Upload
others
View
6
Download
0
Embed Size (px)
Citation preview
Gen
om
ic D
NA
Am
plifi
cati
on
sigma.com/pcr ORDER: 800-325-30�0 TECHNICAL SERVICE: 800-325-58323�
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
Gen
om
ic DN
A A
mp
lificatio
n
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
Gen
om
ic D
NA
Am
plifi
cati
on
sigma.com/pcr ORDER: 800-325-30�0 TECHNICAL SERVICE: 800-325-58323�
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
Figure 2.
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)
Gen
om
ic DN
A A
mp
lificatio
n
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
2
2.5
3
3.5
4
4.5
5
5.5
6
0 1 2 3 Dye 2
AA
BB
AB
Dye
1
384 Customer DNA
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0 1 2 3 Dye 2
AA BB
AB Unscored
Dye
1
(A)
(B)
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)