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NEB/NEBNext? Library Quant Kit for Illumina?/500 reactions/E7630L
  • NEB/NEBNext? Library Quant Kit for Illumina?/500 reactions/E7630L

NEB/NEBNext? Library Quant Kit for Illumina?/500 reactions/E7630L

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貨號(hào): E7630L
品牌: NEB
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    • Figure 1: Typical amplification and standard curves for the NEBNext Library Quant Kit
      Typical results from the NEBNext Library Quant Kit on a Bio-Rad CFX96 Touch (A) and an Applied Biosystems 7500 Fast real-time qPCR instrument (B). Amplification curves are shown on the left and resulting standard curves on the right. All default settings were used on both platforms. ABI 7500 Fast assay included 1X ROX (Low Concentration) and the ROX normalization.
      The NEBNext Library Quant Kit components have been optimized to deliver significant improvements to qPCR-based library quantitation for Illumina sequencing. The kit contains primers which target the P5 and P7 Illumina adaptor sequences, and a set of six high-quality, pre-diluted DNA standards to enable reliable quantitation of diluted DNA libraries between 150–1000 bp. Features
      • Provides more accurate and reproducible quant values than alternative methods and kits
      • Compatible with libraries with a broad range of insert sizes and GC content, made by a variety of methods
      • Allows you to choose the optimal standard curve (four or six standards) for your experimental needs
      • Supplied with a convenient Library Dilution Buffer
      • The NEBNext Library Quant Master Mix requires only the addition of primers
      • Utilizes a single extension time for all libraries, regardless of insert size
      • Library quant values can be easily calculated using NEB’s online tool, at NEBioCalculator.neb.com
      • ROX is included in the kit, for use with qPCR instruments that require a reference dye for normalization
      Each set of reagents is functionally validated together through qPCR-based library quantitation assays. Reagents pass the functional test by adhering to stringent criteria set for assay efficiency and quantitation cycles (Cq) for each DNA standard and no template control reaction.
      Figure 2: NEBNext Library Quant Kit workflow
      Figure 3: qPCR provides more consistent library quantitation results than Bioanalyzer® analysis
      Concentrations of 4 libraries were determined by the NEBNext Library Quant Kit and compared to values measured using the Agilent Bioanalyzer. Compared to NEBNext’s qPCR-based method, the Bioanalyzer concentrations displayed a greater level of variation.
      Figure 4: Greater reproducibility of library quantitation with the NEBNext Library Quant Kit
      Three 340–400 bp libraries were quantitated by 4 different users 2–4 times using either the NEBNext or KapaTM Library Quantification Kit (Universal). A notable improvement in quantitation consistency was observed for concentrations determined by the NEBNext Kit versus those from the Kapa kit.
      Figure 5: Greater lot-to-lot consistency of standards with the NEBNext Library Quant Kit
      Accurate qPCR quantitation requires the use of high-quality DNA standards with known concentrations. The NEBNext Library Quant Kit contains 6 standardsproduced with a high level of both quantitation accuracy and consistency. This figure shows data from >70 total runs from 4 lots of both NEBNext and Kapa standards, with all Cq values plotted. Box and whiskers indicate mean and quartiles. The NEBNext Library Standards displayed much lower variation in Cq, resulting in more consistent quantitation performance.
      Figure 6: The NEBNext Library Quant Kit values enable optimal cluster densities
      Seven different libraries were quantitated using either the NEBNext Library Quant Kit or the Kapa Library Quantification Kit (Universal). Undiluted library concentrations ranged from 2–200 nM. Libraries were diluted to 8 pM and loaded onto a MiSeq® instrument (v2 chemistry; MCS v2.4.1.3). Libraries quantitated with the NEBNext kit resulted in a raw cluster density average of 1160 k/mm2, directly in the optimal range of 900–1300 k/mm2. In contrast, libraries loaded based on the Kapa quantitation averaged only 660 k/mm2.
      Figure 7: With the NEBNext Library Quant Kit, optimal cluster density is achieved from quantitated libraries with a broad range of library size and GC content
      Libraries of 310–963 bp from the indicated sources were quantitated using the NEBNext Library Quant Kit, then diluted to 8 pM and loaded onto a MiSeq (v2 chemistry; MCS v2.4.1.3). Library concentrations ranged from 7–120 nM, and resulting raw cluster density for all libraries was 965–1300 k/mm2 (ave. =1199). Optimal cluster density was achieved using concentrations determined by the NEBNext Library Quant Kit for all library sizes.
      Figure 8: Accurate Library Quantitation is achieved with a broad range of library size and GC content
      A selection of libraries successfully quantitated with the NEBNext Library Quant Kit. Libraries are plotted by size, ranging from smaller libraries (sRNA, FFPE) at 150–230 bp to largest libraries at 980 bp. Various input sources were used, ranging from 20-70% GC content, as indicated by text color. Libraries were prepared using NEBNext, Illumina TruSeq® Nano and Kapa Hyper library prep kits (data not shown). No dependence on size or GC content was observed in library quantitation when using NEBNext.
      This product is related to the following categories:
      NGS Library Quantitation,
      Next Generation Sequencing Library Preparation
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