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PerfeCTa® NGS Quantification Kit – Illumina, ROX 500 reactions

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95151-500 – PerfeCTa® NGS Quantification Kit – Illumina, ROX 500...

SKU: 95155-500 Categories: , , ,

Description

Description

Accurate quantification of the number of amplifiable library molecules is the most critical step in the NGS workflow in obtaining high quality read data with next-generation sequencing technologies. The PerfeCTa NGS Library Quantification Kit uses real-time PCR to specifically quantify library molecules that possess the appropriate adapter tag at each end. These are the suitable template molecules for either emulsion PCR used for the Ion Torrent platform or Bridge PCR used for Illumina NGS platforms. PerfeCTa NGS Quantification kits simplify the library quantification process by providing stabilized, pre-diluted standards, pre-qualified primer sets, and an optimized dilution buffer for your NGS library samples. This minimizes pipetting errors and ensures reproducible and precise qPCR results, even with dilute samples. The robust qPCR performance of PerfeCTa SYBR Green SuperMix provides accurate quantification of NGS libraries with varying fragment sizes or GC content. Kits are available to support all major qPCR instrument platforms. A common problem with some NGS library quantification protocols is the use of DNA standards that are too concentrated and generate qPCR data that are outside of the linear dynamic range for many qPCR instruments. Improper baseline settings result in compressions between the highest concentrated DNA standards that in turn give rise to inflated PCR efficiencies and inaccurate library quantification results. The NGS DNA standards supplied with the PerfeCta NGS Library quantification kits have been carefully selected to avoid these artifacts and produce NGS library standard curves with exceptionally high linear regression correlation coefficients. The DNA standard for Illumina NGS platforms generates a 426-bp amplicon (48.8% GC). Primer sequences correspond to the “P5″ and “”P7” primer sequences for Illumina sequencing libraries: Illumina forward: 5’-AATGATACGGCGACCACCGA-3’ Illumina reverse: 5’-CAAGCAGAAGACGGCATACGA-3’ The Ion Torrent DNA standard produces a 183-bp amplicon (51.9% GC) using primers that target the “A” and “trP1” adaptor sequences: Ion Torrent forward: 5’-CCATCTCATCCCTGCGTGTC-3’ Ion Torrent reverse: 5’-CCTCTCTATGGGCAGTCGGTGAT-3’

Features & Benefits

  • Precise quantification of adapter-tagged library molecules
  • Accurate and sensitive method for NGS library quantification
  • Stabilized, prediluted standards for convenient use
  • Consistency across a broad range of samples

Contents

  • Library dilution buffer (10X)
  • Illumina primer mix
  • Illumina Standards 1-5
  • qPCR reagent (either standard, ROX, or Low-ROX)

Storage Conditions

Kit components are stable for 2 years when stored in a constant temperature freezer at or below -20°C, protected from light. For convenience, they may be stored unfrozen at 4ºC for up to 6 months. Reagent performance is unaffected by repetitive (>20X) freeze thaw. Prevent photobleaching of the SYBR Green I dye by avoiding prolonged exposure to light.

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