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DRAFTAugust27,200212:32pm,4337036A_v1.
1Title.
fmBigDyeTerminatorv1.
1CycleSequencingKitProtocolCopyright2002,AppliedBiosystems.
Allrightsreserved.
ForResearchUseOnly.
Notforuseindiagnosticprocedures.
NoticeToPurchaser:LimitedLicenseAlicenseundertheprocessclaimsofU.
S.
Patents5,332,666and5,821,058ortheirforeigncounterpartclaims,hasanup-frontfeecomponentandarunning-royaltycomponent.
ThepurchasepriceofBigDyeTerminatorv1.
1CycleSequencingKitincludeslimited,non-transferablerightsundertherunning-royaltycomponenttouseonlythisamountoftheproducttopracticetheDNAsequenceandfragmentanalysisprocessesdescribedinsaidpatentswhenthisproductisusedinconjunctionwithanAuthorizedDNAsequenceanalysisinstrumentwhoseuseiscoveredundertheup-frontfeecomponentofthesepatents.
Nootherrightsaregrantedexpressly,byimplication,orbyestoppel,orunderanyotherpatentrightsownedorlicensablebyAppliedBiosystems.
FurtherinformationrelatingtothepurchaseoflicensesforDNAsequenceandfragmentanalysisandotherapplicationsmaybeobtainedbycontactingtheDirectorofLicensingatAppliedBiosystems,850LincolnCentreDrive,FosterCity,CA94404,U.
S.
A.
NoticetoPurchaser:LimitedLicenseThepurchaseoftheBigDyeTerminatorv1.
1CycleSequencingKitincludesalimited,nontransferable,non-exclusivelicense(withouttherighttoresell,repackage,orsublicense)undertheprocessclaimsofoneormoreofU.
S.
Patents5,800,996,5,863,727,and5,945,526,andcorrespondingclaimsinforeigncounterpartpatentsandpatentapplications,tousethisproductsolelywithanAppliedBiosystemscommercialautomatedDNAsequencingmachineorotherauthorizedautomatedDNAsequencingmachinesthathavebeenauthorizedunderthesepatentsbyAppliedBiosystems.
Nolicenseisherebygrantedfortheuseofthiskitorthereagentsthereininanyotherautomatedsequencingmachine.
Suchlicenseisgrantedsolelyforresearchandotherusesthatarenotunlawful.
Nootherlicenseisgrantedexpressly,impliedly,orbyestoppel.
Forinformationconcerningtheavailabilityofadditionallicensestopracticethepatentedmethodologies,contact:DirectorofLicensing,AppliedBiosystems,850LincolnCentreDrive,FosterCity,California94404,USA.
PatentsarependingincountriesoutsidetheUnitedStates.
NoticetoPurchaserAboutLimitedLicenseThiskit(reagent)issoldpursuanttoalimitedsublicensefromAmershamInternationalplcunderoneormoreU.
S.
PatentNos.
5,498,523;4,994,372,U.
S.
PatentApplicationSerialNos.
08/324437;08/337615andcorrespondingforeignpatentsandpatentapplications.
Thepurchaseofthiskit(reagent)includesalimitednon-exclusivesublicense(withouttherighttoresell,repackageorfurthersublicense)undersuchpatentrightstousethisreagentforDNAsequencingorfragmentlengthanalysissolelywithanAppliedBiosystemscommercialautomatedsequencingmachineorotherauthorizedDNAsequencingmachinesthathavebeenauthorizedforsuchusebyAppliedBiosystems,orformanualDNAsequencing.
Nolicenseisherebygrantedforuseofthiskit,orthereagentstherein,inanyotherautomatedsequencingmachine.
Suchsublicenseisgrantedsolelyforresearchorotherusesthatarenotunlawful.
Nootherlicenseisgrantedexpressly,impliedly,orbyestoppel.
Forinformationconcerningtheavailabilityofadditionallicensetopracticethepatentedmethodologies,contact:AmershamLifeScience,Inc.
,VicePresident,RegulatoryAffairs,P.
O.
Box22400,Cleveland,Ohio44122.
PatentsarependingincountriesoutsidetheUnitedStates.
ABIPRISM,AppliedBiosystems,BigDye,GeneScan,MicroAmp,andPrimerIslandareregisteredtrademarksofAppleraCorporationoritssubsidiariesintheU.
S.
andcertainothercountries.
,andABI,Avant,CATALYST,Hi-Di,POP,POP-4,POP-5,andPOP-6aretrademarksofAppleraCorporationoritssubsidiariesintheU.
S.
andcertainothercountries.
.
CentriconisatrademarkofW.
R.
GraceandCo.
Centri-SepisatrademarkofPrincetonSeparations,Inc.
GeneAmpareregisteredtrademarksofRocheMolecularSystems,Inc.
pGEMisaregisteredtrademarkofPromegaCorporation.
Allothertrademarksarethesolepropertyoftheirrespectiveowners.
PartNumber4337036Rev.
A09/2002DRAFTAugust21,20028:36am,4337036A_v1.
1TOC.
fmBigDyeTerminatorv1.
1CycleSequencingKitiiiContentsChapter1IntroductionChapterSummary1-1InThisChapter1-1AbouttheKit1-1NewFormulation1-1FeaturesandCompatibilities1-1BigDyeTerminatorv1.
1CycleSequencingKit1-2Instruments1-3InstrumentPlatforms1-3ThermalCyclers1-3RequiredSoftware1-4Dye/FilterSetsandMatrixStandardsforthe310and377Instruments1-4DyeSetsandSpectralStandardsforthe3700,3100,and3100-AvantInstruments1-4Instructions1-5DyeSet/Primer(Mobility)Files1-6ReagentsandStorage1-6AvailableKits1-6KitReagents1-6StorageandUseoftheKits1-7MaterialsSuppliedbytheUser1-7Overview1-7MaterialsforCycleSequencing1-8MaterialsforPurifyingExtensionProducts1-9OtherEquipment1-9MaterialsforElectrophoresis1-10GeneralSafety1-11DRAFTAugust21,20028:36am,4337036A_v1.
1TOC.
fmivBigDyeTerminatorv1.
1CycleSequencingKitChapter2PreparingtheTemplatesChapterSummary2-1InThisChapter2-1ControlDNATemplates2-1UsingControlDNA2-1ControlDNASequence2-1AnAdditionalControlSoldSeparately2-1TemplatePreparationMethods2-3Single-andDouble-StrandedTemplates2-3BACDNATemplates2-3PCRTemplates2-3PurifyingPCRFragments2-4DNAQuality2-4PoorTemplateQuality2-4Contamination2-5DeterminingDNAQuality2-5DNAQuantity2-6QuantitatingDNA2-6TemplateQuantity2-6Chapter3PerformingCycleSequencingChapterSummary3-1InThisChapter3-1SequencingSingle-andDouble-StrandedDNA3-2Overview3-2PreparingtheReactionsfor96-WellReactionPlatesorMicrocentrifugeTubes3-2UsingBigDyeTerminatorSequencingBuffer3-3PreparingtheReactionsfor384-WellPlates3-4CycleSequencingontheSystem9700,9600,2700,or2400.
.
.
3-5SequencingLargeDNATemplates3-5Overview3-5ThermalCyclers3-5PreparingSequencingReactions3-6PerformingCycleSequencing3-7DRAFTAugust21,20028:36am,4337036A_v1.
1TOC.
fmBigDyeTerminatorv1.
1CycleSequencingKitvChapter4PurifyingExtensionProductsChapterSummary4-1InThisChapter4-1ChoosingaMethodofPurification4-1Purpose4-1PurificationMethods4-1Ethanol/EDTAPrecipitation4-2RecommendedProtocol4-2Precipitatingin96-WellReactionPlates4-2Precipitatingin384-WellReactionPlates4-4Ethanol/EDTA/SodiumAcetatePrecipitation4-6Precipitatingin96-WellReactionPlates4-6Precipitatingin384-WellReactionPlates4-8PlateandSpinColumnPurification4-10Overview4-10RecommendedSpinColumns4-10OptimizingSpinColumnPurification4-10PerformingSpinColumnPurification4-11PlatePurificationUsingSDS/HeatTreatment4-13Overview4-13PreparingExtensionProducts4-13Performing96-WellSpinPlatePurification4-14Recommended96-WellSpinPlates4-14Purifyingwiththe96-WellSpinPlate4-14AppendixASelectingSequencingPrimersSelectingSequencingPrimersA-1OverviewA-1OptimizingPrimerSelectionA-1AppendixBControlDNASequenceControlSequenceB-1PartialSequenceofpGEM-3Zf(B-1DRAFTAugust21,20028:36am,4337036A_v1.
1TOC.
fmviBigDyeTerminatorv1.
1CycleSequencingKitAppendixCSampleElectrophoresisSomeImportantRemindersC-1ElectrophoresisontheABIPRISM3700DNAAnalyzerC-2RequirementsC-2PerformingSampleElectrophoresisC-3ElectrophoresisontheABIPRISM3100and3100-AvantGeneticAnalyzersC-4RequirementsC-4PerformingSampleElectrophoresisC-5ElectrophoresisontheABIPRISM310GeneticAnalyzerC-6RequirementsC-6PerformingSampleElectrophoresisC-7ElectrophoresisontheABIPRISM377DNASequencersC-8RequirementsC-8UsingtheLaneGuideKitC-9UsingLong-ReadGelandBufferFormulationsC-9PerformingSampleElectrophoresisC-9AppendixDTroubleshootingAppendixEObtainingTechnicalSupportServicesandSupportE-1AppliedBiosystemsWebSiteE-1IndexDRAFTAugust27,200212:26pm,01_Introduction.
fmBigDyeTerminatorv1.
1CycleSequencingKit1-11Introduction1ChapterSummaryInThisChapterThefollowingtopicsarecoveredinthischapter:AbouttheKit.
1-1Instruments1-3RequiredSoftware.
1-4ReagentsandStorage1-6MaterialsSuppliedbytheUser1-7GeneralSafety.
1-11AbouttheKitNewFormulationTheBigDyeTerminatorv1.
1CycleSequencingKithasanewformulationthatdelivers:IncreasedrobustnessHighqualitydataImprovedsuccesswithdifficulttemplatesTheprotocolsforcyclesequencinghavebeenmodifiedtooptimizeresultsusingthenewformulation.
FeaturesandCompatibilitiesTheBigDyeTerminatorv1.
1CycleSequencingKitusesv1.
0matrixandspectralfilesanddoesnotrequirenewinstrument(matrix)filesfortheABIPRISM310GeneticAnalyzer,andABIPRISM377DNASequencersornewspectralcalibrationsfortheABIPRISM3700DNAAnalyzer,ABIPRISM3100GeneticAnalyzer,andABIPRISM3100-AvantGeneticAnalyzer.
DRAFTAugust27,200212:26pm,01_Introduction.
fmChapter1Introduction1-2BigDyeTerminatorv1.
1CycleSequencingKitAppliedBiosystemsrecommendsthatyouverifythequalityofyourcurrentmatrixbeforeproceeding.
Ifitisnecessarytogenerateanewmatrix,usetheappropriatematrixand/orsequencingstandardforyourinstrument.
–The310and377instrumentsusethe310/377BigDyeTerminatorv1.
