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蚂蚁淘在线 / 品牌中心 / Kapa biosystems / Kapa生物系统/Kapa hgDNA定量和质控试剂盒/KK4966/200µL

Kapa生物系统/Kapa hgDNA定量和质控试剂盒/KK4966/200µL

价格
￥2100.00
货号：KK4966
浏览量：127
品牌：Kapa biosystems
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商品描述

Description:

10X129bpPrimerPremixonly

KAPAhgDNAQuantificationandQCKit

Qualitymatters.

KAPAhgDNAQuantificationandQCKitscontainallthereagentsneededfortheqPCR-basedquantificationandqualityassessmentofhumangenomicDNAsamplespriortoNGSlibraryconstruction.

KitscontainKAPASYBR®FASTqPCRMasterMix,optimizedforhigh-performanceSYBRGreenI-basedqPCR,aswellasapre-dilutedsetofDNAstandardsandprimerpremixestargetingdifferentportionsofahighlyconservedsingle-copyhumanlocus.Absolutequantificationisachievedwiththeprimerpairdefiningtheshortestfragment,whereastheadditionalprimersareusedtoderiveinformationabouttheamountofamplifiabletemplateintheDNAsample.Qualityscores(orQ-ratios)generatedwiththekitmaybeusedtopredicttheoutcomeoflibraryconstruction,ortailorworkflowsforsamplesofvariablequality,particularlyFFPEDNA.

ThemethodiseasytoautomateandcanbeappliedtoanyprocessorworkflowthatrequiresaccuratequantificationofdiluteDNAsamples,orsamplesthatmaycontainahighproportionofDNAthatisrecalcitranttoPCRamplification.

Downloadour KAPAhgDNAQuantificationandQCDataAnalysisTemplate.

ForResearchUseOnly.Notforuseindiagnosticprocedures.

ProductHighlights

Principle of the hgDNA Quantification and QC assay. A single set of DNA standards is used to generate up to three standard curves using three different primer pairs that amplify targets of 41 bp, 129 bp or 305 bp within a conserved, single-copy human locus. The 41 bp assay is used for absolute quantification of DNA samples. For an assessment of DNA quality, standard curves are generated and samples assayed with the 129 bp and/or 305 bp primer premix(es). Since poor DNA quality has a greater impact on the amplification of longer targets, the relative quality of a DNA sample can be inferred by normalizing the concentration obtained using the 129 bp or 305 bp assay against the concentration obtained from the 41 bp assay. This normalization generates a Q-ratio (with a value between 0 and 1) that is indicative of DNA quality, or the amount of amplifiable material in a DNA sample. Data on file.

Obtainconcentrationandqualityinformationwithasingleassay

  • AllowsforaccuratequantificationofdiluteDNAsamples
  • QuantificationwithanadditionalprimerpairprovidesaQ-ratiothatisindicativeofsamplequality
Conversion rates (% input DNA converted to adapter-ligated library) for libraries prepared for targeted re-sequencing on the Illumina platform. Libraries were prepared in duplicate from 80 – 100 ng FFPE DNA of variable quality (orange) or a commercial hgDNA preparation (grey), using the KAPA LTP Library Preparation Kit. FFPE DNA samples were assessed with the KAPA hgDNA Quantification and QC assay prior to Covaris shearing. Q-ratios were as indicated. The amount of fragmented DNA (average size ~180 bp) used for library construction was determined using the Qubit HS dsDNA assay (Life Technologies), whereas adapter-ligated libraries were quantified using the qPCR-based KAPA Library Quantification Kit. Results indicated that conversion rates for FFPE samples were significantly lower than for high-quality DNA. This limits the diversity of FFPE libraries, and necessitates more cycles of library amplification to generate sufficient material for target capture. This results in higher duplication rates and lower target coverage for FFPE libraries. Data on file.

