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| Product Name | 2× Gold High-Fidelity PCR Master Mix |
| Catalog No. | RESK-0035 |
| Description | This product is a ready-to-use 2× premixed solution containing a gold-grade high-fidelity DNA polymerase, dNTPs, and an optimized buffer system. The premix incorporates a hot-start factor for the polymerase, greatly improving amplification detection rates and product specificity. An added elongation factor enables long-fragment amplification of targets up to 10 kb. The enzyme exhibits 5'→3' DNA polymerase activity and 3'→5' exonuclease (proofreading) activity, with a fidelity 52-fold higher than Taq DNA polymerase and 6-fold higher than conventional Pfu DNA polymerase, and extension rates of up to 15 sec/kb. It is suitable for complex templates and produces blunt-ended products. The master mix is fast, convenient, highly sensitive, specific, and stable: only primers and template need to be added, and a two-step PCR program can be used, streamlining the workflow and saving time. This product contains no loading dye, so loading buffer must be added before electrophoresis. A specific protective agent keeps the premix active after repeated freeze-thaw cycles. |
| Intended Use | High-fidelity PCR amplification for gene cloning, site-directed mutagenesis, and accurate amplification of complex templates, long fragments, and templates of varying GC content. |
| Principle / Technology | Based on a Pfu-derived high-fidelity DNA polymerase whose 3'→5' exonuclease proofreading activity corrects misincorporated bases. A hot-start factor suppresses nonspecific activity before thermal cycling, and an elongation factor supports fast, long-fragment amplification. |
| Sample Type | Genomic DNA (e.g., human, mouse, rice, Arabidopsis), cDNA, plasmid DNA, and λ DNA templates, including complex templates and fragments with GC contents of 30%–70%. |
| Performance Range / Specifications | Fidelity: 52× Taq DNA polymerase and 6× conventional Pfu DNA polymerase; error rate approximately 0.000399% (Taq: ~0.0002, about one mutation per 5 kb) Extension rate: up to 15 sec/kb; extreme speed of 1 sec/kb Amplification length: targets up to 10 kb; human gDNA/cDNA ≤10 kb; λ DNA ≤20 kb; verified up to 20 kb for simple templates and 10 kb for complex templates Sensitivity: amplifies templates as low as 1 pg Annealing temperature: 68°C GC range: 30%–70% Product ends: blunt ends Stability: retains stable activity after repeated freeze-thaw cycles |
| Specificity | Hot-start factor greatly improves amplification specificity and detection rate, yielding a single target band; outperforms comparable high-fidelity polymerases in specificity and yield. |
| Reaction Conditions / Protocol | 2× ready-to-use format: add only primers and template. A two-step PCR program can be used. Recommended annealing temperature: 68°C; extension rate up to 15 sec/kb (extreme 1 sec/kb). No loading dye in the mix; add loading buffer before electrophoresis. |
| Components / Formulation | 2× ready-to-use premixed solution containing gold-grade high-fidelity DNA polymerase, dNTPs, and an optimized buffer system; contains hot-start factor, elongation factor, and a specific protective agent; contains no loading dye. |
| Storage Conditions | Store at -20°C. |
| Shelf Life | 2 years. |
| Package Specifications | 1 mL 100 μL 5× 1 mL |
| Product Form | Master mix (2× premix) |
| Concentration | 2× |
| Source / Origin | Polymerase component genetically engineered from Pfu DNA polymerase. |
| Shipping Conditions | Shipped with ice packs. |
| Expiration Date / Stability | Contains a specific protective agent; the premix maintains stable activity after repeated freeze-thaw cycles. Valid for 2 years when stored at -20°C. |
| Compatibility | Efficiently amplifies fragments of different lengths (1–12 kb demonstrated) and GC contents (30%–70%) from diverse templates, including gDNA, cDNA, plasmid DNA, and λ DNA. Not recommended for direct amplification from yeast colonies unless cells are first disrupted (e.g., boiling, freeze-thawing, or snailase treatment). |
| Recommended Buffer System | Built-in optimized buffer system supplied at 2× concentration. |
| Application Notes / Precautions | Error rate is approximately 0.000399%, giving 52× higher fidelity than Taq (Taq error rate ~0.0002, about one mutation per 5 kb). Fidelity derives from 3'→5' exonuclease proofreading activity. Products are blunt-ended. To add A-tails for TA cloning: purify the PCR product (≥20 ng/μL), combine 14.5 μL of product with 2 μL Taq buffer, 3 μL dNTPs, and 0.5 μL Taq polymerase, incubate at 72°C for 10–20 min, then purify. Not recommended for direct amplification from yeast suspensions; if needed, disrupt the cell wall first by boiling, repeated freeze-thawing, or snailase treatment. Targets within the verified range (up to 20 kb for simple templates, 10 kb for complex templates) are theoretically amplifiable; actual results depend on template and primer quality. |
| Batch-to-Batch Consistency | The premixed format reduces manual pipetting errors and improves experimental throughput and result reproducibility. |
For research use only, not for clinical use.
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