Action 1 Prepare a loading buffer by combining the following in a 5 1 ratio 5 parts deionized formamide to 1 part EDTA with blue dextran Deionized formamide 25 mM EDTA pH 8 0 with blue dextran 50 mg mL CHEMICAL HAZARD Formamide causes eye skin and respiratory tract irritation It is a possible reproductive and birth defect hazard Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves CHEMICAL HAZARD EDTA may cause eye skin and respiratory tract irritation Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Resuspend each sample pellet in loading buffer as follows Volume pL 18 or 36 well Volume pL Template 48 64 or 96 well PCR product plasmid M13 6 8 4 6 Vortex and spin the samples Heat the samples at 95 C for 2 minutes to denature Place on ice until ready to load 5 4 Sample Electropho
36. ng Extension Products a Rapid thermal ramp is 1 C second Comparison to The following changes have been made Original Procedure Original New Rapid thermal ramp to 96 C No change 96 C for 10 seconds No change Rapid thermal ramp to 50 C Rapid thermal ramp to 68 C 50 C for 5 sec Eliminated Rapid thermal ramp to 60 C Eliminated 60 C for 4 minutes 68 C for 2 minutes Note Two steps have been modified and two steps have been eliminated This is a faster procedure because of the change from a 3 step to a 2 step protocol and the use of a higher extension temperature New Cycle Sequencing Protocols 3 3 Cycle Sequencing To sequence single and double stranded DNA on the DNA Thermal on the TC1 or 480 Cycler TC1 or DNA Thermal Cycler 480 Step Action 1 Place the tubes in a thermal cycler and set the volume to 20 pL 2 Repeat the following for 25 cycles Rapid thermal ramp to 96 C 96 C for 30 seconds Rapid thermal ramp to 68 C 68 C for 2 minutes 3 Rapid thermal ramp to 4 C and hold until ready to purify 4 Spin down the contents of the tubes in a microcentrifuge 5 Proceed to Chapter 4 Purifying Extension Products a Rapid thermal ramp is 1 C second Comparison to The following changes have been made Original Procedure Original New Rapid therm
37. ntly 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 hazardous chemical after an MSDS update MSDSs provide you with the safety information you need to store handle transport and dispose of the chemicals safely We strongly recommend that you replace the appropriate MSDS in your files each time you receive a new MSDS packaged with a hazardous chemical Introduction 1 15 NUNNA 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 or from outside the U S and Canada dial 1 858 712 0317 2 Follow the voice instructions to order documents for delivery by fax Note There is a limit of five documents per fax request To order documents by telephone In the U S Dial 1 800 345 5224 and press 1 4 To order in English dial 1 800 668 6913 and press 1 then 2 then 1 To order in French dial 1 800 668 6913 and press 2 In Canada then 2 then 1 From any See the specific region under To Contact Technical other country Support by Tele
38. or Instrument Instrument Dye Filter Set Matrix File Generation 377 DNA Sequencersa Filter Set E ABI Prism BigDye Matrix 373 DNA Sequencers Filter Set A Standards v3 0 with the BigDye Filter P N 4390421 Wheel a 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 b Includes the ABI PRISM 373 and ABI PRISM 373 with XL Upgrade instruments Instructions for For the 377 instruments refer to the product insert for instructions Generating on using the ABI Prism BigDye Matrix Standards v3 0 Matrices P N 4390421 to generate matrices For the 373 instruments contact Technical Support for instructions on using the ABI Prism BigDye Matrix Standards v3 0 P N 4390421 to generate matrices 1 8 Introduction Dye Set Primer Available in Two Places Mobility Files o analyze sequencing data generated with BigDye chemistries v3 0 you need dye set primer mobility files that were created for v3 0 chemistries The dye set primer mobility files can be obtained from two places The files can be installed from the two CD ROMs or one floppy disk enclosed in the ABI PRISM BigDye Matrix Standards v3 0 P N 4390421 The files can be downloaded from the Internet Installing Files from the CD ROMs or Floppy Disk Enclosed in the v3 0 Matrix Standards Refer to the CD ROM or floppy disk labeled PN 4326478 For and you wis