1MatrixStandards(PN4336805)forinstrument(matrix)filegeneration.
–The3700instrumentrequiresthe3700BigDyeTerminatorv1.
1MatrixStandard(PN4336825)forspectralcalibration.
–The3100and3100-Avantinstrumentsrequirethe3100BigDyeTerminatorv1.
1MatrixStandard(PN4336824)forspectralcalibration.
ThealcoholprecipitationmethodsaredifferentfromthoserecommendedfortheoriginalandotherversionsofBigDyeterminators.
Theexistingmobilityfilescanbeusedwiththeirrespectiveplatforms.
Newmobilityfilesarenotnecessary.
BigDyeTerminatorv1.
1CycleSequencingKitTheBigDyeTerminatorv1.
1CycleSequencingKitprovidestherequiredreagentcomponentsforthesequencingreactioninareadyreaction,pre-mixedformat.
Youneedonlyprovideyourtemplateandtemplate-specificprimer.
Thesereagentsaresuitableforperformingfluorescence-basedcyclesequencingreactionsonsingle-strandedordouble-strandedDNAtemplates,onpolymerasechainreaction(PCR)fragments,andonlargetemplates(forexample,BACclones).
Note:ThiskitincludesBigDyeTerminatorv1.
1/3.
1SequencingBuffer(5X),whichhasbeenspecificallyoptimizedforusewiththenewBigDyereadyreactionmixes.
Thisbuffershouldbeusedforanyreactionoptimizationyouundertake.
DRAFTAugust27,200212:26pm,01_Introduction.
fmInstrumentsBigDyeTerminatorv1.
1CycleSequencingKit1-3InstrumentsInstrumentPlatformsTheBigDyeTerminatorv1.
1CycleSequencingKitisforusewiththefollowinginstruments:ABIPRISM3700DNAAnalyzerABIPRISM3100GeneticAnalyzerABIPRISM3100-AvantGeneticAnalyzerABIPRISM310GeneticAnalyzerABIPRISM377DNASequencer(allmodels*)Generalinstructionsaregivenforusingthekitreagentstogeneratesamplesfortheseinstruments.
Formoredetailedinstructions,refertotheappropriateinstrumentuser'smanualorchemistryguide.
ThermalCyclersTheprotocolsprovidedinthisdocumentwereoptimizedusingAppliedBiosystemsthermalcyclers,includingtheGeneAmpPCRSystems9700,9600,2700,and2400.
Note:IfyouuseathermalcyclernotmanufacturedbyAppliedBiosystems,youmayneedtooptimizethermalcyclingconditions.
Rampingtimeisveryimportant.
Ifthethermalrampingtimeistoofast(>1°/sec),poor(noisy)datamayresult.
*IncludestheABIPRISM377,ABIPRISM377-18,ABIPRISM377withXLUpgrade,andtheABIPRISM377with96-LaneUpgradeinstruments.
DRAFTAugust27,200212:26pm,01_Introduction.
fmChapter1Introduction1-4BigDyeTerminatorv1.
1CycleSequencingKitRequiredSoftwareDye/FilterSetsandMatrixStandardsforthe310and377InstrumentsThedye/filtersetsandmatrixstandardsrequiredforthe310and377instrumentsarelistedinthetablebelow.
DyeSetsandSpectralStandardsforthe3700,3100,and3100-AvantInstrumentsIMPORTANT!
SpectralcalibrationsfortheBigDyeterminatorsv1.
1arenotcompatiblewiththev3.
0orv3.
1terminators.
IMPORTANT!
UsersoftheABIPrism3700DNAAnalyzerrefertotheABIPrism3700DNAAnalyzerUser'sManual(PN4306152)forinformationoninstrument(matrix)files.
InstrumentDye/FilterSetStandardsforInstrument(Matrix)FileGeneration310GeneticAnalyzerFilterSetE310/377BigDyeTerminatorv1.
1MatrixStandards(PN4336805)**TheBigDyeTerminatorv1.
1CycleSequencingKitdoesnotrequireanewspectralcalibrationfileifyoucurrentlyhaveaBigDyeTerminatorv1.
0spectralfileonyourinstrument.
377DNASequencerIncludestheABIPRISM377,ABIPRISM377-18,ABIPRISM377withXLUpgrade,andtheABIPRISM377with96-LaneUpgradeinstruments.
FilterSetEInstrumentDye/FilterSetStandardsforInstrument(Matrix)FileGeneration3700DNAAnalyzerFilterSetE3700BigDyeTerminatorv1.
1MatrixStandard(PN4336825)**TheBigDyeTerminatorv1.
1CycleSequencingKitdoesnotrequireanewspectralcalibrationfileifyoucurrentlyhaveaBigDyeTerminatorv1.
0spectralfileonyourinstrument.
3100GeneticAnalyzerFilterSetE3100BigDyeTerminatorv1.
1MatrixStandard(PN4336824)*3100-AvantGeneticAnalyzerDRAFTAugust27,200212:26pm,01_Introduction.
fmRequiredSoftwareBigDyeTerminatorv1.
1CycleSequencingKit1-5InstructionsForGeneratingMatricesForthe377and310instruments,refertotheproductinsert(includedwithmatrixorsequencestandards)forinstructionsonusingtheBigDyeMatrixStandardsv1.
1togeneratematrices.
ForPerformingSpectralCalibrationsForthe3700instrument,refertotheproductinsertforinstructionsonusingthe3700/3730BigDyeTerminatorv1.
1MatrixStandardtoperformspectralcalibration.
Forthe3100and3100-Avantinstruments,refertotheproductinsertforinstructionsonusingthe3100BigDyeTerminatorv1.
1MatrixStandardtoperformspectralcalibration.
DRAFTAugust27,200212:26pm,01_Introduction.
fmChapter1Introduction1-6BigDyeTerminatorv1.
1CycleSequencingKitDyeSet/Primer(Mobility)FilesToanalyzesequencingdatageneratedwithBigDyechemistriesv1.
1,youneeddyeset/primer(mobility)filesthatwerecreatedforv1.
0chemistries.
Thedyeset/primer(mobility)filescanbedownloadedfromtheInternet.
Dyeset/primer(mobility)filescanbedownloadedfromtheAppliedBiosystemswebsite:http://www.
appliedbiosystems.
com/support/softwareIfyoudonothaveaccesstotheInternet,contactAppliedBiosystemsTechnicalSupport,oryourlocalfieldapplicationsspecialist(callyourlocalsalesofficeformoreinformation).
ReagentsandStorageAvailableKitsThefollowingkitsareavailable:TheBigDyeTerminatorv1.
1CycleSequencingKitProtocol(PN4337036)isavailableseparatelyandcanbeorderedatnocharge.
KitReagentsAlistingofthekitcomponentsisgivenbelow.
ReadyReactionMixpGEM-3Zf(+)double-strandedDNAControlTemplate–21M13ControlPrimer(forward)BigDyeTerminatorv1.
1/3.
1SequencingBuffer(5X)KitNumberofReactionsPartNumberBigDyeTerminatorv1.
1CycleSequencingKit1004337450100043374515000433745225,0004337453DRAFTAugust27,200212:26pm,01_Introduction.
fmMaterialsSuppliedbytheUserBigDyeTerminatorv1.
1CycleSequencingKit1-7StorageandUseoftheKitsStorethekitat–15to–25°C.
Note:TheBigDyesequencingbuffercanbestoredat4°C.
Avoidexcess(thatis,nomorethan5to10)freeze-thawcycles.
Aliquotreagentsinsmalleramountsifnecessary.
Beforeeachuseofthekit,allowthefrozenstockstothawatroomtemperature(donotheat).
IMPORTANT!
Mixeachstockthoroughlyandthencentrifugebrieflytocollectalltheliquidatthebottomofeachtube.
Wheneverpossible,keepthawedmaterialsoniceduringuse.
Donotleavereagentsatroomtemperatureforextendedperiods.
MaterialsSuppliedbytheUserOverviewInadditiontothereagentssuppliedinthiskit,otheritemsarerequired.
Thissectionlistsgeneralmaterialsneededfor:CyclesequencingPurifyingextensionproductsNote:Manyoftheitemslistedinthissectionareavailablefrommajorlaboratorysuppliers(MLS)unlessotherwisenoted.
Equivalentsourcesmaybeacceptablewherenoted.
Refertotheindividualinstrumentprotocolsforthespecificitemsneededforeachinstrument.
CHEMICALHAZARD.
Beforehandlingthechemicalreagentsneededforcyclesequencing,readthesafetywarningsonthereagentbottlesandinthemanufacturers'MaterialSafetyDataSheets(MSDSs),andfollowthehandlinginstructions.
Wearappropriateprotectiveeyewear,clothing,andgloves.
Disposeofwasteinaccordancewithalllocal,state/provincial,andnationalenvironmentalandhealthregulations.
DRAFTAugust27,200212:26pm,01_Introduction.
fmChapter1Introduction1-8BigDyeTerminatorv1.
1CycleSequencingKitMaterialsforCycleSequencingThetablebelowliststheplatesortubesrequiredfortherecommendedAppliedBiosystemsthermalcyclers.
ThermalCyclerPlateorTubeAppliedBiosystemsPartNumberGeneAmpPCRSystem9700MicroAmp384-WellReactionPlate4305505MicroAmp96-WellReactionPlateN801-0560MicroAmpReactionTubes,0.
2-LN801-0533MicroAmpCaps,12or8/stripN801-0534orN801-0535ABIPRISMOpticalAdhesiveCoverStarterPackorABIPRISMOpticalAdhesiveCovers4313663or4311971GeneAmpPCRSystem9600MicroAmp96-WellReactionPlateN801-0560MicroAmpReactionTubes,0.
2-LN801-0533MicroAmpCaps,12or8/stripN801-0534N801-0535ABIPRISMOpticalAdhesiveCoverStarterPackorABIPRISMOpticalAdhesiveCovers4313663or4311971GeneAmpPCRSystem2400and2700MicroAmpReactionTubes,0.
2-mLN801-0533MicroAmpCaps,12or8/stripN801-0534N801-0535DRAFTAugust27,200212:26pm,01_Introduction.
fmMaterialsSuppliedbytheUserBigDyeTerminatorv1.
1CycleSequencingKit1-9MaterialsforPurifyingExtensionProductsOtherEquipmentYouwillalsoneedavariablespeedcentrifugewithmicrotiterplateholderscapableofreachaspinspeedofatleast1400*g.
AppliedBiosystemsrecommendsaBeckmanAllegra6AcentrifugewithaGH-3.
8Arotor.
MethodMaterialSupplierEthanol/EDTAPrecipitationEthanol(EtOH),200proof,MolecularBiologygradeEDTA,125mMSealingtapedMLSMLSCostar6570ThermowellSealingTapeEthanol/SodiumAcetatePrecipitationEthanol(EtOH),200proof,MolecularBiologygradeSodiumacetate(NaOAc),3M,pH5.
