Description
High-fidelity PCR master mix
GenieFusion Flash High-Fidelity 2X Master Mix is the ready-to-use master-mix format of the GenieFusion Flash enzyme. It pairs high fidelity (~106× lower error rate than Taq DNA polymerase) with fast 5 sec/kb extension, so fragments up to 10 kb are amplified in about an hour. Just add primers and template.
The mix contains GenieFusion Flash High-Fidelity DNA Polymerase, dNTPs and an optimised buffer. Hot-start technology and specificity-enhancing factors raise the success rate, and the premixed format cuts pipetting steps for better throughput and reproducibility. It amplifies genomic DNA, cDNA, dU-containing DNA and crude samples, including GC-rich templates. Products are blunt-ended and ready for blunt-end, seamless or TOPO cloning, such as with the GenieClone Ultra DNA Assembly Kit. For research use only.
Key Features
| High fidelity | Error rate ~106× lower than Taq DNA polymerase. | |
| Flash amplification | Extension time as short as 5 sec/kb for fragments up to 10 kb. | |
| Robust performance | Hot-start formulation gives high specificity, sensitivity and yield. | |
| Broad compatibility | Works with complex and crude samples and GC-rich templates. |
Specifications
| Format | 2X ready-to-use master mix (polymerase, dNTPs, optimised buffer) |
| Fidelity | ~106× higher than Taq DNA polymerase |
| Extension speed | 5 sec/kb (≤10 kb); 10 sec/kb (>10 kb) |
| Templates | Genomic DNA, cDNA, dU-containing DNA and crude samples |
| Product ends | Blunt |
| Shelf life | 2 years |
Components
| Components | Amount |
| GenieFusion Flash High-Fidelity 2X Master Mix | 1 ml / 5 × 1 ml / 15 × 1 ml |
Storage
Store at -30 ~ -15°C.
- Prepare the reaction system
- Work on ice. Thaw, mix and briefly centrifuge each component before use, and return the mix to -20°C promptly after use.
- Set up a 50 μl reaction as follows:
| Components | Volume |
| ddH2O | Up to 50 μl |
| GenieFusion Flash High-Fidelity 2X Master Mix | 25 μl |
| Primer 1 (10 μM) | 2 μl |
| Primer 2 (10 μM) | 2 μl |
| Template DNA* | x μl |
*Recommended template amount in a 50 μl reaction:
| Template type | Amount |
| Genomic DNA | 10–500 ng |
| Plasmid or virus DNA | 10 pg - 20 ng |
| cDNA | 1–5 μl (≤1/10 of the total PCR volume) |
- Run the reaction program
- Use either the standard or the fast program; both give equivalent performance.
Standard program
| Step | Temperature | Time | Cycles |
| Initial denaturation | 98°C | 30 sec | 1 |
| Denaturation | 98°C | 10 sec | 28–35 |
| Annealinga | Tm + 5°C | 5 sec | 28–35 |
| Extensionb | 72°C | 5–10 sec/kb | 28–35 |
| Final extension | 72°C | 1 min | 1 |
Fast program
| Step | Temperature | Time | Cycles |
| Denaturation | 98°C | 10 sec | 28–35 |
| Annealinga | Tm + 5°C | 5 sec | 28–35 |
| Extensionb | 72°C | 5–10 sec/kb | 28–35 |
a Use an annealing temperature of primer Tm + 5°C, no higher than 68°C.
b Extension time: 5 sec/kb for fragments ≤10 kb; 10 sec/kb for fragments >10 kb.
Q1: What should I do if there is no amplification or the yield is low?
- Optimise primer design; run an annealing temperature gradient to find the optimum; increase primer concentration; extend the extension time to 10–15 sec/kb; increase cycles to 36–40; use high-purity template; and increase the template amount within the recommended range.
Q2: How can I reduce nonspecific products or smeared bands?
- Optimise primer design; raise the annealing temperature or run a temperature gradient; decrease primer concentration; reduce cycles to 25–30; use high-purity template; and decrease the template amount within the recommended range.
Q3: Why are PCR products stuck in the agarose gel wells?
- This is usually caused by excess template, too many cycles, a suboptimal annealing temperature or aerosol contamination. Clean the work area thoroughly (or replace reagents and consumables) before re-amplifying, decrease the template amount, reduce cycles to 25–30 and run an annealing temperature gradient.
Q4: Which extension time should I use?
- Use 5 sec/kb for fragments up to 10 kb and 10 sec/kb for fragments longer than 10 kb.
Q5: Can I use the PCR products for TA cloning?
- The products are blunt-ended because the polymerase has strong proofreading activity. For TA cloning, purify the product first and then add an A-tail.
