Description
High-fidelity DNA polymerase for PCR
GenieFusion Flash High-Fidelity DNA Polymerase combines high fidelity (~106× lower error rate than Taq DNA polymerase) with rapid 5 sec/kb amplification, so fragments up to 10 kb are completed in about one hour. It is supplied as a standalone enzyme with its own buffer, which keeps reaction setup simple.
The enzyme was developed by directed evolution. Hot-start technology and specificity-enhancing factors significantly improve specificity and success rate. It is broadly compatible with crude templates, high GC/AT templates (overall GC 24%–79%, local GC 22%–88%) and methylated templates, and amplifies genomic DNA, cDNA, dU-containing DNA and crude samples. Products are blunt-ended and ready for seamless or TOPO cloning, for example with the GenieClone Ultra DNA Assembly Kit. For research use only.
Key Features
| Flash amplification | Fragments up to 10 kb are amplified in about 1 hour at 5 sec/kb. | |
| High fidelity | Error rate ~106× lower than Taq DNA polymerase. | |
| Broad compatibility | Handles crude, methylated and high GC/AT templates (overall GC 24%–79%, local GC 22%–88%). | |
| Convenient setup | Standalone enzyme plus a 2× buffer that already contains Mg2+ and dNTPs. |
Specifications
| Format | Standalone hot-start DNA polymerase with 2× buffer (contains Mg2+ and dNTPs) |
| 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 |
| GC tolerance | Overall GC 24%–79%, local GC 22%–88% |
| Product ends | Blunt |
| Shelf life | 2 years |
Components
| Components | 100 U | 500 U | 1,000 U |
| GenieFusion Flash High-Fidelity DNA Polymerase | 100 μl | 5 × 100 μl | 10 × 100 μl |
| 2× GenieFusion Flash Buffer | 2 × 1.25 ml | 10 × 1.25 ml | 20 × 1.25 ml |
| 10× DNA Loading Buffer | 1.25 ml | 5 × 1.25 ml | 10 × 1.25 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 reagents to -20°C promptly after use.
- Set up a 50 μl reaction as follows:
| Components | Volume |
| ddH2O | Up to 50 μl |
| 2× GenieFusion Flash Buffera | 25 μl |
| Forward primer (10 μM) | 2 μl |
| Reverse primer (10 μM) | 2 μl |
| GenieFusion Flash High-Fidelity DNA Polymerase | 1 μl |
| Template DNAb | x μl |
a The 2× buffer already contains Mg2+ (final concentration 2 mM) and dNTPs (final concentration 0.4 mM).
b 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?
- Clean the work area thoroughly to avoid aerosol contamination (or replace reagents and consumables) before re-amplifying; decrease the template amount; reduce cycles to 25–30; and run an annealing temperature gradient to find the optimum.
Q4: Do I need to add Mg2+ or dNTPs?
- No. The 2× buffer already contains Mg2+ (final concentration 2 mM) and dNTPs (final concentration 0.4 mM).
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.