2SealingtapeMLSAppliedBiosystems(PN400320)Costar6570ThermowellSealingTapePlateColumnPurificationNote:For96-wellreactionplates96-WellcolumnsforpurificationSealingtapeSee"Recommended96-WellSpinPlates"onpage4-14Costar6570ThermowellSealingTapeSpinColumnPurificationCentri-Sepspincolumn,1-mL,32columns,100columnsSealingtapeAppliedBiosystemsPN401763,PN401762Costar6570ThermowellSealingTapeDRAFTAugust27,200212:26pm,01_Introduction.
fmChapter1Introduction1-10BigDyeTerminatorv1.
1CycleSequencingKitMaterialsforElectrophoresisInstrumentMaterialSupplierABIPRISM3700DNAAnalyzerHi-DiFormamide,25-mLbottleAppliedBiosystems(PN4311320)3700BigDyeTerminatorv1.
1MatrixStandardAppliedBiosystems(PN4336825)3700/3730BigDyeTerminatorv1.
1SequencingStandardAppliedBiosystems(PN4336799)ABIPRISM3100and3100-AvantGeneticAnalyzersHi-DiFormamide,25-mLbottleAppliedBiosystems(PN4311320)3100BigDyeTerminatorv1.
1MatrixStandardAppliedBiosystems(PN4336824)BigDyeTerminatorv1.
1SequencingStandardAppliedBiosystems(PN4336791)ABIPRISM310GeneticAnalyzerTemplateSuppressionReagent(TSR)AppliedBiosystems(PN401674)EDTAMLS310/377BigDyeTerminatorv1.
1MatrixStandardsAppliedBiosystems(PN4336805)ABIPRISM377DNASequencerFormamideMLSEDTAMLS25mMEDTAwith50mg/mLbluedextranAppliedBiosystems(PN402055)310/377BigDyeTerminatorv1.
1MatrixStandardsAppliedBiosystems(PN4336805)DRAFTAugust27,200212:26pm,01_Introduction.
fmGeneralSafetyBigDyeTerminatorv1.
1CycleSequencingKit1-11GeneralSafetyDocumentationUserAttentionWordsFiveuserattentionwordsappearinthetextofallAppliedBiosystemsuserdocumentation.
Eachwordimpliesaparticularlevelofobservationoractionasdescribedbelow.
Note:Callsattentiontousefulinformation.
IMPORTANT!
Indicatesinformationthatisnecessaryforproperinstrumentoperation,accuratechemistrykituse,orsafeuseofachemical.
Indicatesapotentiallyhazardoussituationwhich,ifnotavoided,mayresultinminorormoderateinjury.
Itmayalsobeusedtoalertagainstunsafepractices.
Indicatesapotentiallyhazardoussituationwhich,ifnotavoided,couldresultindeathorseriousinjury.
Indicatesanimminentlyhazardoussituationwhich,ifnotavoided,willresultindeathorseriousinjury.
Thissignalwordistobelimitedtothemostextremesituations.
SitePreparationandSafetyGuideAsitepreparationandsafetyguideisaseparatedocumentsenttoallcustomerswhohavepurchasedanAppliedBiosystemsinstrument.
Refertotheguidewrittenforyourinstrumentforinformationonsitepreparation,instrumentsafety,chemicalsafety,andwasteprofiles.
ChemicalSafetyChemicalHazardWarningCHEMICALHAZARD.
SomeofthechemicalsusedwithAppliedBiosystemsinstrumentsandprotocolsarepotentiallyhazardousandcancauseinjury,illness,ordeath.
Readandunderstandthematerialsafetydatasheets(MSDSs)providedbythechemicalmanufacturerbeforeyoustore,handle,orworkwithanychemicalsorhazardousmaterials.
Minimizecontactwithchemicals.
Wearappropriatepersonalprotectiveequipmentwhenhandlingchemicals(e.
g.
,safetyglasses,gloves,orprotectiveclothing).
Foradditionalsafetyguidelines,consulttheMSDS.
DRAFTAugust27,200212:26pm,01_Introduction.
fmChapter1Introduction1-12BigDyeTerminatorv1.
1CycleSequencingKitMinimizetheinhalationofchemicals.
Donotleavechemicalcontainersopen.
Useonlywithadequateventilation(e.
g.
,fumehood).
Foradditionalsafetyguidelines,consulttheMSDS.
Checkregularlyforchemicalleaksorspills.
Ifaleakorspilloccurs,followthemanufacturer'scleanupproceduresasrecommendedontheMSDS.
Complywithalllocal,state/provincial,ornationallawsandregulationsrelatedtochemicalstorage,handling,anddisposal.
ChemicalWasteHazardWarningCHEMICALWASTEHAZARD.
WastesproducedbyAppliedBiosystemsinstrumentsarepotentiallyhazardousandcancauseinjury,illness,ordeath.
Readandunderstandthematerialsafetydatasheets(MSDSs)providedbythemanufacturersofthechemicalsinthewastecontainerbeforeyoustore,handle,ordisposeofchemicalwaste.
Handlechemicalwastesinafumehood.
Minimizecontactwithchemicals.
Wearappropriatepersonalprotectiveequipmentwhenhandlingchemicals(e.
g.
,safetyglasses,gloves,orprotectiveclothing).
Foradditionalsafetyguidelines,consulttheMSDS.
Minimizetheinhalationofchemicals.
Donotleavechemicalcontainersopen.
Useonlywithadequateventilation(e.
g.
,fumehood).
Foradditionalsafetyguidelines,consulttheMSDS.
Afteremptyingthewastecontainer,sealitwiththecapprovided.
Disposeofthecontentsofthewastetrayandwastebottleinaccordancewithgoodlaboratorypracticesandlocal,state/provincial,ornationalenvironmentalandhealthregulations.
AboutMSDSsSomeofthechemicalsusedwiththisinstrumentmaybelistedashazardousbytheirmanufacturer.
Whenhazardsexist,warningsareprominentlydisplayedonthelabelsofallchemicals.
Chemicalmanufacturerssupplyacurrentmaterialsafetydatasheet(MSDS)beforeorwithshipmentsofhazardouschemicalstonewcustomersandwiththefirstshipmentofahazardouschemicalafteranMSDSupdate.
MSDSsprovideyouwiththesafetyinformationyouneedtostore,handle,transportanddisposeofthechemicalssafely.
DRAFTAugust27,200212:26pm,01_Introduction.
fmChemicalSafetyBigDyeTerminatorv1.
1CycleSequencingKit1-13WestronglyrecommendthatyoureplacetheappropriateMSDSinyourfileseachtimeyoureceiveanewMSDSpackagedwithahazardouschemical.
CHEMICALHAZARD.
BesuretofamiliarizeyourselfwiththeMSDSsbeforeusingreagentsorsolvents.
OrderingMSDSsYoucanorderfreeadditionalcopiesofMSDSsforchemicalsmanufacturedordistributedbyAppliedBiosystemsusingthecontactinformationbelow.
Toorderdocumentsbyautomatedtelephoneservice:ToobtaindocumentsthroughtheAppliedBiosystemsWebsite:ForchemicalsnotmanufacturedordistributedbyAppliedBiosystems,callthechemicalmanufacturer.
1.
FromtheU.
S.
orCanada,dial1.
800.
487.
6809.
2.
Followthevoiceinstructionstoorderdocuments(fordeliverybyfax).
Note:Thereisalimitoffivedocumentsperfaxrequest.
1.
Gotohttp://docs.
appliedbiosystems.
com/msdssearch.
html2.
IntheSEARCHfield,typeinthechemicalname,partnumber,orotherinformationthatwillappearintheMSDSandclickSEARCH.
Note:Youmayalsoselectthelanguageofyourchoicefromthedrop-downlist.
3.
WhentheSearchResultspageopens,findthedocumentyouwantandclickonittoopenaPDFofthedocument.
DRAFTAugust27,200212:26pm,01_Introduction.
fmChapter1Introduction1-14BigDyeTerminatorv1.
1CycleSequencingKitDRAFTAugust27,200210:06am,02_TemplatePrep.
fmBigDyeTerminatorv1.
1CycleSequencingKit2-12PreparingtheTemplates2ChapterSummaryInThisChapterThefollowingtopicsarecoveredinthischapter:ControlDNATemplates2-1TemplatePreparationMethods2-3DNAQuality2-4DNAQuantity2-6ControlDNATemplatesUsingControlDNAIncludeacontrolDNAtemplateasoneofthetemplatesinasetofsequencingreactions.
Theresultsfromthecontrolcanhelpdeterminewhetherfailedreactionsaretheresultofpoortemplatequalityorsequencingreactionfailure.
ControlDNASequenceAppliedBiosystemsrecommendsM13mp18asasingle-strandedcontrolandpGEM-3Zf(+)asadouble-strandedcontrol.
AllAppliedBiosystemsDNAsequencingkitsprovidepGEMcontrolDNA.
Alldyeterminatorcyclesequencingkitsincludea–21M13forwardprimerforuseinperformingcontrolreactions.
ThepartialsequenceofpGEM-3Zf(+)fromthe–21M13forwardprimer,followedbytheensuing1000basesisshowninAppendixA,"ControlDNASequence.
"AnAdditionalControlSoldSeparatelyTheBigDyeTerminatorv1.
1SequencingStandardprovidesanadditionalcontroltohelpintroubleshootingelectrophoresisruns.
Thisstandardcontainslyophilizedsequencingreactionsthatrequireonlyresuspensionanddenaturationbeforeuse.
DRAFTAugust27,200210:06am,02_TemplatePrep.
fmChapter2PreparingtheTemplates2-2BigDyeTerminatorv1.
1CycleSequencingKitTherearetwoformsofthev1.
0andv1.
1sequencingstandard,asshowninthetablebelow.
Pleaseusethecorrectsequencingstandardforyourinstrument.
Refertotheproductinsertsforinstructionsonusingeachsequencingstandard.
InstrumentKitPNABIPRISM3700DNAAnalyzer3700/3730BigDyeTerminatorv1.
1SequencingStandard4336799ABIPRISM3100GeneticAnalyzerBigDyeTerminatorv1.
1SequencingStandard4336791ABIPRISM3100-AvantGeneticAnalyzerABIPRISM310GeneticAnalyzerABIPRISM377DNASequencers**IncludestheABIPRISM377,ABIPRISM377-18,ABIPRISM377withXLUpgrade,andtheABIPRISM377with96-LaneUpgradeinstruments.
DRAFTAugust27,200210:06am,02_TemplatePrep.
fmTemplatePreparationMethodsBigDyeTerminatorv1.
1CycleSequencingKit2-3TemplatePreparationMethodsSingle-andDouble-StrandedTemplatesRefertotheAutomatedDNASequencingChemistryGuide(PN4305080)forinformationonpreparingsingle-anddouble-strandedtemplates.
BACDNATemplatesWithlargerDNAtargetssuchasbacterialartificialchromosomes(BACs),thequalityofDNAtemplateisimportanttothesuccessofthesequencingreaction.
Twomethodshavegivengoodsequencingresults:Alkalinelysis,*withextraphenolextractionandisopropanolprecipitationifverycleanDNAisdesiredCesiumchloride(CsCl)bandingCommercialKitsCommercialkitsarealsoavailableforBACDNApreparation:QIAGEN-tip100(QIAGEN:PN10043,25reactions;10045,100reactions)QIAGEN-tip500(QIAGEN:PN10063,25reactions;10065,100reactions)PCRTemplatesCyclesequencingprovidesthemostreproducibleresultsforsequencingPCRtemplates.