39. phone or Fax Outside North America To view download or order documents through the Applied Biosystems web site Step Action 1 Go to http www appliedbiosystems com 2 Click SERVICES amp SUPPORT at the top of the page click Documents on Demand then click MSDS 3 Click MSDS Index search through the list for the chemical of interest to you then click on the MSDS document number for that chemical to open a pdf of the MSDS For chemicals not manufactured or distributed by Applied Biosystems call the chemical manufacturer 1 16 Introduction Preparing the Templates Chapter Summary In This Chapter The following topics are covered in this chapter Topic See Page Control DNA Templates 2 2 Template Preparation Methods 2 3 Use of the Primer Island Transposition Kit 2 4 DNA Quantity 2 5 Preparing the Templates 2 1 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 We recommend 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 se
40. quencing kits include a 21 M13 forward primer for use to perform control reactions The partial sequence of pGEM 3Zf from the 21 M13 forward primer followed by the ensuing 1000 bases is shown in Appendix B Control DNA Sequence The BigDye terminator v3 0 sequencing standard provides an additional control to help in troubleshooting electrophoresis runs It contains lyophilized sequencing reactions that require only resuspension and denaturation before use Refer to the product insert for instructions on using the sequencing standard Instrument Kit Part Number ABI Prism 377 DNA ABI Prism BigDye Terminator 4390303 Sequencers v3 0 Sequencing Standard ABI Prism 373 DNA Sequencers with the BigDye Filter Wheelb a 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 b Includes the ABI PRISM 373 and ABI PRISM 373 with XL Upgrade instruments 2 2 Preparing the Templates Template Preparation Methods Single and Double Stranded Templates PCR Templates Importance of Purifying Product Purifying PCR Fragments Refer to Automated DNA Sequencing Chemistry Guide P N 4305080 for information on preparing single and double stranded templates Cycle sequencing provides the most reproducible results for sequencing PCR templates Although PCR fragments can be difficult to denature with traditional s
41. r moderate injury It may also be used to alert against unsafe practices PNG Indicates a potentially hazardous situation which if not avoided could result in death or serious injury PN icis Indicates an imminently hazardous situation which if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations VN he CHEMICAL HAZARD Some of the chemicals used with Applied Biosystems instruments and protocols are potentially hazardous and can cause injury illness or death Y 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 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 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 and disposal Chemical Waste Hazard Warning Site Preparation and Safety Guide
42. resis To resuspend and load the samples continued Step Action 5 Load each sample into a separate lane of the gel as follows Volume pL Volume pL Template 18 or 36 well 48 64 or 96 well PCR product 0 75 2 0 0 5 1 5 plasmid M13 Note If a weak signal is obtained on the ABI Prism 377 DNA Sequencer with XL Upgrade rerun the samples using a CCD gain of 4 Refer to the ABI PRISM 377 DNA Sequencer XL Upgrade User s Manual P N 904412 for more information Sample Electrophoresis 5 5 Electrophoresis on the 373 Instrument with BigDye Filter Wheel Requirements General guidelines are provided below for running the ABI PRISM 373 DNA Sequencers with the ABI PRISM BigDye Filter Wheel installed For more detailed instructions please refer the user s manual for your 373 instrument or to user bulletin Using the ABI Prism 373 BigDye Filter Wheel P N 4304367 Gel For 48 cm well to read wtr we recommended 5 Long Ranger gel New Dye Set Primer Mobility Files Gel Formulation Dye Set Primer Mobility File 5 Long Ranger gel 48 cm wtra DT373 BDv3 v1 mob a If you are running other wtr lengths these are currently being tested Please contact Technical Support Matrix Standards IMPORTANT Instrument matrix file for the dGTP BigDye terminators v3 0 cannot be used for the BigDye terminators original BigDye terminators v2 0 dRhodamine terminators or