Employqualityscores intheanalysisofNGSlibraryconstructionworkflows

  • Q-ratioscanprovidevaluableinsightsintothebottlenecksinNGSlibraryconstructionworkflows
  • ForFFPEsamples,libraryandsequencequalityisprimarilylimitedbyinefficientconversionofinputDNAtoadapter-ligatedlibrary
0.4yieldedlibrariesthatmetminimumsequencequalityrequirements.Dataonfile.">Q(129/41) ratios for correlate with key sequencing metrics, such as duplication rate. Libraries were prepared manually from 250 ng Covaris-sheared FFPE DNA (n=14) or control DNA, extracted from cultured cells, fresh-frozen tissue or blood (n=6). Samples are arranged on the x-axis (left to right) in order of increasing Q(129/41)-ratio (designated by orange bars for FFPE samples and blue bars for control DNAs). Libraries were constructed with NEBNext reagents (New England Biolabs), whereas KAPA HiFi was used for pre- and post-capture amplification (10 cycles). Target capture was performed with a custom Roche NimbleGen SeqCap EZ panel (300 genes, 0.9 MB). Sequencing (2 x 100 bp) was performed on an Illumina HiSeq. The average duplication rate (grey dots) were 2X higher for FFPE (75% vs. 38% for control samples). FFPE samples with a Q(129/41) ratio >0.4yieldedlibrariesthatmetminimumsequencequalityrequirements.Dataonfile.
0.4yieldedlibrariesthatmetminimumsequencequalityrequirements.Dataonfile.">Q(129/41) ratios for correlate with key sequencing metrics, such as mean target coverage. Libraries were prepared manually from 250 ng Covaris-sheared FFPE DNA (n=14) or control DNA, extracted from cultured cells, fresh-frozen tissue or blood (n=6). Samples are arranged on the x-axis (left to right) in order of increasing Q(129/41)-ratio (designated by orange bars for FFPE samples and blue bars for control DNAs). Libraries were constructed with NEBNext reagents (New England Biolabs), whereas KAPA HiFi was used for pre- and post-capture amplification (10 cycles). Target capture was performed with a custom Roche NimbleGen SeqCap EZ panel (300 genes, 0.9 MB). Sequencing (2 x 100 bp) was performed on an Illumina HiSeq. The average mean target coverage (grey dots) was 4X higher for control DNA (1,247X, vs. 301X for FFPE samples). FFPE samples with a Q(129/41) ratio >0.4yieldedlibrariesthatmetminimumsequencequalityrequirements.Dataonfile.
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Obtainactionabledataforsamplepreparation

  • Q-ratioscorrelatewithkeysequencingmetricssuchasduplicationratesandmeantargetcoverage
  • FFPEsampleswithaQscore>0.4yieldlibrariesofacceptablequalitywhenprocessedinstandardsamplepreparationsfortargetcapture

Applications:

Applications
  • FFPEsamples
  • Free-circulatingDNAfromplasmaorserum
  • Samplesobtainedbylaser-capturemicrodissectionoffresh,frozenorFFPEtissue
  • Forensicsamples
  • Cellscollectedbyflowcytometry
  • Anyotherlimitingorpreciousclinicalsample

KitSpecificationsandContents/Storage:

KitSpecificationsandContents/Storage

Kitscanbestoredforupto12months at-20˚C.

Complete(MasterMix)kitsincludeKAPASYBRFASTqPCRMasterMix(2X),PrimerPremix(41bp,129bpand305bp,10X)andasetof5DNAStandards.PrimerPremixesandDNAStandardsarealsosoldseparately.

Components

Specifications

Spec
Description
CompatIBLePlatform
AllNGSplatforms
CompatibleSamples
HumangenomicDNA
Samplesources
FFPEtissueCellscollectedbylaser-capturemicrodissectionFlow-sortedcellsFree-circulatingDNAfromplasmaorserumForensicsamplesClinicalsamples
Standardcurveconcentrationrange
2.5ng/µL–10pg/µLor3,080–12copies
SequencingApplications
WholeGenomeSequencingWholeExomeSequencingTargetedSequencing(custompanels)AmpliconSequencing
Kapa是世界上著名的生物技术公司。总部位于美国波士顿, 公司的主要理念是运用分子和遗传学技术来打造新一代的生物科学和医学诊断产品。Kapa公司目前主要是在“直接进化（directed evolution technology platform）”的技术平台上来筛选和生产高品质的生物酶。目前生物医药领域所使用的大部分生物酶都只是天然存在形式，即使有些酶经过基因工程改造，但是由于酶分子自身的结构和功能限制，要想得到真正满足科研工作人员需要的产品，却是一件费时费力的事情，而且常常达不到预期的要求。“直接进化”技术正是针对这一现实情况，运用遗传，基因工程，生物信息学等技术来在分子水平上设计真正满足不同实际要求的生物酶。通过创新，Kapa为广大的科研人员提供了一系列拥有多项技术的产品，不仅能够满足普通科研需要，同时为高通量，基因芯片等高新技术的普及创造了条件。 运用“直接进化”技术，Kapa目前已经开发