AlthoughPCRfragmentscanbedifficulttodenaturewithtraditionalsequencingmethods,cyclesequencingprovidesseveralchancestodenatureandextendthetemplate,ensuringadequatesignalinthesequencingreaction.
ImportanceofPurifyingProductForoptimumresults,purifythePCRproductbeforesequencing.
Ingeneral,anymethodthatremovesdNTPsandprimersshouldwork.
WerecommendCentricon-100columns(PNN930-2119).
Theprotocolforusingthesecolumnsisprovidedin"PurifyingPCRFragments"onpage2-4RefertotheAutomatedDNASequencingChemistryGuideforinformationonsequencingPCRtemplates.
*Marra,M.
,Weinstock,L.
A.
,andMardis,E.
R.
1966.
Endsequencedeterminationfromlargeinsertcloningusingenergytransferfluorescentprimers.
GenomicMethods6:1118-1122.
DRAFTAugust27,200210:06am,02_TemplatePrep.
fmChapter2PreparingtheTemplates2-4BigDyeTerminatorv1.
1CycleSequencingKitPurifyingPCRFragmentsDNAQualityPoorTemplateQualityPoortemplatequalityisthemostcommoncauseofsequencingproblems.
Thefollowingarecharacteristicsofpoorqualitytemplates:NoisydataorpeaksunderpeaksNousablesequencedataWeaksignalAlwaysfollowrecommendedprocedurestopreparetemplates.
TopurifyPCRfragmentsbyultrafiltration:1.
AssembletheCentricon-100columnaccordingtothemanufacturer'srecommendations.
2.
Load2mLdeionizedwaterontothecolumn.
3.
Addtheentiresampletothecolumn.
4.
Spinthecolumnat3000*ginafixed-anglecentrifugefor10minutes.
Note:Themanufacturerrecommendsamaximumspeedof1000*g,but3000*ghasworkedwellinAppliedBiosystemslaboratories.
Ifyouarefollowingthemanufacturer'sguidelines,increasethetimetocompensate.
5.
Removethewastereceptacleandattachthecollectionvial.
6.
Invertthecolumnandspinitat270*gfor2minutestocollectapproximately40–60Lofsample.
7.
AdddeionizedwatertobringthepurifiedPCRfragmentstotheoriginalvolume.
DRAFTAugust27,200210:06am,02_TemplatePrep.
fmDNAQualityBigDyeTerminatorv1.
1CycleSequencingKit2-5ContaminationPotentialcontaminantsinclude:ProteinsRNAChromosomalDNAExcessPCRprimers,dNTPs,enzyme,andbuffercomponents(fromaPCRamplificationusedtogeneratethesequencingtemplate)ResidualsaltsResidualorganicchemicalssuchasphenol,chloroform,andethanolResidualdetergentsDeterminingDNAQualityThefollowingmethodscanbeusedtoexamineDNAquality:AgarosegelelectrophoresisPurifiedDNAshouldrunasasinglebandonanagarosegel.
Note:UncutplasmidDNAcanrunasthreebands:supercoiled,nicked,andlinear.
SpectrophotometryTheA260/A280ratioshouldbe1.
7to1.
9.
Smallerratiosusuallyindicatecontaminationbyproteinororganicchemicals.
AgarosegelsrevealthepresenceofcontaminatingDNAsandRNAs,butnotproteins.
Spectrophotometrycanindicatethepresenceofproteincontamination,butnotDNAandRNAcontamination.
UsethesemethodstogethertogetthemostinformationaboutyourDNAbeforesequencing.
DRAFTAugust27,200210:06am,02_TemplatePrep.
fmChapter2PreparingtheTemplates2-6BigDyeTerminatorv1.
1CycleSequencingKitDNAQuantityQuantitatingDNAIfpossible,quantitatetheamountofpurifiedDNAbymeasuringtheabsorbanceat260nmorbysomeothermethod.
TemplateQuantityThetablebelowshowstheamountoftemplatetouseinacyclesequencingreaction.
Note:Ingeneral,higherDNAquantitiesgivehighersignalintensities.
HigherDNAquantitiesmayalsogiveshorterreadlengthsandtop-heavydata.
Thetemplatequantitiesgivenaboveshouldworkwithallprimers.
YoumaybeabletouseevenlessDNAwhenusingcapillaryinstrumentsfordetection.
TheamountofPCRproducttouseinsequencingalsodependsonthelengthandpurityofthePCRproduct.
TemplateQuantityPCRproduct:100–200bp200–500bp500–1000bp1000–2000bp>2000bp1–3ng3–10ng5–20ng10–40ng20–50ngSingle-stranded25–50ngDouble-stranded150–300ngCosmid,BAC0.
5–1.
0gBacterialgenomicDNA2–3gDRAFTAugust27,20029:59am,03_CycleSequencing.
fmBigDyeTerminatorv1.
1CycleSequencingKit3-13PerformingCycleSequencing3ChapterSummaryInThisChapterThefollowingtopicsarecoveredinthischapter:SequencingSingle-andDouble-StrandedDNA3-2SequencingLargeDNATemplates3-5DRAFTAugust27,20029:59am,03_CycleSequencing.
fmChapter3PerformingCycleSequencing3-2BigDyeTerminatorv1.
1CycleSequencingKitSequencingSingle-andDouble-StrandedDNAOverviewThissectiondescribeshowtopreparereactionsandperformcyclesequencingonavarietyoftemplates,includingM13,plasmids,andPCRproducts.
PreparingtheReactionsfor96-WellReactionPlatesorMicrocentrifugeTubesThetypeoftuberequireddependsonthethermalcyclerthatyouareusing.
Referto"MaterialsforCycleSequencing"onpage1-8.
Topreparethereactionmixtures:1.
Foreachreactionaddthefollowingreagentstoaseparatetube:ReagentQuantityTerminatorReadyReactionMix**See"UsingBigDyeTerminatorSequencingBuffer"below.
8.
0LTemplateSeethetablein"TemplateQuantity"onpage2-6.
Primer3.
2pmolDeionizedwaterq.
s.
TotalVolume20L2.
Mixwellandspinbriefly.
DRAFTAugust27,20029:59am,03_CycleSequencing.
fmSequencingSingle-andDouble-StrandedDNABigDyeTerminatorv1.
1CycleSequencingKit3-3UsingBigDyeTerminatorSequencingBufferTheBigDyeTerminatorv1.
1/3.
1SequencingBuffer(5X)*issuppliedata5Xconcentration.
Ifyouuseitforsequencingreactions,besurethefinalreactionvolumeisataconcentrationof1X.
Forexample,forahalfreactionin20Lfinalvolume,youwoulduse4Lofreadyreactionpremixand2LofBigDyesequencingbufferasshownbelow.
Note:Theuseofthisbufferwithoutoptimizationmayresultindeteriorationofsequencequality.
AppliedBiosystemsdoesnotsupportdilutedreactionsorguaranteetheperformanceofBigDyechemistrywhenitisdiluted.
*TheBigDyeTerminatorv1.
1/3.
1SequencingBufferisintendedforuseonlywithBigDyeTerminatorv1.
1/3.
lCycleSequencingKits.
ReagentConcentrationVolumeReadyReactionPremix2.
5X4LBigDyeSequencingBuffer5X2LPrimer—3.
2pmolTemplate—See"TemplateQuantity"onpage2-6.
Water—to20LFinalVolume1X20LDRAFTAugust27,20029:59am,03_CycleSequencing.
fmChapter3PerformingCycleSequencing3-4BigDyeTerminatorv1.
1CycleSequencingKitPreparingtheReactionsfor384-WellPlatesThetypeoftuberequireddependsonthethermalcyclerthatyouareusing.
Referto"MaterialsforCycleSequencing"onpage1-8.
Note:Thewellsina384-wellreactionplatehaveavolumecapacityof35L.
Therefore,AppliedBiosystemsrecommendsdoinga10Lreaction.
Thisallowsthepost-reactioncleanupsteptobeperformedinthesamewell.
Topreparethereactionmixtures:1.
Foreachreactionaddthefollowingreagentstoaseparatetube:ReagentQuantityTerminatorReadyReactionMix**Note:ForinstructionsonusingBigDyesequencingbuffer,see"UsingBigDyeTerminatorSequencingBuffer"onpage3-3.
4.
0LTemplateSeethetablein"TemplateQuantity"onpage2-6.
Primer3.
2pmolDeionizedwaterq.
s.
TotalVolume10L2.
Mixwellandspinbriefly.
3.
UseonaGeneAmpPCRSystem9700Dual384-WellSampleBlockModule.
DRAFTAugust27,20029:59am,03_CycleSequencing.
fmSequencingLargeDNATemplatesBigDyeTerminatorv1.
1CycleSequencingKit3-5CycleSequencingontheSystem9700,9600,2700,or2400SequencingLargeDNATemplatesOverviewThissectiondescribeshowtopreparereactionsandperformcyclesequencingonlargeDNAtemplatessuchas:BACDNACosmidDNAGenomicDNAThermalCyclersOnlythefollowingthermalcyclerscanbeusedwiththisprotocol:GeneAmpPCRSystem9600GeneAmpPCRSystem9700(in9600emulationmode)Tosequencesingle-anddouble-strandedDNAontheGeneAmpPCRSystem9700(in9600emulationmode),9600,2700,or2400:1.
Placethetubesinathermalcyclerandsettothecorrectvolume.
2.
Performaninitialdenaturation.
a.
Rapidthermalrampto96°Cb.
96°Cfor1min3.
Repeatthefollowingfor25cycles:Rapidthermalramp*to96°C96°Cfor10seconds.
Rapidthermalrampto50°C50°Cfor5seconds.
Rapidthermalrampto60°C60°Cfor4minutes*Rapidthermalrampis1°C/second.
4.
Rapidthermalrampto4°Candholduntilreadytopurify.
5.
Spindownthecontentsofthetubesinamicrocentrifuge.
6.
ProceedtoChapter4,"PurifyingExtensionProducts.
"DRAFTAugust27,20029:59am,03_CycleSequencing.
fmChapter3PerformingCycleSequencing3-6BigDyeTerminatorv1.
1CycleSequencingKitReoptimizethisprotocolforuseonotherthermalcyclers.
PreparingSequencingReactionsThetypeoftuberequireddependsonthethermalcyclerthatyouareusing.
Referto"MaterialsSuppliedbytheUser"onpage1-7.
*Note:ForinstructionsonusingBigDyesequencingbuffer,see"UsingBigDyeTerminatorSequencingBuffer"onpage3-3.
Topreparethesequencingreaction:1.
Foreachreaction,addthefollowingreagentstoaseparatetube:2.
Mixwellandspinbriefly.
ReagentQuantityTerminatorReadyReactionMix*8.
0LDNATemplateSeethetablein"DNAQuantity"onpage2-6Primer3.
2pmolDeionizedwaterq.
s.
TotalVolume20LDRAFTAugust27,20029:59am,03_CycleSequencing.
fmSequencingLargeDNATemplatesBigDyeTerminatorv1.
1CycleSequencingKit3-7PerformingCycleSequencingToperformcyclesequencingonBACDNA:1.
Placethetubesinathermalcyclerandsetthevolumeto20L.
2.
