For 100 x 25 µl PCR reactions: 1 x 1.3 ml UCP HiFidelity MasterMix (2x); 1 x 200 μl UCP Template Tracer, 25x; 1 x 50 μl UCP Master MixTracer, 125x; 1 x 1.9 ml UCP PCR Water
The dyes run at approximately 50 bp (orange) and 4000 bp (blue) on a 1% agarose gel.
When the blue template is added to the orange PCR Master Mix, the color changes to green, confirming that sample was added.
18S and 16S fragments were successfully amplified with annealing temperatures ranging from 55-72°C, using constant denaturation (98°C) and extension temperatures (72°C) over 35 cycles. The UCP HiFidelity PCR Kit demonstrates the tremendous flexibility of its reaction buffer.
Templates with high, medium and low GC content [85% (MCART), 65% (PROM1) and 25% (PROM410)] were successfully amplified using the same standard protocol and buffer conditions. The UCP HiFidelity PCR Kit prevents amplification bias due to GC content with no optimization required.
Seven targets varying in size and composition were amplified directly from 1 µl whole blood with no reaction optimization.
A 2 kb fragment of human HRPT1 was amplified using the UCP HiFidelity PCR Kit and competitor master mixes, following the recommended protocols and using a 25 µl final reaction volume. Reactions were amplified using 10 ng human gDNA. Parallel reactions were run, with no inhibitor (N.I.), with 25 ng humic acid (H.A.), 25 µM Heparin (H), 25 ng bile salt (B.S.) and with no template control (NTC). Analysis was performed using the QIAxcel. Only the UCP HiFidelity PCR Kit amplified the specific product in the presence of all inhibitors.
The UCP HiFidelity PCR Kit outperformed the competition with fidelity, 70x greater than Taq, in the rigorous Lacl Fidelity test.
As little as 0.01 pg E. coli and 0.05 pg Candida were successfully detected and NTC showed no background.