repliQa HiFi ToughMix
- High Fidelity – >90x wild type Taq
- Extreme Speed – up to 3x faster PCR results with extension rates as fast as 1 kb/sec*
- Tough Tested – tolerant to a wide range of PCR inhibitors
- Superior Sensitivity – higher yields from lower inputs as little as 2 pg
- Long Range – amplify +24 kb gDNA, +40 kb λDNA
*for fragments less than 1 kb in size
repliQa HiFi ToughMix is intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.
repliQa HiFi ToughMix
Description
The repliQa HiFi ToughMix is a 2x, ready-to-use master mix that contains all the components for high fidelity PCR amplification, including a genetically modified DNA polymerase coupled with hot start antibodies. This unique, next generation master mix provides 90x higher fidelity compared to Taq, while reducing time to PCR results by 2-3x. The extreme speed is enabled by extension times as fast as 1-10 kb/sec depending on target length. The enzyme is coupled with the industry leading ToughMix which is tolerant to a wide variety of inhibitors making it suitable for routine PCR, cloning, amplicon sequencing and site directed mutagenesis.
Details
2x reaction buffer containing optimized concentrations of MgCl2, dNTP’s and proprietarily formulated HiFi polymerase, hot start antibodies and ToughMix chemistry
Performance Data
Extreme Speed: Up to 3x faster results
Comparison of speed. A 2 kb fragment was amplified in 50 μl reaction volumes according to the recommended protocol. Following a 30 s activation at 98°C; 30 cycles of PCR were performed: 98°C, 10 s; 60°C, 10 s; 68°C, 5–30 s. The thermal cycler had a ramp rate of 5°C/s.
Tough Tested: Tolerant to a wide range of PCR inhibitors
Strong Inhibitor Resistance. A 2 kb λ DNA template was amplified using each manufacturer’s recommended cycling conditions with different amounts io inhibitors. This experiment was run in duplicate.
Superior Yield and Sensitivity
Comparison of yield. A gDNA template was amplified with varying GC-content and length targets using each manufacturer’s recommended cycling conditions. The experiment was run using 8 different targets, in duplicate.
Long Amplification and Consistent GC Tolerance
A Long Range Capabilities. A range from 0.25 to 24 kb of gDNA templates were amplified with varying GC-content and lengths using recommended cycling
conditions. B/C Wide GC-content tolerance range. gDNA templates were amplified with varying GC-content and lengths using recommended cycling conditions.
Resources
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Application Notes
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repliQa HiFi ToughMix
I evaluated the repliQa® HiFi ToughMix® using the L segment of the virus that I’m working on (~9 kb) as a long-template test. PCR reactions were prepared in 20 µL total volume (10 µL of 2× ToughMix, 1.2 µL each of forward and reverse primers at 5 µM, 2 µL of template, and nuclease-free water). I used the three-step cycling protocol with an added initial denaturation at 98 °C for 15 s, followed by 30 cycles of 98 °C for 10 s, annealing for 15 s at the lowest primer Tm, and 68 °C for 50 s (~5 s per kb plus extra), and 5 Min final extension step. The reaction produced strong, clean amplification in only about 1 hour 20 minutes, compared to nearly 4 hours 30 minutes with Phusion. Although the manufacturer reports fidelity about twice that of Phusion and ~90× higher than Taq, I plan to confirm this experimentally by sequencing to ensure its accuracy given the exceptionally fast performance.
I tested on two constructs, mix worked on both constructs at a range of Tm +/- 3 ˚C (did not test any other temperatures). Reaction was completed in 40 minutes (30 cycles) which is much faster than what I get with other reagents. Was able to scale down reaction to 20 µL total volume and it still worked. Overall, I am happy with the project.