New England Biolabs, M0544L, NEBNext® Ultra™ II Q5® Master Mix

NEBNext Ultra II Q5 Related Categories DNA Library Prep for Illumina,, Analysis of DNA Methylation by Bisulfite Conversion,, Automation for NEBNext® NGS Library Prep, Applications PCR FAQ Q: What’s New About NEBNext Ultra II Q5 Master Mix? A: NEBNext Ultra II Q5 Master Mix has been optimized for increased PCR yields of NGS libraries, with minimal GC bias. Its aptamer-based, hot start nature makes it ideal for automation, and it is compatible with many carboxylated, tosylated and streptavidin magnetic beads often used with NGS workflows. Q: Is NEBNext Ultra II Q5 Master Mix a hotstart? A: Yes, the NEBNext Ultra II Q5 Mast Mix is a hotstart. You can set up the PCR reaction on ice or at ambient temperature. Q: Is NEBNext Ultra II Q5 Master Mix the same as the Q5 Hot Start High-Fidelity 2X Master Mix (M0494)? A: No. Although both master mixes contain Q5 Hot Start High-Fidelity DNA Polymerase, the master mix composition is different. NEBNext Ultra II Q5 Master Mix has been specifically optimized for robust amplification of NGS libraries, while Q5 Hot Start High-Fidelity 2X Master Mix has been optimized for amplification of a variety of templates including longer amplicons. Q: What is the difference between NEBNext Ultra II Q5 Master Mix and NEBNext Q5 Hot Start HiFi PCR Master Mix (M0543)? A: Although both master mixes contain Q5 Hot Start High-Fidelity DNA Polymerase, the master mix composition is different. NEBNext Ultra II Q5 Master Mix has been specifically optimized to faithfully amplify NGS libraries with the least amount of amplification bias. Additionally, the NEBNext Ultra II Q5 Master Mix does not form a precipitate. In contrast, NEBNext Q5 Hot Start HiFi PCR Master Mix sometimes forms a precipitate and requires additional mixing. The recommended number of PCR cycles are different for each formulation and it is important that the correct cycling guidelines are followed. Q: What is the difference between NEBNext Ultra II Q5 Master Mix and NEBNext High-Fidelity 2X PCR Master Mix (M0541)? A: Although both master mixes contain Q5 High-Fidelity DNA Polymerase, the master mix composition is different. NEBNext Ultra II Q5 Master Mix has been specifically optimized for to faithfully amplify NGS libraries with the least amount of amplification bias. Additionally, amplification of NGS libraries in the presence or absence of magnetic beads is more robust, and it is a hot start, making it ideal for workflows involving automation. Q: What cycling conditions should I use for NEBNext Ultra II Q5 Master Mix? A: We have optimized the cycling condition for robust, low-bias amplification of NGS libraries. We recommend an extension temperature of 65°C for this application. If using primers other than NEBNext primers, you may need to optimize annealing temperature. Q: Can I scale up the PCR reaction volume in order to increase library yields? A: We recommend a PCR volume of 50μl. Simply scaling up the PCR reaction does not increase the yield. You may gradually increase the PCR cycle number and/or increase the primer concentration in a 50μl PCR reaction to achieve higher yields. Please note that it is common for libraries with yields of 2μg or higher tend to show an amplification bias. Q: How many cycles of PCR should I perform with NEBNext Ultra II Q5 Master Mix? A: This depends on input amount. For use with NEBNext kits, consult the guidelines included in the protocol card or with the specific library kit protocol. Q: Can I use NEBNext Ultra II Q5 Master Mix to amplify low quality DNA, such as FFPE DNA? A: Yes. However, as with most products, you may expect a lower yield with severely damaged DNA such as DNA from FFPE samples. Q: Will NEBNext Ultra II Q5 Master Mix incorporate dUTPs? A: No. NEBNext Ultra II Q5 Master Mix will not incorporate dUTPs and is not compatible with bisulfite-treated DNA. For protocols employing bisulfite-treated DNA, NEBNext(r) Q5U(r) Master Mix (or EpiMark(tm) Hot Start DNA Polymerase) can be used. Q: Can I use NEBNext Ultra II Q5 Master Mix to amplify bisulfite-converted libraries? A: No. NEBNext Ultra II Q5 Master Mix contains a family B-type DNA polymerase that cannot tolerate uracil/dUTP. NEBNext Ultra II Q5 Master Mix is hence not compatible bisulfite-converted DNAs and cannot incorporate dUTP. For protocols employing bisulfite-treated DNA, EpiMark™ Hot Start DNA Polymerase can be used. Q: 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? A: As with all Q5 products, NEBNext Ultra II Q5 Master Mix produces blunt ends. Q: Is NEBNext Ultra II Q5 Master Mix compatible with beads typically used in NGS workflows? A: Yes, PCR reactions using NEBNext Ultra II Q5 Master Mix can be set up in the presence of various carboxylated or tosylated beads used in typical NGS workflows, including AMPure beads and streptavidin beads. Q: Can I use NEBNext Ultra II Q5 Master Mix to do simple end-point PCR experiments? A: Yes. We also offer another Q5 master mix, M0494, which is optimized for typical end-point PCR. If using NEBNext Ultra II Q5 Master Mix for typical end-point PCR, we recommend using the M0494 cycling conditions that has an extension temperature of 72°C. More information can be found here.