43. room temperature for 15 minutes to precipitate the extension products Note Precipitation times 15 minutes will result in the loss of very short extension products Precipitation times gt 24 hours will increase the precipitation of unincorporated dye terminators Purifying Extension Products 4 11 4 12 To precipitate in microcentrifuge tubes continued Step Action 5 Place the tubes in a microcentrifuge and mark their orientations Spin the tubes for 20 minutes at maximum speed IMPORTANT Proceed to the next step immediately If this is not possible then spin the tubes for 2 minutes more immediately before performing the next step Carefully aspirate the supernatants with a separate pipette tip for each sample and discard Pellets may or may not be visible IMPORTANT The supernatants must be removed completely as unincorporated dye terminators are dissolved in them The more residual supernatant left in the tubes the more unincorporated dye terminators will remain in the samples Add 250 uL of 70 ethanol to the tubes and vortex them briefly Place the tubes in the microcentrifuge in the same orientation as in step 5 and spin for 10 minutes at maximum speed Aspirate the supernatants carefully as in step 6 Dry the samples in a vacuum centrifuge for 10 15 minutes or to dryness Do not over dry Purifying Extension Products Spin Column Purification Overview
44. s water from the atmosphere gradually decreasing its concentration This can lead to inaccurate final concentrations of ethanol which can affect some protocols To precipitate in 96 well reaction plates Step Action 1 Remove the 96 well MicroAmp reaction plate from the thermal cycler Remove the caps from each tube Purifying Extension Products 4 3 To precipitate in 96 well reaction plates continued Step Action 2 Prepare the ethanol sodium acetate solution by combining the following for each sample Make enough to precipitate all samples in your experiment 3 0 uL of 3 M sodium acetate NaOAc pH 4 6 62 5 uL of non denatured 95 ethanol EtOH 14 5 uL of deionized water The final volume should be 80 uL for each sample CHEMICAL HAZARD 3M sodium acetate may cause eye skin and respiratory tract irritation Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves CHEMICAL HAZARD Ethanol is a flammable liquid and vapor It may cause eye skin and upper respiratory tract irritation Prolonged or repeated contact may dry the skin Exposure may cause central nervous system depression and liver damage Keep away from heat sparks and flame Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Add 80 pL of this ethanol sodium acetate solution to 20 uL
45. sposons about 2 4 and antibiotics 2 4 inserting 2 4 Index 2 Applied KS Biosystems 850 Lincoln Centre Drive Foster City California 94404 1128 USA P N 4390038 Rev D
46. stranded DNA Control Template 0 2 ug uL 21 M13 Control Primer forward 0 8 pmol uL Storage and Use of the Kit Y Store the kit at 15 to 25 C Avoid excessive e no more than 5 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 thawed materials should be kept 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 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
47. stry is not appropriate for use with the dGTP v3 0 chemistry The 377 and 373 instruments require the ABI PRISM BigDye Matrix Standards v3 0 P N 4390421 for instrument matrix file generation The dRhodamine Matrix Standards and Matrix Standard Set DS 01 are not compatible with dGTP BigDye terminators v3 0 BigDye terminators v3 0 or BigDye primers v3 0 4 There are new v3 0 mobility files for all existing platforms The basecallers are the same We have developed the ABI PRISM dGTP BigDye Terminator v3 0 Ready Reaction Cycle Sequencing Kit for use with difficult templates where the standard terminator kits give data with early signal loss This kit uses dGTP in the deoxynucleoside triphosphate mix instead of the dITP used in standard ABI PRISM dye terminator cycle sequencing kits The dITP is used in dye terminator kits to minimize band compressions However this substitution can lead to early signal loss in some sequence data IMPORTANT Because of compressions see page 1 6 we do not recommend using the dGTP BigDye Terminator v3 0 Kit for routine sequencing It should be used only where the standard terminator kits do not give good data Kit Format Cycle Sequencing with AmpliTaq DNA Polymerase FS The kit combines AmpliTaq9 DNA Polymerase FS the BigDye terminators v3 0 and the following required components for the sequencing reaction The following items are premixed into a single tube of Read
