New England Biolabs, M0543L, NEBNext® Q5® Hot Start HiFi PCR Master Mix

Related Categories Master Mixes,, Next Generation Sequencing Library Preparation Applications Polymerases for NGS Library Preparation,, Hot Start PCR,, Fast PCR, Specification Heat Inactivation No FAQ Q: What’s New About NEBNext® Q5 Hot Start HiFi PCR Master Mix? A: NEBNext Q5 Hot Start HiFi PCR Master Mix has been optimized for increased PCR yields during PCR amplification of NGS libraries. 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: What types of beads can be used with NEBNext® Q5 Hot Start HiFi PCR Master Mix? A: NEBNext Q5 Hot Start HiFi PCR Master Mix can be used in workflows involving carboxylated magnetic beads such as Agencourt® AMPure® XP (Beckman Coulter, Inc), Sera-MagTM SpeedBeadsTM (GE Healthcare Life Sciences) and Mag-Bind® RXNPure (Omega Bio-tek, Inc), along with carboxylated streptavidin beads such as Dynabeads® MyOneTM Streptavidin C1 and Dynabeads® M-270 Streptavidin (Life Technologies). It can also be used with tosylated streptavidin beads such as Dynabeads® MyOne TM Streptavidin T1 and Dynabeads® Streptavidin M-280. (Life Technologies.) Because bead manufacturers vary and bead concentrations are not always disclosed, we recommend minimizing the quantity of beads present in the PCR where possible. Q: Is NEBNext® Q5 Hot Start HiFi PCR 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 Q5 Hot Start HiFi PCR 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: Is NEBNext® Q5 Hot Start HiFi PCR Master Mix the same as NEBNext® High-Fidelity 2X PCR Master Mix (M0541)? A: No. Although both master mixes contain Q5 High-Fidelity DNA Polymerase, the master mix composition is different. NEBNext Q5 Hot Start HiFi PCR Master Mix has been specifically optimized for even more robust amplification of NGS libraries in the presence or absence of magnetic beads and its hot start nature makes it ideal for workflows involving automation. Q: Is NEBNext® Q5 Hot Start HiFi PCR Master Mix the same as NEBNext® High-Fidelity 2X PCR Master Mix (M0541), or Q5 Hot Start High-Fidelity 2X PCR Master Mix (M0494)? A: No. Although all three master mixes contain Q5 High-Fidelity DNA Polymerase, only NEBNext Q5 Hot Start HiFi PCR Master Mix has been optimized for especially robust, low bias amplification. Its hot-start nature and ability to amplify in the presence of magnetic beads give it advantages over its predecessor, NEBNext High-Fidelity 2X PCR Master Mix, in NGS applications. Q5 Hot Start High-Fidelity 2X PCR Master Mix also contains the hot start aptamer for convenient room temperature setup, but it has been optimized for amplification of a variety of templates including longer amplicons. Q: Do I need to make any changes to the cycling conditions for my NGS library amplification I have previously been using with Phusion High-Fidelity PCR Master Mix? A: Yes. Cycling conditions for NEBNext Q5 Hot Start HiFi PCR Master Mix differ from those recommended for Phusion High-Fidelity PCR Master. NEBNext Q5 Hot Start HiFi PCR Master Mix has been optimized for robust, low-bias amplification of NGS libraries. We recommend using an extension temperature of 65C for this application. Please consult the guidelines included in the NEBNext kit product manuals for more specifics. Q: How many cycles of PCR should I perform with NEBNext® Q5 Hot Start HiFi PCR Master Mix? A: This depends on input amount. For use with NEBNext kits, consult the guidelines included in the product manuals. 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 Q5 Hot Start HiFi PCR Master Mix produces blunt ends. Q: What do I do if I see precipitation in the NEBNext Q5 Hot Start HiFi PCR Master Mix? A: A precipitate can form upon thawing of the NEBNext Q5 Hot Start HiFi PCR Master Mix. To ensure optimal performance, allow the master mix to reach room temperature. Once thawed, gently mix by inverting the tube several times. Q: What are the advantages of using NEBNext® Q5 Hot Start HiFi PCR Master Mix? A: NEBNext Q5 Hot Start HiFi PCR Master Mix provides a unique combination of ultra-high fidelity and robust amplification of NGS libraries, regardless of GC content. It gives even higher PCR yields than its predecessor, NEBNext High-Fidelity 2X PCR Master Mix. In addition, due to its hot start nature, NEBNext Q5 Hot Start HiFi PCR Master Mix is ideal for workflows involving automation. It is compatible with a variety of carboxylated, tosylated and streptavidin beads that may be carried over or included in the PCR step of library construction protocols. Q: Will NEBNext® Q5 Hot Start HiFi PCR Master Mix incorporate dUTPs? A: No. NEBNext Q5 Hot Start HiFi PCR Master Mix will not incorporate dUTPs and is not compatible with bisulfite-treated DNA. For protocols employing bisulfite-treated DNA, EpiMark™ Hot Start DNA Polymerase can be used.