Genomic DNA Amplification
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Genomic DNA Amplification sigma.com/pcr ORDER: 800-325-300 TECHNICAL SERVICE: 800-325-5832 3 Genomic DNA Amplification Genomic DNA Amplification Product Listing Catalog Number Product Description Page D8312 REDTaq Genomic DNA Polymerase with 103 Reaction Buffer containing MgCl 2 34 D2812 REDTaq Genomic DNA Polymerase with 103 Reaction Buffer without MgCl 2 34 UVS1 Universal Vectorette System 36 REDTaq ® Genomic DNA Polymerase REDTaq Genomic DNA Polymerase is a special formulation of REDTaq DNA Polymerase designed to provide enhanced amplification of more complex genomic templates. REDTaq Genomic DNA Polymerase is more sensitive, produces higher yields and is more capable of generating longer product lengths. It has all the advantages of REDTaq DNA Polymerase, such as easy visualization of enzyme addition and complete reaction mixing, and direct loading to an agarose gel. The dye migrates slightly faster than bromophenol blue at the same rate as a 125 base pair fragment. The inert red dye has no effect on automated or manual sequencing, restriction digestions, ligation or other down- stream applications. However, if dye removal is desired, this can easily be accomplished using any standard purification method. Features and Benefits n Enhanced amplification on genomic and difficult DNA templates n No loading buffers or tracking dyes required. The PCR product is loaded directly onto an agarose gel after amplification n Quick recognition when the REDTaq has been added to the reaction tube n Confirms proper mixing at a glance for greater consistency across reactions n PCR samples can be easily re-amplified as in nested PCR n The unique red dye migrates slightly faster than bromophenol blue Higher Yields from Genomic Templates with REDTaq Genomic DNA Polymerase M 1 2 3 4 M Higher Yields from Genomic Templates with REDTaq Genomic DNA Polymerase. PCR reactions were set up using 1 µl of mouse genomic DNA and 1 unit of polymerase. The resulting amplicon is a specific 1181 bp fragment. Each sample was prepared in duplicate, and conditions for both sets were identical with the exception of the enzyme used. Lanes M: 1 kb DNA Ladder (D3937) Lanes 1, 2: REDTaq DNA Polymerase Lanes 3, 4: REDTaq Genomic DNA Polymerase Components: REDTaq Genomic DNA Polymerase 103 PCR Buffer or 103 PCR Buffer without MgCl 2 Separate vial of 25 mM MgCl 2 included with D2812 Unit definition: One unit incorporates 10 nmol of total dNTPs into acid-precipitable DNA in 30 min at 74 °C Concentration: 1 unit per µl Storage: –20 °C Shipped in wet ice Ordering Information Cat. No. Product Description Quantity D8312 REDTaq Genomic DNA 250 units Polymerase with 103 1,000 units Reaction Buffer 2,500 (10 × 250) units containing MgCl 2 D2812 REDTaq Genomic DNA 250 units Polymerase with 103 1,000 units Reaction Buffer 2,500 (10 × 250) units without MgCl 2 . Includes a separate tube of 25 mM MgCl 2
Transcript of Genomic DNA Amplification
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Genomic DNA AmplificationGenomic DNA Amplification Product Listing
Catalog Number Product Description PageD8312 REDTaqGenomicDNAPolymerasewith103ReactionBuffercontainingMgCl2 34
D2812 REDTaqGenomicDNAPolymerasewith103ReactionBufferwithoutMgCl2 34
UVS1 UniversalVectoretteSystem 36
REDTaq® Genomic DNA PolymeraseREDTaqGenomicDNAPolymeraseisaspecialformulationofREDTaqDNAPolymerasedesignedtoprovideenhancedamplificationofmorecomplexgenomictemplates.REDTaqGenomicDNAPolymeraseismoresensitive,produceshigheryieldsandismorecapableofgeneratinglongerproductlengths.IthasalltheadvantagesofREDTaqDNAPolymerase,suchaseasyvisualizationofenzymeadditionandcompletereactionmixing,anddirectloadingtoanagarosegel.Thedyemigratesslightlyfasterthanbromophenolblueatthesamerateasa125basepairfragment.
Theinertreddyehasnoeffectonautomatedormanualsequencing,restrictiondigestions,ligationorotherdown-streamapplications.However,ifdyeremovalisdesired,thiscaneasilybeaccomplishedusinganystandardpurificationmethod.
Features and Benefitsn EnhancedamplificationongenomicanddifficultDNA
templates
n Noloadingbuffersortrackingdyesrequired.ThePCRproductisloadeddirectlyontoanagarosegelafteramplification
n QuickrecognitionwhentheREDTaqhasbeenaddedtothereactiontube
n Confirmspropermixingataglanceforgreaterconsistencyacrossreactions
n PCRsamplescanbeeasilyre-amplifiedasinnestedPCR
n Theuniquereddyemigratesslightlyfasterthanbromophenolblue
Higher Yields from Genomic Templates with REDTaq Genomic DNA Polymerase
M 1 2 3 4 M
Higher Yields from Genomic Templates with REDTaq Genomic DNA Polymerase. PCRreactionsweresetupusing1µlofmousegenomicDNAand1unitofpolymerase.Theresultingampliconisaspecific1181bpfragment.Eachsamplewaspreparedinduplicate,andconditionsforbothsetswereidenticalwiththeexceptionoftheenzymeused.