48. 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 14 Purifying Extension Products Sample Electrophoresis Chapter Summary In This Chapter The following topics are covered in this chapter Topic See Page Before You Begin 5 2 Electrophoresis on the ABI PRISM 377 DNA Sequencers 5 3 Electrophoresis on the 373 Instrument with BigDye Filter 5 6 Wheel Sample Electrophoresis 5 1 Before You Begin Important Dye set primer mobility file names for the dGTP BigDye Reminders terminators v3 0 are different from those for the dRhodamine terminators and BigDye terminators original and v2 0 Ifa mobility file for the wrong sequencing chemistry is used some bases may be miscalled This is due to different dye labeling for the different chemistries In addition there are differences in the mobility shifts between the dRhodamine and BigDye terminator v3 0 chemistries Use the same dye set primer mobility files for BigDye terminators v3 0 and dGTP BigDye terminators v3 0 Note See Dye Set Primer Mobility Files on page 1 9 for information on obtaining the v3 0 dye set primer mobility files 5 2 Sample Electrophoresis Electrophoresis on the ABI PRISM 377 DNA Sequencers Requirements Electrophores
49. the purchaser No license under these patents to use the PCR Process is conveyed expressly or by implication to the purchaser by the purchase of this product A license to use the PCR Process for certain research and development activities accompanies the purchase of certain reagents from licensed suppliers such as Applied Biosystems when used in conjunction with an Authorized Thermal Cycler or is available from Applied Biosystems Further information on purchasing licenses to practice the PCR Process may be obtained by contacting the Director of Licensing at Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 ABI PRISM Applied Biosystems GeneScan and MicroAmp are registered trademarks of Applied Biosystems or its subsidiaries in the U S and certain other countries and AB Design ABI PRISM and Applera are trademarks of Applied Biosystems or its subsidiaries in the U S and certain other countries AmpliTaq and GeneAmp are registered trademarks of Roche Molecular Systems Inc Centri Sep is a trademark of Princeton Separations Inc Long Ranger is a trademark of FMC Corporation Macintosh is a registered trademark of Apple Computer Inc pGEM is a registered trademark of Promega Corporation Windows NT is a registered trademark of the Microsoft Corporation All other trademarks are the sole property of their respective owners Applera Corporation is committed to providing the world s leading technology and informat
50. ubes then invert the plate a few times to mix 12 Spin the plate for 10 minutes at maximum speed see step 7 above 13 Repeat steps 8 and 9 14 Remove the plate and discard the paper towel Note Pellets may or may not be visible Vacuum drying of the samples is not necessary Purifying Extension Products 4 5 Ethanol Sodium Acetate Precipitation in Microcentrifuge Tubes Recommended Protocol Precipitating in Microcentrifuge Tubes With the dGTP BigDye terminators v3 0 the ethanol sodium acetate precipitation method in microcentrifuge tubes produces consistent signal while minimizing unincorporated dyes A final 70 ethanol wash is required Note While this method produces the cleanest signal it may cause loss of small molecular weight fragments IMPORTANT Use non denatured 95 ethanol rather than absolute 100 ethanol Absolute ethanol absorbs water from the atmosphere gradually decreasing its concentration This can lead to inaccurate final concentrations of ethanol which can affect some protocols To precipitate in microcentrifuge tubes Step Action 1 IMPORTANT If the TC1 or DNA Thermal Cycler 480 was used for thermal cycling remove the reactions from the tubes as described below To remove reactions run on the TC1 or DNA Thermal Cycler 480 Place the pipette tip into the bottom of the reaction and carefully remove the reaction from the oil Transfer as little oil as possible Oil
51. um Acetate Precipitation in Microcentrifuge Tubes 4 6 Recommended Protocol 4 6 Precipitating in Microcentrifuge Tubes 4 6 Ethanol Precipitation in 96 Well Reaction Plates 4 9 Unincorporated Terminators 0 0 serenan eee eee eee 4 9 Precipitating in 96 Well Reaction Plates o ooooo 4 9 Ethanol Precipitation in Microcentrifuge Tubes 4 11 Unincorporated Terminators 2 0 2 0 0 0 0 0 c eee eee eee 4 11 Precipitating in Microcentrifuge Tubes 4 11 Spin Column Purification 0 0 0 0 0 eee eee 4 13 OVER AO A ELE gu ES 4 13 Recommended Spin Columns 0 00 00 o 4 13 Optimizing Spin Column Purification 4 13 Performing Spin Column Purification 4 14 5 Sample Electrophoresis Chapter Summary 2 0 0 eee ccc In 5 1 In This Chapter tra n EA UE 5 1 Betore You Begin yu s yp ae eee LEM LUCERE Ge 5 2 Important Reminders 5 2 Electrophoresis on the ABI PRISM 377 DNA Sequencers 5 3 Requirements c secre Pus uqu eee ee es 5 3 Using the Lane Guide Kit decoy dae eis ete i a ee 5 4 Using Long Read Gel and Buffer Formulations 5 4 Resuspending and Loading the Samples 5 4 Electrophoresis on the 373 Instrument with BigDye Filter Wheel 5 6 Requirements Antes