Heatthetubesat95°Cfor5minutes.
3.
Repeatthefollowingfor30cycles:*Rapidthermalrampto95°C95°Cfor30seconds.
Rapidthermalrampto50–55°C(dependingontemplate)50–55°Cfor10seconds.
Rapidthermalrampto60°C60°Cfor4minutes.
*Somelaboratorieshavefoundthatincreasingthenumberofcyclesgivesbetterresults.
Rapidthermalrampis1°C/sec.
4.
Rapidthermalrampto4°Candholduntilreadytopurify.
5.
Spindownthecontentsofthetubesinamicrocentrifuge.
6.
ProceedtoChapter4,"PurifyingExtensionProducts.
"DRAFTAugust27,20029:59am,03_CycleSequencing.
fmChapter3PerformingCycleSequencing3-8BigDyeTerminatorv1.
1CycleSequencingKitDRAFTAugust27,20029:57am,04_PurifyExtension.
fmBigDyeTerminatorv1.
1CycleSequencingKit4-14PurifyingExtensionProducts4ChapterSummaryInThisChapterThefollowingtopicsarecoveredinthischapter:ChoosingaMethodofPurification.
4-1Ethanol/EDTAPrecipitation4-2Ethanol/EDTA/SodiumAcetatePrecipitation.
4-6PlateandSpinColumnPurification4-10PlatePurificationUsingSDS/HeatTreatment4-13Performing96-WellSpinPlatePurification4-14ChoosingaMethodofPurificationPurposeThebestresultsareobtainedwhenunincorporateddyeterminatorsarecompletelyremovedpriortoelectrophoresis.
Excessdyeterminatorsinsequencingreactionsobscuredataintheearlypartofthesequenceandcaninterferewithbasecalling.
PurificationMethodsThemethodsrecommendedbelowhaveproducedcleansequencingdataforBigDyeTerminatorv1.
1CycleSequencingKitproducts:Ethanol/EDTAprecipitationEthanol/EDTA/sodiumacetateprecipitationPlateandSpincolumnpurificationEarliermethodsdevelopedforothersequencingkitsmaynotremoveunincorporateddyesefficiently.
Usethemethodthatworksbestforyourparticularapplication.
DRAFTAugust27,20029:57am,04_PurifyExtension.
fmChapter4PurifyingExtensionProducts4-2BigDyeTerminatorv1.
1CycleSequencingKitNote:Theseprecipitationprotocolshavebeenoptimizedforusewiththev1.
1formulationatthespecifiedsequencingreactionvolumes(20Lin96-wellformatand10Lin384-wellformat)andarenotrecommendedforotherversions.
IMPORTANT!
Tocleanupsequencingreactionsatvolumeslessthanthosespecified,reduceeachcomponentoftheprecipitationprotocolproportionately.
Ethanol/EDTAPrecipitationRecommendedProtocolWiththeBigDyeterminatorsv1.
1,theethanol/EDTAprecipitationmethodproducesconsistentsignal,whileminimizingunincorporateddyes.
Itisparticularlygoodatgettingridofunincorporateddye-labelledterminatorsthatobscuredataintheearlypartofthesequence.
Note:Whilethismethodproducesthecleanestsignal,itmaycauselossofsmallmolecularweightfragments.
IMPORTANT!
Absoluteethanolabsorbswaterfromtheatmosphere,graduallydecreasingitsconcentrationandleadingtoinaccuratefinalconcentrationsofethanol,whichcanaffectsomesequencingresults.
IMPORTANT!
95%ethanolisusable,butyoumustmakesurethefinalethanolconcentrationforprecipitationremainsthesame(67–71%).
Precipitatingin96-WellReactionPlatesCHEMICALHAZARD.
EDTA.
Exposurecauseseyeirritation.
ReadtheMSDS,andfollowthehandlinginstructions.
Wearappropriateprotectiveeyewear,clothing,andgloves.
CHEMICALHAZARD.
Ethanolisaflammableliquidandvapor.
Exposurecauseseye,skin,andrespiratorytractirritationandmaycausecentralnervoussystemdepressionandliverdamage.
ReadtheMSDS,andfollowthehandlinginstructions.
Wearappropriateprotectiveeyewear,clothing,andgloves.
DRAFTAugust27,20029:57am,04_PurifyExtension.
fmEthanol/EDTAPrecipitationBigDyeTerminatorv1.
1CycleSequencingKit4-3Toprecipitate20Lsequencingreactionsin96-wellreactionplates:1.
Removethe96-wellreactionplatefromthethermalcyclerandbrieflyspin.
2.
Add5Lof125mMEDTAtoeachwell.
Note:MakesuretheEDTAreachesthebottomofthewells.
3.
Add60Lof100%ethanoltoeachwell.
4.
Sealtheplatewithaluminumtapeandmixbyinverting4times.
5.
Incubateatroomtemperaturefor15min.
6.
IMPORTANT!
Proceedtothenextstepimmediately.
Ifthisisnotpossible,thenspintheplateforanadditional2minbeforeperformingthenextstep.
7.
Inverttheplateandspinupto185*g,thenremovefromthecentrifuge.
8.
Add60Lof70%ethanoltoeachwell.
9.
Withthecentrifugesetto4°C,spinat1650*gfor15min.
10.
Inverttheplateandspinupto185*gfor1min,thenremovefromthecentrifuge.
Note:Starttimingwhentherotorstartsmoving.
Ifyouareusing.
.
.
Then.
.
.
aBeckmanAllegra6AcentrifugewithaGH-3.
8Arotorsetitat4°Candspintheplateat1650*gfor45min.
anyothercentrifugeuseaplateadapterandspintheplateatthemaximumspeedasfollows:1400–2000*gfor45minor2000–3000*gfor30minDRAFTAugust27,20029:57am,04_PurifyExtension.
fmChapter4PurifyingExtensionProducts4-4BigDyeTerminatorv1.
1CycleSequencingKitPrecipitatingin384-WellReactionPlatesCHEMICALHAZARD.
EDTA.
Exposurecauseseyeirritation.
ReadtheMSDS,andfollowthehandlinginstructions.
Wearappropriateprotectiveeyewear,clothing,andgloves.
CHEMICALHAZARD.
Ethanolisaflammableliquidandvapor.
Exposurecauseseye,skin,andrespiratorytractirritationandmaycausecentralnervoussystemdepressionandliverdamage.
ReadtheMSDS,andfollowthehandlinginstructions.
Wearappropriateprotectiveeyewear,clothing,andgloves.
11.
Tocontinue,resuspendthesamplesininjectionbuffer.
Tostore,coverwithaluminumfoil,andstoreat4°C.
Note:Makesurethewellsaredry.
YoumayuseaSpeed-Vacfor15mintodrytheplate.
IMPORTANT!
Makesurethesamplesareprotectedfromlightwhiletheyaredrying.
Toprecipitate20Lsequencingreactionsin96-wellreactionplates:(continued)Toprecipitatein384-wellreactionplates:1.
Removethe384-wellreactionplatesfromthethermalcycler.
Removethesealfromeachplateandbrieflyspintheplates.
2.
Add2.
5Lof125mMEDTAtoeachwell.
Note:MakesuretheEDTAreachesthebottomofthewells.
3.
Add25Lof100%ethanoltoeachwell.
4.
Sealtheplateswithaluminumtapeandmixbyinverting4times.
5.
Incubateatroomtemperaturefor15min.
DRAFTAugust27,20029:57am,04_PurifyExtension.
fmEthanol/EDTAPrecipitationBigDyeTerminatorv1.
1CycleSequencingKit4-56.
IMPORTANT!
Proceedtothenextstepimmediately.
Ifthisisnotpossible,thenspintheplateforanadditional2minbeforeperformingthenextstep.
7.
Inverttheplateandspinupto185*g,thenremovefromthecentrifuge.
8.
Add30Lof70%ethanoltoeachwell.
9.
Withthecentrifugesetto4°C,spinat1650*gfor15min.
10.
Inverttheplateandspinupto185*gfor1min,thenremovefromthecentrifuge.
Note:Starttimingwhentherotorstartsmoving.
11.
Tocontinue,resuspendthesamplesininjectionbuffer.
Tostore,coverwithaluminumfoil,andstoreat4°C.
Note:Makesurethewellsaredry.
YoumayuseaSpeed-Vacfor15mintodrytheplate.
IMPORTANT!
Makesurethesamplesareprotectedfromlightwhiletheyaredrying.
Toprecipitatein384-wellreactionplates:(continued)Ifyouareusing.
.
.
Then.
.
.
aBeckmanAllegra6AcentrifugewithaGH-3.
8Arotorsetitat4°Candspintheplateat1650*gfor45min.
anyothercentrifugeuseaplateadapterandspintheplateatthemaximumspeedasfollows:1400–2000*gfor45minor2000–3000*gfor30minDRAFTAugust27,20029:57am,04_PurifyExtension.
fmChapter4PurifyingExtensionProducts4-6BigDyeTerminatorv1.
1CycleSequencingKitEthanol/EDTA/SodiumAcetatePrecipitationNote:Ethano/EDTA/sodiumacetateprecipitationisrecommendedwhengoodsignalfrombase1isrequired.
However,forreactionscontaininghighconcentrationsofunincorporatedterminators,someresidualterminatorsmaybecarriedthroughtheprecipitation.
Tocompletelyremoveexcessterminatorsinthesecases,ethanol/EDTAprecipitationisrecommended(seepage4-2).
Precipitatingin96-WellReactionPlatesCHEMICALHAZARD.
EDTA.
Exposurecauseseyeirritation.
ReadtheMSDS,andfollowthehandlinginstructions.
Wearappropriateprotectiveeyewear,clothing,andgloves.
CHEMICALHAZARD.
Ethanolisaflammableliquidandvapor.
Exposurecauseseye,skin,andrespiratorytractirritationandmaycausecentralnervoussystemdepressionandliverdamage.
ReadtheMSDS,andfollowthehandlinginstructions.
Wearappropriateprotectiveeyewear,clothing,andgloves.
Toprecipitate20Lsequencingreactionsin96-wellreactionplates:1.
Removethe96-wellreactionplatefromthethermalcyclerandbrieflyspin.
2.
Add2Lof125mMEDTAtoeachwell.
Note:MakesuretheEDTAreachesthebottomofthewells.
3.
Add2Lof3Msodiumacetatetoeachwell.
Note:Makesurethesodiumacetatereachesthebottomofthewells.
4.
Add50Lof100%ethanoltoeachwell.
5.
Sealtheplatewithaluminumtapeandmixbyinverting4times.
6.
Incubateatroomtemperaturefor15min.
DRAFTAugust27,20029:57am,04_PurifyExtension.
fmEthanol/EDTA/SodiumAcetatePrecipitationBigDyeTerminatorv1.
1CycleSequencingKit4-77.
IMPORTANT!
Proceedtothenextstepimmediately.
Ifthisisnotpossible,thenspintheplateforanadditional2minbeforeperformingthenextstep.
8.
Inverttheplateandspinupto185*g,thenremovefromthecentrifuge.
9.
Add70Lof70%ethanoltoeachwell.
10.
Withthecentrifugesetto4°C,spinat1650*gfor15min.
11.
