NEBNext® Ultra™ II Q5® Master Mix

Description







 
The NEBNext Ultra II Q5® Master Mix is a new formulation of Q5 DNA polymerase that has been optimized for robust, high-fidelity amplification of next-generation sequencing (NGS) libraries. This new formulation further improves the uniformity of amplification of libraries, including superior performance with GC-rich regions.

The polymerase component of the master mix, Q5 High-Fidelity DNA Polymerase, is a novel thermostable DNA polymerase that possesses 3´→5´ exonuclease activity, and is fused to a processivity-enhancing Sso7d domain. Q5 also has the highest fidelity available (> 100-fold higher than that of Taq DNA Polymerase and ~12-fold higher than that of Pyrococcus furiosus (Pfu) DNA Polymerase), resulting in ultra-low error rates.

The NEBNext Ultra II Q5 Master Mix is an aptamer-based hot start formulation that allows convenient room temperature reaction set up. The convenient 2X master mix format contains dNTPs, Mg++ and a proprietary buffer, and requires only the addition of primers and DNA template for robust amplification. NEBNext Ultra II Q5 Master Mix is also included in the NEBNext Ultra II DNA Library Prep Kit for Illumina®.

NEBNext Ultra II Q5 Master Mix provides improved coverage of known low coverage regions of the human genome.
Libraries were prepared from Human NA19240 genomic DNA. One library was not amplified. The other two libraries were amplified using 5 cycles of PCR with NEBNext Q5 Hot Start HiFi PCR Master Mix (NEB #M0543) or with NEBNext Ultra II Q5 Master Mix (NEB #M0544). Libraries were sequenced on an Illumina® NextSeq® 500. 420 million 75 bp reads were randomly extracted from each dataset, representing an average coverage of 10X. Reads were mapped to the GRCh37 reference genome using Bowtie 2.2.4. Reads on each region were counted using bedtools v2.19.1. A: The number of reads overlapping distinct low coverage regions of the human genome (1) are shown for each library. B: From the 420 million 75 bp reads randomly extracted from each dataset, 10X coverage was expected. The % of difficult regions covered at > 10X are shown for each library. The NEBNext Ultra II Q5 Master Mix provides improved coverage of these known low coverage regions, without drop-outs, and shows similar coverage to the unamplified sample.

Ultra II provides the highest and most uniform coverage of difficult sequence regions.
Indexed libraries were prepared from 100 ng of Human NA19240 genomic DNA using a PCR-free workflow or the library prep kits shown, following manufacturers’ recommendations. The PCR-free library was prepared using NEBNext Ultra II. Libraries were sequenced on the Illumina NextSeq® 500. 420 million reads were randomly extracted from each dataset, to produce an average coverage of 10X. Reads were mapped to the GRCh37 reference genome using Bowtie 2.2.4. Reads on each region were counted using bedtools v2.19.1. The number of reads overlapping distinct difficult, low-coverage regions of the human genome (1) are shown for each library. Ultra II provides the highest and most uniform coverage of these difficult regions, and provides the coverage closest to that obtained with a PCR-free protocol.
NEBNext Ultra II Q5 Master Mix provides uniform GC coverage for microbial genomic DNA with a broad range of GC composition.


Libraries were made using 100 ng of the genomic DNAs shown and the NEBNext Ultra II DNA Library Prep Kit. Libraries were amplified using the NEBNext Ultra II Q5 Master Mix, and sequenced on an Illumina MiSeq. GC coverage information was calculated using Picard’s CollectGCBiasMetrics (v1.117). Expected normalized coverage of 1.0 is indicated by the horizontal grey line, the number of 100 bp regions at each GC% is indicated by the vertical grey bars, and the colored lines represent the normalized coverage for each library. NEBNext Ultra II Q5 Master Mix provides uniform GC coverage regardless of the GC content of the DNA.
Stability: Stability testing using up to 30 freeze/ thaw cycles has shown no negative effect on master mix performance. The NEBNext Ultra II Q5 Master Mix may be stored at –20°C for up to 18 months.

Source
An E. coli strain that carries the Q5 High-Fidelity DNA Polymerase gene.

Reaction Conditions
NEBNext Ultra II Q5 Master Mix, DNA template and 1 μM primers in a total reaction volume of 50 μl.

Advantages and Features

Applications

  • Next generation sequencing library construction
  • High-fidelity PCR
  • Difficult amplification
  • High-throughput PCR

Properties and Usage

Storage Temperature

-20°C

References

  1. Aird, D. et al. (2011). Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries. Genome Biology. 12(2), R18.

FAQs

  1. What’s New About NEBNext Ultra II Q5 Master Mix?
  2. Is NEBNext Ultra II Q5 Master Mix a hotstart?
  3. Is NEBNext Ultra II Q5 Master Mix the same as the Q5 Hot Start High-Fidelity 2X Master Mix (M0494)?
  4. What is the difference between NEBNext Ultra II Q5 Master Mix and NEBNext Q5 Hot Start HiFi PCR Master Mix (M0543)?
  5. What is the difference between NEBNext Ultra II Q5 Master Mix and NEBNext High-Fidelity 2X PCR Master Mix (M0541)?
  6. What cycling conditions should I use for NEBNext Ultra II Q5 Master Mix?
  7. How many cycles of PCR should I perform with NEBNext Ultra II Q5 Master Mix?
  8. Can I scale up the PCR reaction volume in order to increase library yields?
  9. Can I use NEBNext Ultra II Q5 Master Mix to amplify low quality DNA, such as FFPE DNA?
  10. Is NEBNext Ultra II Q5 Master Mix compatible with other NGS library kits?
  11. Will NEBNext Ultra II Q5 Master Mix incorporate dUTPs?
  12. Can I use NEBNext Ultra II Q5 Master Mix to amplify bisulfite-converted libraries?
  13. Are the DNA products produced by NEBNext Q5 Hot Start HiFi PCR Master Mix blunt-ended or do they have a single base 3’ overhang?
  14. Is NEBNext Ultra II Q5 Master Mix compatible with beads typically used in NGS workflows?
  15. Can I use NEBNext Ultra II Q5 Master Mix to do simple end-point PCR experiments?

Protocols

  1. Protocol for a PCR reaction using NEBNext® Ultra™ II Q5® Master Mix (M0544)
  2. KLD Enzyme Mix Reaction Protocol (M0554)

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

Quality Control

Quality Control Assays

The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page. Further information regarding NEB product quality can be found here.
  • Functional Test (Multiplex PCR, Bead Inhibition):
    The polymerase master mix is tested in a multiplex polymerase chain reaction (PCR) using a control template, specific primers and carboxylated magnetic beads, resulting in the expected products.
  • Non-Specific DNase Activity (16 hour):
    The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.
  • Phosphatase Activity (PNPP):
    The product is tested in a reaction containing a p-nitrophenyl phosphate (PNPP), a chromogenic substrate for most phosphatases. After incubation the percent degradation is determined by spectrophotometric analysis of released p-nitrophenol at 405 nm.

Safety Data Sheet

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.