YALEPIC^® Pfu High-Fidelity PCR Master Mix

The fidelity of this product is approximately 108 times that of regular Taq DNA polymerase. The high fidelity comes from the polymerase’s strong 3′ to 5′ exonuclease activity (i.e., proofreading function), which can excise incorrectly incorporated nucleotides, ensuring highly accurate amplified sequences with an error rate far lower than conventional Taq enzyme.

The DNA polymerase used in this product has been genetically engineered with greatly enhanced processivity. The extension speed can reach 10 sec/kb, meaning a 10 kb fragment can be extended in 100 seconds, significantly faster than traditional high-fidelity enzymes (which typically require 30-60 sec/kb).

This product is available in two versions: YEM51007-A (non-Dye, standard) – does not contain tracking dye, suitable for applications requiring fluorescence signal detection (such as qPCR, NGS library amplification); YEM51007-B (Dye version) – contains electrophoresis tracking dye, allowing direct agarose gel electrophoresis after PCR, simplifying operation and saving time.

Yes. This product’s formulation has been optimized with strong complex template compatibility, capable of directly amplifying high GC content fragments within a certain range. For extremely high GC content difficult templates, the following optimization strategies are recommended: (1) Appropriately increase denaturation temperature or extend denaturation time; (2) Use gradient annealing temperature to test optimal conditions. For further optimization, contact YALI Biotech technical support for professional guidance.

This product can efficiently amplify genomic fragments up to 40 kb. For long fragment PCR (especially fragments >10 kb), the keys to success are: Template quality: using high-purity, intact genomic DNA is critical. Avoid template degradation or inhibitors. Primer design: for amplicons exceeding 20 kb, primer GC content should be above 50%, Tm above 60 degrees C, and length at least 24 nucleotides. Experimental technique: handle gently when preparing the reaction system to avoid physical shearing of long-chain DNA templates.

Possible causes and solutions: Low annealing temperature – increase annealing temperature; gradient annealing temperature optimization recommended (typically test 50-65 degrees C range). Poor primer design – redesign primers, ensure 3′ end specificity, avoid self-complementary primers. Primer concentration too high – reduce primer concentration (0.2-0.5 microM final concentration recommended). Inappropriate template amount – reduce template usage; 50-200 ng genomic DNA is typically appropriate. Too many cycles – reduce cycle number; 25-35 cycles typically sufficient for most amplification needs. If smearing persists, use freshly extracted high-quality template DNA and check the storage condition of all reagents.

No. The MasterMix may show small amounts of precipitate after low-temperature storage or repeated freeze-thaw, which is a normal phenomenon. Before use, remove the MasterMix and thaw at room temperature, gently inverting to mix every 10-15 minutes until the precipitate is fully dissolved, then briefly centrifuge to collect. Performance testing shows that multiple freeze-thaw cycles (up to 30) have no significant effect on PCR amplification performance. For long-term storage, aliquot after first use to reduce repeated freeze-thaw.

Follow these best practices to minimize PCR contamination risk: Spatial separation: designate a dedicated PCR setup area in the lab, strictly separated from template handling and PCR product analysis areas. Dedicated equipment: equip the setup area with dedicated pipettes, disposable filtered tips, and PCR tubes; avoid cross-use. Proper technique: briefly centrifuge PCR reagent tubes before opening to settle liquid to the bottom, reducing contamination of gloves and pipettes. Template management: dilute DNA templates at your own bench, bringing only the required dilutions into the setup area. Product isolation: never bring PCR tubes containing amplification products into the setup area after PCR; perform subsequent electrophoresis detection in designated areas. Set up controls: set up no-template negative controls (NTC) for every PCR reaction to monitor for DNA contamination.