Inverttheplateandspinupto185*gfor1min,thenremovefromthecentrifuge.
Note:Starttimingwhentherotorstartsmoving.
12.
Tocontinue,resuspendthesamplesininjectionbuffer.
Tostore,coverwithaluminumfoil,andstoreat4°C.
Note:Makesurethewellsaredry.
YoumayuseaSpeed-Vacfor15mintodrytheplate.
IMPORTANT!
Makesurethesamplesareprotectedfromlightwhiletheyaredrying.
Toprecipitate20Lsequencingreactionsin96-wellreactionplates:(continued)Ifyouareusing.
.
.
Then.
.
.
aBeckmanAllegra6AcentrifugewithaGH-3.
8Arotorsetitat4°Candspintheplateat1650*gfor45min.
anyothercentrifugeuseaplateadapterandspintheplateatthemaximumspeedasfollows:1400–2000*gfor45minor2000–3000*gfor30minDRAFTAugust27,20029:57am,04_PurifyExtension.
fmChapter4PurifyingExtensionProducts4-8BigDyeTerminatorv1.
1CycleSequencingKitPrecipitatingin384-WellReactionPlatesCHEMICALHAZARD.
EDTA.
Exposurecauseseyeirritation.
ReadtheMSDS,andfollowthehandlinginstructions.
Wearappropriateprotectiveeyewear,clothing,andgloves.
CHEMICALHAZARD.
Ethanolisaflammableliquidandvapor.
Exposurecauseseye,skin,andrespiratorytractirritationandmaycausecentralnervoussystemdepressionandliverdamage.
ReadtheMSDS,andfollowthehandlinginstructions.
Wearappropriateprotectiveeyewear,clothing,andgloves.
Toprecipitate10Lsequencingreactionsin384-wellreactionplates:1.
Removethe384-wellreactionplatefromthethermalcyclerandbrieflyspin.
2.
Add1Lof125mMEDTAtoeachwell.
Note:MakesuretheEDTAreachesthebottomofthewells.
3.
Add1Lof3Msodiumacetatetoeachwell.
Note:Makesurethesodiumacetatereachesthebottomofthewells.
4.
Add25Lof100%ethanoltoeachwell.
5.
Sealtheplatewithaluminumtapeandmixbyinverting4times.
6.
Incubateatroomtemperaturefor15min.
DRAFTAugust27,20029:57am,04_PurifyExtension.
fmEthanol/EDTA/SodiumAcetatePrecipitationBigDyeTerminatorv1.
1CycleSequencingKit4-97.
IMPORTANT!
Proceedtothenextstepimmediately.
Ifthisisnotpossible,thenspintheplateforanadditional2minbeforeperformingthenextstep.
8.
Inverttheplateandspinupto185*g,thenremovefromthecentrifuge.
9.
Add35Lof70%ethanoltoeachwell.
10.
Withthecentrifugesetto4°C,spinat1650*gfor15min.
11.
Inverttheplateandspinupto185*gfor1min,thenremovefromthecentrifuge.
Note:Starttimingwhentherotorstartsmoving.
12.
Tocontinue,resuspendthesamplesininjectionbuffer.
Tostore,coverwithaluminumfoil,andstoreat4°C.
Note:Makesurethewellsaredry.
YoumayuseaSpeed-Vacfor15mintodrytheplate.
IMPORTANT!
Makesurethesamplesareprotectedfromlightwhiletheyaredrying.
Toprecipitate10Lsequencingreactionsin384-wellreactionplates:(continued)Ifyouareusing.
.
.
Then.
.
.
aBeckmanAllegra6AcentrifugewithaGH-3.
8Arotorsetitat4°Candspintheplateat1650*gfor45min.
anyothercentrifugeuseaplateadapterandspintheplateatthemaximumspeedasfollows:1400–2000*gfor45minor2000–3000*gfor30minDRAFTAugust27,20029:57am,04_PurifyExtension.
fmChapter4PurifyingExtensionProducts4-10BigDyeTerminatorv1.
1CycleSequencingKitPlateandSpinColumnPurificationOverviewThissectiondescribestherecommendedplateandspincolumnsforpurifyingextensionproducts.
IMPORTANT!
Extracautionisrequiredwhendispensingsamplesontothecolumnbed.
Residualdyepeakswillresultifsamplesflowthroughthesidesofthecolumn.
RecommendedSpinColumnsWerecommendCentri-Sepspincolumns(AppliedBiosystems,PN401763for32columnsandPN401762for100columns).
OptimizingSpinColumnPurificationIMPORTANT!
WhenusingtheBigDyeterminatorsv1.
1,hydratethecolumnfor2hours(seebelow).
Tipsforoptimizingspincolumnpurificationwhenusingindividualcolumns:Donotprocessmorecolumnsthanyoucanhandleconvenientlyatonetime.
Loadthesampleinthecenterofthecolumnbedslowly.
Makesurethatthesampledoesnottouchthesidesofthecolumnandthatthepipettipdoesnottouchthegelsurface.
Note:Ifsamplesarenotproperlyloaded,peaksfromunincorporateddyeterminatorscanresult.
Spinthecolumnat325–730*gforbestresults.
Usethefollowingformulatocalculatethebestspeedforyourcentrifuge:g=11.
18*r*(rpm/1000)2where:g=relativecentrifugalforcer=radiusoftherotorincmrpm=revolutionsperminuteDonotspinformorethan2minutes.
Performtheentireprocedurewithoutinterruptiontoensureoptimalresults.
Donotallowthecolumntodryout.
DRAFTAugust27,20029:57am,04_PurifyExtension.
fmPlateandSpinColumnPurificationBigDyeTerminatorv1.
1CycleSequencingKit4-11PerformingSpinColumnPurificationToperformspincolumnpurification:1.
Gentlytapthecolumntocausethegelmaterialtosettletothebottomofthecolumn.
2.
Removetheupperendcapandadd0.
8mLofdeionizedwater.
3.
Replacetheupperendcapandvortexorinvertthecolumnafewtimestomixthewaterandgelmaterial.
4.
Allowthegeltohydrateatroomtemperatureforatleast2hours.
Note:Hydratedcolumnscanbestoredforafewdaysat2–6°C.
Longerstorageinwaterisnotrecommended.
Allowcolumnsstoredat2–6°Ctowarmtoroomtemperaturebeforeuse.
5.
Removeanyairbubblesbyinvertingortappingthecolumnandallowingthegeltosettle.
6.
Removetheupperendcapfirst,thenremovethebottomcap.
Allowthecolumntodraincompletelybygravity.
Note:Ifflowdoesnotbeginimmediately,applygentlepressuretothecolumnwithapipettebulb.
7.
Insertthecolumnintothewashtubeprovided.
8.
Spinthecolumninamicrocentrifugeat730*gfor2minutestoremovetheinterstitialfluid.
9.
Removethecolumnfromthewashtubeandinsertitintoasamplecollectiontube(forexample,a1.
5-mLmicrocentrifugetube).
10.
Removetheextensionreactionmixturefromitstubeandloaditcarefullyontothecenterofthegelmaterial.
DRAFTAugust27,20029:57am,04_PurifyExtension.
fmChapter4PurifyingExtensionProducts4-12BigDyeTerminatorv1.
1CycleSequencingKit11.
Spinthecolumninamicrocentrifugeat730*gfor2minutes.
Note:Ifusingacentrifugewithafixed-anglerotor,placethecolumninthesameorientationasitwasinforthefirstspin.
Thisisimportantbecausethesurfaceofthegelwillbeatanangleinthecolumnafterthefirstspin.
12.
Discardthecolumn.
Thesampleisinthesamplecollectiontube.
13.
Drythesampleinavacuumcentrifugefor10–15minutes,oruntildry.
Donotover-dry.
Toperformspincolumnpurification:(continued)DRAFTAugust27,20029:57am,04_PurifyExtension.
fmPlatePurificationUsingSDS/HeatTreatmentBigDyeTerminatorv1.
1CycleSequencingKit4-13PlatePurificationUsingSDS/HeatTreatmentOverviewThissectionprovidesinstructionsforaddingasodiumdodecylsulfate(SDS)/heattreatmenttothespinplatepurificationmethod.
ThisSDS/heattreatmenteffectivelyeliminateshighconcentrationsofunincorporateddyeterminatorsfromcyclesequencingreactions.
PreparingExtensionProductsUsethisproceduretoprepareextensionproductsfor96-wellspinplatepurification.
CHEMICALHAZARD.
Sodiumdodecylsulfate(SDS).
Exposurecauseseye,skin,andrespiratorytractirritation.
Exposuremaycausesevereallergicrespiratoryandskinreaction.
ReadtheMSDS,andfollowthehandlinginstructions.
Wearappropriateprotectiveeyewear,clothing,andgloves.
Toprepareextensionproducts:1.
Prepareasolutionof2.
2%SDSindeionizedwater.
ThisSDSsolutionisstableatroomtemperature.
2.
Addanappropriateamountofthe2.
2%SDSsolutiontoeachsampletobringthefinalSDSconcentrationto0.
2%.
Forexample:Add2Lof2.
2%SDSsolutiontoeach20-Lcompletedcyclesequencingreaction.
3.
Sealthetubesandmixthoroughly.
4.
Heatthetubesto98°Cfor5min,thenallowthetubestocooltoambienttemperaturebeforeproceedingtothenextstep.
Note:Aconvenientwaytoperformthisheating/coolingcycleistoplacethetubesinathermalcyclerandsetitasfollows:98°Cfor5min25°Cfor10min5.
Spindownthecontentsofthetubesbriefly.
6.
Continuewith96-wellplatepurification.
DRAFTAugust27,20029:57am,04_PurifyExtension.
fmChapter4PurifyingExtensionProducts4-14BigDyeTerminatorv1.
1CycleSequencingKitPerforming96-WellSpinPlatePurificationRecommended96-WellSpinPlatesForlarge-scaleprocedures,youcanuse96-wellspinplates,suchastheGelFiltrationKitfromEdgeBiosystems.
Note:Otherspinplatesystemsmaybeusedtosuccessfullyremoveunincorporateddyeterminators.
However,duetothelargenumberofvariablesassociatedwithusingspinplatesystems,youwillneedtooptimizetheperformanceofyoursysteminyourownlaboratory.
Purifyingwiththe96-WellSpinPlateToperformpurificationwiththespinplate:1.
Preparetheextensionproductsaccordingto"PreparingExtensionProducts"onpage4-13,ifdesired.
2.
Preparethe96-wellspinplateperthemanufacturer'sinstructions.
3.
Performthepurificationperthemanufacturer'sinstructions.
IMPORTANT!
WhenusingtheEdgeBiosystemsgelfiltrationkitonly,centrifugeat850*gfor2min.
DRAFTAugust15,200211:28am,AppA_SelectingPrimers.
fmBigDyeTerminatorv1.
1CycleSequencingKitA-1ASelectingSequencingPrimersASelectingSequencingPrimersOverviewThechoiceofsequencingprimersequence,methodofprimersynthesis,andapproachtoprimerpurificationcanhaveasignificanteffectonthequalityofthesequencingdataobtainedindyeterminatorcyclesequencingreactionswiththiskit.
Thesedecisionsareparticularlyimportantwhensequencingisdoneonreal-timedetectionsystemswheresignalstrengthiscritical.
