Get precise results with Q5's high fidelity (~280x Taq)
Reduce turnaround time with fast cycling conditions (5–15 seconds/kb extension times)
Optimized formulation enables robust, reliable amplification of diverse targets (from high AT to high GC) up to 20 kb and supports amplification of simple templates up to 30 kb
Improve reaction specificity with a hot start aptamer that allows convenient room temperature setup
Compatible with a 62°C universal annealing temperature protocol. Learn more in our application note
When DNA targets are long or complex, or when speed is a priority, Q5‑XT Hot Start High-Fidelity 2X Master Mix provides a faster, more robust amplification solution while maintaining the performance you expect of Q5. With Q5-XT, spend less time troubleshooting, have confidence in the accuracy of your amplicon, and accelerate your workflows.
The Q5‑XT Hot Start High‑Fidelity 2X Master Mix contains Q5 DNA Polymerase, a high‑fidelity, thermostable enzyme with 3′→5′ exonuclease activity, fused to an Sso7d-derived domain to enhance processivity. Its optimized buffer system delivers high yields when amplifying long targets, strong performance on short targets, and fast thermocycling with extension times of 5–15 seconds per kb. An aptamer‑based hot start inhibitor enables convenient room‑temperature setup while reducing non‑specific amplification.
With an error rate approximately 280‑fold lower than Taq DNA Polymerase, Q5 is well suited for applications that require exceptionally accurate DNA amplification. The 2X master mix includes dNTPs, Mg++, and a broadly compatible buffer designed to support robust amplification across a wide range of AT and GC content. At a 1X working concentration, Q5‑XT provides 1.6 mM Mg++.
Q5‑XT is compatible with a 62 °C universal annealing temperature. For optimal results and performance, primer‑specific annealing temperatures are recommended. To determine the optimal annealing temperatures for a given set of primers, use of the NEB Tm Calculator is highly recommended.
Figure 1: Q5-XT enables amplification on long DNA targets
Amplification of specific sequences from human genomic DNA (A) or Lambda phage DNA (B) using Q5-XT High-Fidelity 2X Master Mix. Amplicon sizes are indicated above the gel. Ladder (L) is Quick-Load® 1 kb Extend DNA Ladder (NEB #N3239). All PCR products were visualized by ethidium bromide staining on 1.2% TAE agarose gel.
Figure 2: Q5-XT performs well on a broad range of DNA sizes
PCR assays were performed on six targets with human genomic DNA, utilizing various hot start, high-fidelity PCR master mixes according to each manufacturer's recommended protocols. Ladder (L) is Quick-Load® 100 bp DNA Ladder (NEB #N0467). All PCR products were visualized by ethidium bromide staining on 1.2% TAE agarose gel.
Figure 3: Q5-XT is compatible with a universal annealing temperature
PCR assays were conducted using a universal thermocycling protocol with a single annealing temperature of 62°C for all nine amplicons regardless of the calculated Ta as obtained using the NEB Tm Calculator. An extension time of 10 seconds was used. All PCR products were visualized by ethidium bromide staining on 1.2% TAE agarose gel. The data highlights that a single annealing temperature and extension time can be used successfully with Q5-XT to support multiple different PCR assays simultaneously, offering significant assay flexibility and convenience. However, when the experimental annealing temperature deviates from the optimal primer annealing temperature, reduced product yield (i.e., reaction efficiency) or poor specificity may occur. As a result, when trying to achieve the best performance in PCR, we recommend using the optimal annealing temperature as determined by a temperature gradient or Tm Calculator. However, when convenience is desired or necessary, a single annealing temperature for multiple different primer sets can be used. Learn more in our application note. Ladder (L) is Quick-Load® 100 bp DNA Ladder (NEB #N0467).
Figure 4: Q5-XT supports robust DNA amplification on a broad range of GC content
For targets over 65% GC content, the addition of 5X Q5 GC Enhancer, available as a component of Q5 Reaction Buffer Pack (NEB #B9027), is recommended.
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