Figure 1: Typical amplification and standard curves for the NEBNext Library Quant KitTypical 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 workflowFigure 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 KitThree 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 KitAccurate 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 densitiesSeven 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 contentA 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.
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