Someoftherecommendationsgivenherearebasedoninformationthatisgeneralknowledge,whileothersarebasedonpracticalexperiencegainedbyAppliedBiosystemsscientists.
OptimizingPrimerSelectionThefollowingrecommendationsareprovidedtohelpoptimizeprimerselection:Primersshouldbeatleast18baseslongtoensuregoodhybridization.
Avoidrunsofanidenticalnucleotide,especiallyguanine,whererunsoffourormoreGsshouldbeavoided.
KeeptheG-Ccontentintherange30–80%.
Forcyclesequencing,primerswithmeltingtemperatures(Tm)above45°CproducebetterresultsthanprimerswithlowerTm.
ForprimerswithaG-Ccontentlessthan50%,itmaybenecessarytoextendtheprimersequencebeyond18basestokeeptheTm>45°C.
Useofprimerslongerthan18basesalsominimizesthechanceofhavingasecondaryhybridizationsiteonthetargetDNA.
Avoidprimersthathavesecondarystructureorthatcanhybridizetoformdimers.
Severalcomputerprogramsforprimerselectionareavailable.
TheycanbeusefulinidentifyingpotentialsecondarystructureproblemsanddeterminingifasecondaryhybridizationsiteexistsonthetargetDNA.
DRAFTAugust15,200211:28am,AppA_SelectingPrimers.
fmAppendixASelectingSequencingPrimersA-2BigDyeTerminatorv1.
1CycleSequencingKitDRAFTAugust15,200211:26am,AppB_ControlSeq.
fmBigDyeTerminatorv1.
1CycleSequencingKitB-1BControlDNASequenceBControlSequencePartialSequenceofpGEM-3Zf(+)ThepGEM-3Zf(+)sequencebelowisthesequenceofthe–21M13forwardprimer,followedbytheensuing1000bases.
TGTAAAACGACGGCCAGT(–21M13primer)GAATTGTAATACGACTCACTATAGGGCGAATTCGAGCTCG40GTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAA80GCTTGAGTATTCTATAGTGTCACCTAAATAGCTTGGCGTA120ATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCG160CTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGT200GTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATT240AATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAAC280CTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGG320GGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTC360GCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGA400GCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCA440CAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAA480AGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTG520CTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATC560ACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGAC600AGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCC640DRAFTAugust15,200211:26am,AppB_ControlSeq.
fmAppendixBControlDNASequenceB-2BigDyeTerminatorv1.
1CycleSequencingKitCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGAT680ACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTC720TCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTC760GTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTC800AGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGA840GTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCA880GCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCG920GTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTA960CACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAG1000DRAFTAugust26,200211:39am,AppC_Electrophoresis.
fmBigDyeTerminatorv1.
1CycleSequencingKitC-1CSampleElectrophoresisCSomeImportantRemindersDyeset/primer(mobility)filesfortheBigDyeterminatorsv1.
1arethesameasthoseforBigDyeterminatorsoriginal,butdifferentthanthoseforthedRhodamineterminatorsandBigDyeterminatorsv3.
1.
Ifamobilityfileforthewrongsequencingchemistryisused,somebasesmaybemiscalledduetodifferentdyelabelingforthedifferentchemistries.
Note:See"RequiredSoftware"onpage1-4forinformationonobtainingthev1.
1dyeset/primer(mobility)files.
DRAFTAugust26,200211:39am,AppC_Electrophoresis.
fmAppendixCSampleElectrophoresisC-2BigDyeTerminatorv1.
1CycleSequencingKitElectrophoresisontheABIPRISM3700DNAAnalyzerRequirementsRunModulesDyeSet/Primer(Mobility)FilesStandardsIMPORTANT!
UseDyeSetE.
Note:Refertotheproductinsertforinstructionsonusingthestandardsforthisinstrument.
ConfigurationRunModulePOP-5polymer,50-cmSeq1_1POP5DefaultModuleSeq1_2POP5DefaultModulePOP-6polymer,50-cmSeq1_1POP6DefaultModuleSeq1_2POP6DefaultModulePolymerDyeSet/Primer(Mobility)FilePOP-5polymerDT3700POP5{BD}v3.
mobPOP-6polymerDT3700POP6{BD}v5.
mobDyeSetStandardsE3700BigDyeTerminatorv1.
1MatrixStandard(PN4336825)DRAFTAugust26,200211:39am,AppC_Electrophoresis.
fmElectrophoresisontheABIPRISM3700DNAAnalyzerBigDyeTerminatorv1.
1CycleSequencingKitC-3PerformingSampleElectrophoresisForinformationonhowtoperformsampleelectrophoresisonthe3700instrument,refertothefollowingmanuals:ABIPRISM3700DNAAnalyzerSequencingChemistryGuide(PN4309125)ABIPRISM3700DNAAnalyzerUser'sManual(PN4306152)DRAFTAugust26,200211:39am,AppC_Electrophoresis.
fmAppendixCSampleElectrophoresisC-4BigDyeTerminatorv1.
1CycleSequencingKitElectrophoresisontheABIPRISM3100and3100-AvantGeneticAnalyzersRequirementsElectrophoresisanddataanalysisofsamplesontheABIPRISM3100and3100-AvantGeneticAnalyzersrequirethefollowing:RunModulesDyeSet/Primer(Mobility)FilesStandardsIMPORTANT!
UseDyeSetE.
Note:Refertotheproductinsertforinstructionsonusingthestandardsforthisinstrument.
ConfigurationRunModulePOP-4polymer,36-cmUltraSeq_POP4DefaultModulePOP-4polymer,80-cmLongSeq80_POP4DefaultModulePOP-6polymer,36-cmRapidSeq36_POP6DefaultModulePOP-6polymer,50-cmStdSeq50_POP6DefaultModulePolymerDyeSet/Primer(Mobility)FilePOP-4polymerDT3100POP4LR{BD}v1.
mobPOP-6polymerDT3100POP6{BD}v1.
mobDyeSetStandardsE3100BigDyeTerminatorv1.
1MatrixStandard(PN4336824)DRAFTAugust26,200211:39am,AppC_Electrophoresis.
fmElectrophoresisontheABIPRISM3100and3100-AvantGeneticAnalyzersBigDyeTerminatorv1.
1CycleSequencingKitC-5PerformingSampleElectrophoresisForinformationonhowtoperformsampleelectrophoresisonthe3100instrument,refertothefollowingmanuals:ABIPRISM3100GeneticAnalyzerSequencingChemistryGuide(PN4315831)ABIPRISM3100GeneticAnalyzerUser'sManual(PN4315834)DRAFTAugust26,200211:39am,AppC_Electrophoresis.
fmAppendixCSampleElectrophoresisC-6BigDyeTerminatorv1.
1CycleSequencingKitElectrophoresisontheABIPRISM310GeneticAnalyzerRequirementsElectrophoresisanddataanalysisofsamplesontheABIPRISM310GeneticAnalyzerrequiresthefollowing:FilterSetERunModulesDyeSet/Primer(Mobility)FilesConfigurationRunModulePOP-4polymer,1-mLsyringe,47-cm,50-mi.
d.
capillary,Ld=36cmP4StdSeq(1mL)EPOP-4polymer,RapidSequencing,1-mLsyringe,47-cm,50-mi.
d.
capillary,Ld=36cmP4RapidSeq(1mL)EPOP-6polymer,1-mLsyringe,61-cm,50-mi.
d.
capillarySeqPOP6(1mL)EPOP-6polymer,RapidSequencing,1-mLsyringe,47-cm,50-mi.
d.
capillarySeqPOP6Rapid(1mL)EPolymerOperatingSystemDyeSet/Primer(Mobility)FilePOP-4polymerNTDT310POP4{BD}v2.
mobPOP-6polymerDT310POP6{BD-LR}v3.
mobDT310POP6{BD}.
mobPOP-4polymerMacintoshDT310POP4{BD}v2.
mobPOP-6polymerDTPOP6{BDSet-AnyPrimer}DRAFTAugust26,200211:39am,AppC_Electrophoresis.
fmElectrophoresisontheABIPRISM310GeneticAnalyzerBigDyeTerminatorv1.
1CycleSequencingKitC-7StandardsIMPORTANT!
Theinstrument(matrix)filefortheBigDyeterminatorsv1.
1cannotbeusedfortheBigDyeterminatorsv3.
0orv3.
1.
Note:Refertotheproductinsertforinstructionsonusingthestandardsforthisinstrument.
PerformingSampleElectrophoresisForinformationonhowtoperformsampleelectrophoresisonthe310instrument,refertothefollowingmanuals:ABIPRISM310GeneticAnalyzerSequencingChemistryGuide(PN4303189)ABIPRISM310GeneticAnalyzerUser'sManual(PN4317588)Dye/FilterSetStandardsforInstrument(Matrix)FileGenerationE310/377BigDyeTerminatorv1.
1MatrixStandards(PN4336805)DRAFTAugust26,200211:39am,AppC_Electrophoresis.
fmAppendixCSampleElectrophoresisC-8BigDyeTerminatorv1.
1CycleSequencingKitElectrophoresisontheABIPRISM377DNASequencersRequirementsElectrophoresisanddataanalysisofsamplesontheABIPRISM377DNASequencers(allmodels*)requirethefollowing:FilterSetERunModulesDyeSet/Primer(Mobility)Files*IncludestheABIPRISM377,ABIPRISM377-18,ABIPRISM377withXLUpgrade,andtheABIPRISM377with96-LaneUpgradeinstruments.
Configuration**AnyplatecheckandprerunmodulecanbeusedontheABIPRISM377DNASequencers.
RunModule36-cmwtr,1200scans/hr,anycombSeqRun36E-120036-cmwtr,2400scans/hr,anycombSeqRun36E-240048-cmwtr,1200scans/hr,anycombSeqRun48E-1200GelFormulationOperatingSystemDyeSet/Primer(Mobility)File4.
5%acrylamide(29:1)or5%LongRangergelNTDT377{BD}.
mobMacintoshDT{BDSetAny-Primer}DRAFTAugust26,200211:39am,AppC_Electrophoresis.
fmElectrophoresisontheABIPRISM377DNASequencersBigDyeTerminatorv1.
1CycleSequencingKitC-9StandardsIMPORTANT!
Theinstrument(matrix)filefortheBigDyeterminatorsv1.
1cannotbeusedfortheBigDyeterminatorsv3.
0orv3.
1.
Note:Refertotheproductinsertforinstructionsonusingthestandardsforthisinstrument.
UsingtheLaneGuideKitIfyouareusingtheBigDyechemistriesv1.
1onthe377instrumentinconjunctionwiththeABIPRISMLaneGuideLaneIdentificationKit,refertothatkit'sprotocol(PN4313804)forinstructionsonresuspendingandloadingsamples.
UsingLong-ReadGelandBufferFormulationsForlongersequencingreadlengthsfollowthegelandbufferformulationsdescribedintheuserbulletinentitledAchievingLongerHighAccuracyReadsonthe377Sequencer(PN4315153).
PerformingSampleElectrophoresisForinformationonhowtoperformsampleelectrophoresisonthe377instrument,refertothefollowingmanuals:AutomatedDNASequencingChemistryGuide(PN4305080)ABIPRISM377DNASequencerUser'sManual(PN4307164)Dye/FilterSetStandardsforInstrument(Matrix)FileGenerationE310/377BigDyeTerminatorv1.