LanesM: 1kbDNALadder(D3937)Lanes1,2: REDTaqDNAPolymeraseLanes3,4: REDTaqGenomicDNAPolymerase
Components: REDTaqGenomicDNAPolymerase103PCRBufferor103PCRBufferwithoutMgCl2Separatevialof25mMMgCl2includedwithD2812
Unit definition: Oneunitincorporates10nmoloftotaldNTPsintoacid-precipitableDNAin30minat74°C
Concentration: 1unitperµl
Storage: –20°CShippedinwetice
Ordering InformationCat. No. Product Description Quantity
D8312 REDTaqGenomicDNA 250units Polymerasewith103 1,000units ReactionBuffer 2,500(10×250)units containingMgCl2D2812 REDTaqGenomicDNA 250units
Polymerasewith103 1,000units ReactionBuffer 2,500(10×250)units withoutMgCl2. Includesaseparate tubeof25mMMgCl2
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35Our Innovation, Your Research — Shaping the Future of Life Science
The Universal Vectorette™ System A PCR-based method for DNA walking and mappingTheVectorettesystemisaPCR-basedmethodforDNAwalkingandmappingthatusesaformofunidirectionalPCRforamplify-ingandsequencingunknowngenomicorlargeconstructDNA.Thesystemeliminatesthetime-consumingprocessofmakingandscreeninglibrariestoobtainoverlappingclonesandusingconventionalnucleicacidpurificationandscreeningprocedures.AVectoretteunitisemployed,whichconsistsofadoublestrandedlinkerwithaninternalmismatchedregionandastickyend.
TheUniversalVectorettesystemusesthreesimplestepstoobtainDNAsequenceinformation(Fig.1):
Step 1: GenomicorlargeconstructDNAcontainingthetargetsequenceisdigestedwitharestrictionenzymeandVectoretteunitsareligatedtothe5’and3’endstocreateaVectorettelibrary.
Step 2: PCRisperformedontheVectorettelibraryusingaprimercomplementarytothemismatchedregionoftheVectoretteunit(Vectoretteprimerprovided)andaprimerspecifictotheknownDNAsequence.InthefirstPCRcycle,primerextensionoccursonlyfromthespecificPCRprimerthathybridizestotheknownsequenceintheDNAfragmentwithintheVectorettelibrary.ExtensionfromthisprimergeneratesauniquesequenceasthepolymerasereadsthroughthemismatchedportionoftheVectorette.SubsequentPCRcyclesgenerateaDNAfragmentbetweentheknownsequenceandtheVectoretteunitontheendofthefragment.AnyVectorettefragmentthatdoesnotcontainasequencethatiscomplementarytothespecificprimerwillnotgenerateaPCRproduct.
Step 3: Aseparatesequencingprimerisincluded(slightlynested)thatcanbeusedtoperformasequencingreactionfromtheVectoretteend.PCRproductsaretypicallyobtainedfromasinglePCRrun,however,nestedprimersareincludedtoincreasespecificitywhenamplifyingmorecomplextemplates.ThePCRproductsgeneratedbytheVectorettesystemcanbeuseddirectlyforcyclesequencingorclonedintocommerciallyavailablevectorsforfurthercharacterization.
Figure 1. Vectorette System Process
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Genomic DNA AmplificationTheUniversalVectoretteSystemofferstheflexibilitytogenerateVectorettelibrariesfrompurifiedgenomicDNAbyBam HI,Cla I,Eco RI,HindIIIorbluntrestrictionenzymedigests.Thissystemcanbeusedwith1µgtemplategenomicDNAorless,andprovidesatimesavingalternativetotradi-tionallibraryconstructionandscreening.Theprotocolcanalsobemodifiedforhighthroughputapplications.
Ideal for: GenomewalkingSequencingofyeastartificialchromosome(YAC)terminiSequencingofcosmidinsertterminiMappingofpromoters,introns,microsatellites,SSRsandSTRsSequencingoflargecloneswithoutsub-cloningMappingofregionscontainingdeletions,insertionsandtranslocationsGap-fillingingenomemappingprojectsIdentificationofflankinggenomicsequencesoftransgenesintransgenicorganisms
Features and Benefitsn Cell-freegenemanipulationreplacescloningandsubclon-
inginmanymoleculargeneticsprojects
n Twoandthree-stepprocedurescanbeperformedinasingleday
n Highfidelity,highlyspecificamplificationsupto20kbfromgenomicDNA
n EliminatestheneedfornestedPCRinmostapplications
Storage: –20°CShippedinwetice
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Figure 2. ThreedifferentprimerswereusedonaCla IhumangenomicDNAVectorettelibrarytogeneratethreedifferentsizesofamplicons.Thefragmentsgeneratedarefromdifferentregionsofthehumanglobingene.
