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| Product Name | 2× Hot-Start PCR Genotyping Master Mix (With Dye) |
| Catalog No. | RESK-0048 |
| Description | The 2× Hot-Start PCR Genotyping Master Mix (With Dye) is a ready-to-use PCR premix containing a hot-start Taq DNA polymerase, dNTPs, and an optimized buffer system. Only primers and template need to be added, greatly streamlining the workflow, supporting high-throughput operation, and improving the reproducibility of results. The hot-start Taq DNA polymerase is modified with a temperature-responsive ligand that regulates polymerase activity: the enzyme is fully blocked at room temperature and its activity is released only after heating at 95°C. Activation requires just 2-3 minutes, making the mix fully compatible with existing PCR programs. By preventing non-specific amplification during reaction setup and the initial temperature ramp, the mix delivers reliable performance in genotyping assays. A tracking dye is included for direct gel loading, and PCR products carry 3'-dA overhangs suitable for TA cloning. |
| Intended Use | PCR-based genotyping, including mouse genotyping and multiplex PCR (3-4 targets). |
| Principle / Technology | Ligand-modified hot-start Taq DNA polymerase: a temperature-responsive ligand completely blocks enzyme activity at room temperature and releases it upon heating at 95°C. Activation takes only 2-3 minutes and is compatible with standard PCR programs, preventing non-specific amplification during sample preparation and the ramping phase. |
| Sample Type | Genotyping samples such as mouse tissue (e.g., mouse tail) and crude bacterial or cell lysates; templates can be amplified without purification. |
| Performance Range / Specifications | Extension speed: as fast as 1 sec/kb (standard recommendation 30 sec/kb). Amplicon length: up to 1 kb. GC compatibility: 30-60% GC content. Annealing temperature: 50-60°C. Stability: full performance retained after 7 days at 4°C or 37°C. |
| Specificity | Hot-start mechanism prevents non-specific amplification during reaction setup and the temperature ramp, enabling accurate genotyping results. |
| Reaction Conditions / Protocol | Hot-start activation: 95°C for 2-3 minutes. Annealing temperature: 50-60°C. Extension time: 30 sec/kb (as fast as 1 sec/kb for short targets such as 200-500 bp fragments). Add only primers and template to the 2× mix. |
| Components / Formulation | Ligand-modified hot-start Taq DNA polymerase, dNTPs, optimized buffer system, enzyme stabilizers, and a tracking dye for direct gel loading. |
| Storage Conditions | Store at -25°C to -15°C. |
| Shelf Life | 2 years when stored at -25°C to -15°C. |
| Package Specifications | 1 mL; 5× 1 mL; 50× 1 mL; 100× 1 mL |
| Product Form | Premixed master mix (Mix) |
| Quality Control | Performance verified by amplification of 200 bp and 500 bp targets at an extension speed of 1 sec/kb; stability confirmed after 7 days at 4°C and 37°C. |
| Concentration | 2× |
| Source / Origin | Thermostable Taq DNA polymerase modified with a temperature-responsive hot-start ligand. |
| Expiration Date / Stability | Stable for 2 years at -25°C to -15°C. Contains enzyme stabilizers; full amplification performance retained after 7 days at 4°C or 37°C. |
| Compatibility | Compatible with existing PCR programs (2-3 min hot-start activation at 95°C); PCR products carry 3'-dA overhangs suitable for TA cloning; contains a tracking dye for direct gel loading. |
| Application Notes / Precautions | Not recommended for PAGE electrophoresis, as components in the mix may interfere with polyacrylamide gels. Reaction volumes can be scaled down proportionally, reducing primers and template accordingly. Both dye-containing and dye-free products are suitable for downstream sequencing, since sequencing providers purify the PCR products first. Direct restriction digestion of PCR products is not recommended, as some components may affect digestion efficiency. The enzyme has no 3'-5' exonuclease activity; products carry 3'-dA overhangs and are suitable for TA cloning. |
| Batch-to-Batch Consistency | Formulated with enzyme stabilizers; consistent amplification performance maintained after 7 days at 4°C or 37°C. |
For research use only, not for clinical use.
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