1MatrixStandards(PN4336805)DRAFTAugust26,200211:39am,AppC_Electrophoresis.
fmAppendixCSampleElectrophoresisC-10BigDyeTerminatorv1.
1CycleSequencingKitDRAFTAugust27,200210:01am,AppD_Troubleshooting.
fmBigDyeTerminatorv1.
1CycleSequencingKitD-1DTroubleshootingDObservationPossibleCausesRecommendedActionsNorecognizablesequenceInsufficienttemplateQuantitateDNAtemplateIncreaseamountofDNAinthesequencingreactionsInhibitorycontaminantinthetemplateCleanupthetemplateInsufficientprimerQuantitatetheprimerIncreaseamountofprimerinthesequencingreactionsPrimerhasnoannealingsiteUseaprimerthatiscomplementarytothetemplatePoorprimerdesignorincorrectprimersequenceRedesigntheprimerMissingreagentRepeatthereactions,carefullyfollowingtheprotocolOldormishandledreagentsUsefreshreagentsThermalcyclerpowerfailureRepeatthereactionsThermalcyclingconditionsincorrectCalibratethethermalcyclerregularlyUsecorrectthermalcyclingparametersUsecorrecttubeforyourthermalcyclerSetramprateto1°C/secExtensionproductslostduringreactioncleanupMakesureyouusethecorrectcentrifugationspeedsandtimesforprecipitationandspincolumnproceduresMakesuretheethanolconcentrationiscorrectfortheprecipitationprotocolsNorecognizablesequence(continued)ExtensionproductsnotresuspendedCarefullyresuspendthesamplepelletinloadingbufferLanetrackingfailure(377instrument)Checkthelanetracking.
Ifnecessary,retrackandreextractthelanesElectrokineticinjectionfailure(capillaryinstruments)RepeattheinjectionsNoisydatathroughoutthesequencewithlowsignalstrengthInsufficientDNAinthesequencingreactionsUsemoreDNAinthesequencingreactionsLoadorinjectmoreoftheresuspendedsequencingreactionsDegradedtemplatePreparefreshDNAandrepeatthereactionsOldormishandledreagentsUsefreshreagentsThermalcyclingconditionsincorrectCalibratethethermalcyclerregularlyUsecorrectthermalcyclingparametersUsecorrecttubeforyourthermalcyclerSetramprateto1°C/secLanetrackingfailure(377instrument)Checkthelanetracking.
Ifnecessary,retrackandreextractthelanesElectrokineticinjectionfailure(capillaryinstruments)RepeattheinjectionsNoisydatathroughoutthesequencewithgoodsignalstrengthInhibitorycontaminantinthetemplateCleanupthetemplateMultipletemplatesinthesequencingreactionExaminetemplateonanagarosegeltobesureonlyonetemplateispresent.
MultipleprimingsitesMakesureprimerhasonlyoneprimingsite.
Ifnecessary,redesignprimerMultipleprimerswhensequencingPCRproductsPurifyyourPCRtemplatetoremoveexcessprimersObservationPossibleCausesRecommendedActionsDRAFTAugust27,200210:01am,AppD_Troubleshooting.
fmBigDyeTerminatorv1.
1CycleSequencingKitD-3Noisydatathroughoutthesequencewithgoodsignalstrength(continued)PrimerwithN-1contaminationUseHPLC-purifiedprimerHighsignalsaturatingthedetectorUselessDNAinthesequencingreactionsLoadorinjectlessoftheresuspendedsequencingreactionsIncorrectrunmoduleUsecorrectrunmoduleIncorrectinstrument(matrix)fileUsecorrectinstrumentfileforterminatorchemistryNoiseuptoorafteraspecificpointinthesequenceMixedplasmidseparationMakesureyouhaveonlyonetemplateMultiplePCRproductsMakesureyouhaveonlyonetemplatePrimer-dimercontaminationinPCRsequencingOptimizeyourPCRamplificationMakesurethereisnosequencecomplementaritybetweenthetwoPCRprimersMakesureyoursequencingprimerdoesnotoverlapthesequencesofthePCRprimersUseaHotStarttechniquesuchaswithAmplitaqGoldpolymeraseSlippageafterrepeatregionintemplateTryanalternatesequencingchemistryUseananchoredprimerPoormobilitycorrectionIncorrectdyeset/primer(mobility)fileUsecorrectdyeset/primerfileIncorrectPeak1locationfordataanalysisSelectanewPeak1locationGelwithverydifferentseparationpropertiesthanthegelmatricesusedtoconstructthedyeset/primer(mobility)fileUsecorrectdyeset/primerfileforyourgeltypeObservationPossibleCausesRecommendedActionsDRAFTAugust27,200210:01am,AppD_Troubleshooting.
fmAppendixDTroubleshootingD-4BigDyeTerminatorv1.
1CycleSequencingKitExcessdyepeaksatbeginningofsequencePoorremovalofunincorporateddyeterminatorsUseethanol/EDTAprecipitationprotocoltoremoveunincorporateddyeterminatorsWithCentri-Sepspincolumns,takecaretoloadsampleonthecenterofthegelsurfaceNote:Donottouchthegelsurfacewiththepipettip.
IMPORTANT!
Besureyouhydratethecolumnforatleast2hours.
Spinsamplesforrecommendedtimes(spinningtoolongprecipitatesmoredyeswiththesample)Withmicrofugetubes,aspiratethesupernatantratherthandecanting(decantingleavesexcessethanolonthesidesofthetube)SelectthestartpointfordataanalysistoexcludeexcessdyepeaksPull-uppeaksorbleedthroughTotalsignalstrengthover4000QuantitateDNAtemplateDecreaseamountofDNAinthesequencingreactionsLoadorinjectlessoftheresuspendedsequencingreactionsObservationPossibleCausesRecommendedActionsDRAFTAugust20,20022:48pm,AppE_TechSupport.
fmBigDyeTerminatorv1.
1CycleSequencingKitE-1EObtainingTechnicalSupportEServicesandSupportAppliedBiosystemsWebSiteAservicesandsupportpageisavailableontheAppliedBiosystemsWebsite.
Toaccessthis,goto:http://www.
appliedbiosystems.
comandclickthelinkforservicesandsupport.
Attheservicesandsupportpage,youcan:Searchthroughfrequentlyaskedquestions(FAQs)SubmitaquestiondirectlytoTechnicalSupportOrderAppliedBiosystemsuserdocuments,MSDSs,certificatesofanalysis,andotherrelateddocumentsDownloadPDFdocumentsObtaininformationaboutcustomertrainingDownloadsoftwareupdatesandpatchesInaddition,theservicesandsupportpageprovidesworldwidetelephoneandfaxnumberstocontactAppliedBiosystemsTechnicalSupportandSalesfacilities.
DRAFTAugust20,20022:48pm,AppE_TechSupport.
fmAppendixEObtainingTechnicalSupportE-2BigDyeTerminatorv1.
1CycleSequencingKitDRAFTAugust27,200210:02am,4337036A_v1.
1IX.
fmBigDyeTerminatorv1.
1CycleSequencingKitIndex-1IndexNumerics310requirementsmobilityfilesC-6runmodulesC-6sequencingstandardsC-73100requirementsmobilityfilesC-4runmodulesC-4spectralcalibrationstandardsC-43700requirementsmobilityfilesC-2runmodulesC-2spectralcalibrationstandardsC-2377requirementsmatrixfilestandardsC-9mobilityfilesC-8runmodulesC-8Aattentionwords1-11Bbacterialartificialchromosomes2-3Ccentrifuge,recommended1-9components,sequencingkits1-6contaminants,oftemplates2-5controldouble-stranded2-1electrophoresis2-1single-stranded2-1controlDNA,reasonfor2-1customersupport.
SeetechnicalsupportE-1cyclesequencingBACDNA3-7protocolforGeneAmp3-5Ddisposablescyclesequencing1-8purifying1-9DNAquantitycharacteristics2-6quantitytouse2-6dyeset/primerfiles,downloading1-6dye/filterset31001-437001-43771-4Eethanol/EDTAprecipitation384-well4-496-well4-3ethanol/EDTA/sodiumacetateprecipitationin384-well4-8precipitationin96-well4-6Fformulation,new1-1freeze-thaw,limits1-7Hhazardschemical1-11waste1-12DRAFTAugust27,200210:02am,4337036A_v1.
1IX.
fmIndex-2BigDyeTerminatorv1.
1CycleSequencingKitIinstrumentssequencing1-3thermalcyclers1-3LLaneGuideC-9longreadlengthsC-9Mmatrixstandards3101-43771-4generalrequirements1-2mobilityfiles1-2aboutC-1downloading1-6MSDSs,ordering1-13PpGEM-3Zf(+)sequenceB-1primersimportanceofselectionA-1optimizingselectionA-1purificationSDSand96-well4-13spinplate4-14purifyingmethods2-3,4-1methodsforPCR2-3PCRbyultrafiltration2-4Qqualityproblems,template2-5quality,examiningDNA2-5quantitatingDNA2-6RreactionmixturesBACDNA3-7for384-well3-4for96-well3-2formicrocentrifugetubes3-2reagentselectrophoresis1-10purifying1-9requiredfilesmatrix1-1spectral1-1Ssafetychemical1-11to1-13guide1-11to1-13SDS,andpurification4-13sequencingbufferoptimizing1-2sequencingkits,ordering1-6sequencingproblems2-4sequencingstandardsforeachinstrument2-2generalrequirements1-2shorterreadlengths,andDNAquantity2-6signalintensities,andDNAquantity2-6sitepreparationandsafetyguide1-11spectralstandards31001-437001-4spincolumnpurificationoptimizing4-10performing4-11storagetemperatures1-7DRAFTAugust27,200210:02am,4337036A_v1.
1IX.
fmBigDyeTerminatorv1.
1CycleSequencingKitIndex-3TtechnicalsupportE-1templatesBACDNA2-3PCR2-3poorqualitycharacteristics2-4preparing2-3top-heavydata,andDNAquantity2-6Uuserattentionwords1-11Wwarningsymbols1-11DRAFTAugust27,200210:02am,4337036A_v1.
1IX.
fmIndex-4BigDyeTerminatorv1.
1CycleSequencingKitHeadquarters850LincolnCentreDriveFosterCity,CA94404USAPhone:+1650.
638.
5800TollFree(InNorthAmerica):+1800.
345.
5224Fax:+1650.
638.
5884WorldwideSalesandSupportAppliedBiosystemsvastdistributionandservicenetwork,composedofhighlytrainedsupportandapplicationspersonnel,reaches150countriesonsixcontinents.
Forsalesofficelocationsandtechnicalsupport,pleasecallourlocalofficeorrefertoourWebsiteatwww.
appliedbiosystems.
com.
www.
appliedbiosystems.
comAppleraCorporationiscommittedtoprovidingtheworld'sleadingtechnologyandinformationforlifescientists.
AppleraCorporationconsistsoftheAppliedBiosystemsandCeleraGenomicsbusinesses.
PrintedinUSA,09/2002PartNumber4337036Rev.
A