LaneM:1kbDNALadder(D3937)Lane1: 3kbampliconLane2: 1.9kbampliconLane3: 1.3kbamplicon
Figure 3.
M � 2 3 � 5
Figure 3. PositivecontrolPCRresultsfor5differentVectorettelibraries.ThisgelillustratesacommonprimertoaknownsequencegeneratingdifferentampliconsizefragmentsonfivedifferentVectorettelibraries.
LaneM:1kbDNALadder(D3937)Lane1: BamHIVectoretteamplicon,1.9kbLane2: ClaIVectoretteamplicon,8.1kbLane3: EcoRIVectoretteamplicon,3kbLane4: HindIIIVectoretteamplicon,1.1kb
Lane5: SmaIVectoretteamplicon,4.8kb
Ordering InformationCat. No. Product Description Quantity
UVS1 UniversalVectoretteSystem 1kit 1kitsufficientfor25ligation reactionsand20PCRreactions (50µlreactionvolume)
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3�Our Innovation, Your Research — Shaping the Future of Life Science
Human Genomic DNA Human Random Control DNA Panels for use as reference standardsSigma-AldrichandECACChaveteamedtogethertoprovideresearcherswithcontrolpopulationsofhumangenomicDNAforgeneregulationandquantitativePCRresearch.TherangeofHumanRandomControl(HRC)DNAsamplesrepresentsacontrolpopulationof480UKCaucasianblooddonors.TheHRCDNAisextractedfromlymphoblastoidcelllinesderivedbyEpsteinBarrVirus(EBV)thatcanbecontinu-ouslypropagatedinculture.ThisensuresaninfinitesupplyoftheunvaryingDNApanels.Thecompositionofeachpaneliscompletelydefinedandstandardizedsothateachlotwillbeidentical.Therefore,theHRCDNAPanelscanbeusedasreferencestandardsforroutinequalitycontrolinthelaboratory.
Features and Benefitsn Consistentcontrolsamples–theDNAisextractedfrom
immortalizedcelllinesheldascryopreservedbanks,sothereisnobatch-to-batchvariation
n Convenientandreadytouse–idealformoststandardgeneticresearchapplicationsandavoidserrorsinprepar-ingcontrols
n Costeffective–the96×2µgformatprovidesenoughpurifiedHRCDNAforthousandsofPCRassays
ThepurifiedHRCDNAisavailablein5differentpanels(HRC1through5)consistingof96individualsandcontaining2µgDNAeachataconcentrationof100ng/µl.Forconve-nience,allofthepanelsareavailableinaPCRcompatible96-wellformat.
Human Genetic Disease DNA Panel for Breast CancerAlsoavailableisaHumanGeneticDiseaseDNAPanelforBreastCancer(HGDBC1).Thesamplesaretakenfromfemalepatientsthathaveallbeendiagnosedashavingbreastcancer.TheHGDBC1panelisprovidedina96-wellplateandcanbeuseddirectlyinautomatedgeneanalysissystems.
Working in Partnershipecacc.org.uk sigma-aldrich.com
Increased Effectiveness and Reliability of HRC DNA Panels
ECACC DNA Quality Control
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Increased Effectiveness and Reliability of HRC DNA Panels. DemonstrationofhowDNAqualitycanaffectassayperformance.Theresultsofthe5’nucleaseassayusing96ECACCHumanRandomControl(HRC)DNAsamples(plotA)and384CustomerDNAsamples(plotB).Homozygotesforallele1(AA,topleft),heterozygotesforalleles1and2(AB,middle),homozygotesforallele2(BB,lowerright).InplotA,usingtheECACCHRC1DNA,thelackofscatterandtightclusteringofdatapointsmakesscoringthetwoallelesquiteclear.WhereasinplotB,thewidescatterandlackofclustering,makescoringthedifferentallelesverydifficult,showinghowDNAqualitycanaffectassayperformance.DataprovidedcourtesyofMRCGeneservice,Cambridge.
Ordering InformationCat. No. Product Description Quantity
HRC1 HumanRandomControlPanel1 2µg Concentration:100ng/µl
HRC2 HumanRandomControlPanel2 2µg Concentration:100ng/µl
HRC3 HumanRandomControlPanel3 2µg Concentration:100ng/µl
HRC4 HumanRandomControlPanel4 2µg Concentration:100ng/µl
HRC5 HumanRandomControlPanel5 2µg Concentration:100ng/µl
HGDBC1 HumanGeneticDisease– 1each BreastCancer Concentration:100ng/µl 96-wellplate(20µl/well)