52. v3 0 Use strong denaturants such as 1 M betaine refer to pages 7 32 and 7 33 of the Automated DNA Sequencing Chemistry Guide for additional methods for improving template denaturation Secondary structure dGTP BigDye terminator v3 0 Certain sequence dGTP BigDye terminator v3 0 contexts or motifs Note In these cases the reason for the stop may not be apparent The figure below shows data from a DNA clone of the HV2 region of the mitochondria D loop sequenced using the ABI Prism BigDye Terminator v3 0 Ready Reaction Cycle Sequencing Kit This clone contains a difficult to sequence region as shown in the electropherogram Figure 1 1 TGGCAGAGATGIGTTTAAGT GCI GIGGCCAGAAGCGGGGGGAG GGGGGGGGITT GGT GGAAATTTTTTGINAT NAT GI CTG NGNG GAANGC GEN 210 40 250 260 270 280 290 a _ Figure 1 1 Mitochondrial DNA sequenced with BigDye terminators v3 0 Introduction 1 5 Compressions 1 6 Introduction Figure 1 2 below shows data from the same template but sequenced using the dGTP BigDye terminators v3 0 Sequence beyond the stop region is obtained using this chemistry balon o hl Figure 1 2 Mitochondrial DNA sequenced with dGTP BigDye terminators v3 0 Band compressions in DNA sequencing data result from the formation of secondary structures in the DNA fragments that are not eliminated by the denaturing conditions of the gel The fragments do not migrate according to their size and more t
53. y Reaction Mix BigDye terminators v3 0 dATP dCTP dGTP and dUTP AmpliTaq DNA Polymerase FS rTth DNA polymerase Magnesium chloride Buffer o 9 9 The user provides the needed templates and primers This kit formulation contains the sequencing enzyme AmpliTaq DNA Polymerase FS This enzyme is a variant of Thermus aquaticus DNA polymerase that contains a point mutation in the active site This results in less discrimination against dideoxynucleotides which leads to a much more even peak intensity pattern This enzyme also has a second mutation in the amino terminal domain that virtually eliminates the 5 3 nuclease activity of AmpliTag DNA Polymerase The enzyme has been formulated with a thermally stable inorganic pyrophosphatase to eliminate problems associated with pyrophosphorolysis Cycle sequencing protocols that rely on the use of AmpliTaq DNA Polymerase FS offer the following advantages over traditional sequencing methods Less hands on operation No alkaline denaturation step required for double stranded DNA Same protocol for both single and double stranded templates Less starting template needed 9 9 More reproducible results Introduction 1 3 dGTP BigDye Terminator v3 0 Appearance on the 377 or 373 Instrument Gel Image Difficult Templates 1 4 Introduction The dye base relationships and colors of the dGTP BigDye terminators v3 0 as they appear on the gel image are shown below for the 3
54. ying Extension Products Precipitation Note For 96 well reaction plates and microcentrifuge tubes Sodium acetate NaOAc 3 M pH 4 6 Aluminum foil tape adhesive backed Method Material Supplier Ethanol Sodium Ethanol EtOH MLS Acetate non denatured 9596 Applied Biosystems P N 400320 3M Scotch Tape P N 431 or 439 a Ethanol Precipitation Note For 96 well reaction plates and microcentrifuge tubes Ethanol EtOH non denatured 9596 Aluminum foil tape adhesive backed MLS 3M Scotch Tape P N 431 or 439 a Spin Column Purification Centri Sep spin column 1 mL 32 columns 100 columns Aluminum foil tape adhesive backed Applied Biosystems P N 401763 P N 401762 3M Scotch Tape P N 431 or 439 a Contact 3M in the USA at 800 364 3577 for your local 3M representative Use of other tapes may result in leakage or contamination of the sample Introduction 1 13 Safety Documentation User Attention Words Chemical Hazard Warning 1 14 Introduction Five user attention words appear in the text of all Applied Biosystems user documentation Each word implies a particular level of observation or action as described below Note Calls attention to useful information IMPORTANT Indicates information that is necessary for proper instrument operation Me une Indicates a potentially hazardous situation which if not avoided may result in minor o

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