Description
Taq DNA polymerase for routine PCR
GenieTaq DNA Polymerase is a standard Taq enzyme for routine PCR, supplied with a Mg2+-containing 10× buffer. Its optimised buffer system makes it easy to get clean amplification results quickly, making it ideal for colony PCR, genotyping and general screening where a reliable, cost-effective result is needed.
The enzyme is the Thermus aquaticus DNA polymerase expressed in Escherichia coli and refined through multi-step purification, and it is free of endonuclease, exonuclease and bacterial DNA contamination. It has 5'→3' polymerase and 5'→3' exonuclease activity but no 3'→5' exonuclease activity, so PCR products carry a 3'-dA overhang for direct TA cloning into T vectors. Products are also compatible with seamless and TOPO cloning kits. It amplifies DNA from animal, plant and microbial sources. For research use only.
Key Features
| Classic, reliable performance | An optimised buffer system makes it easy to get good amplification results quickly. | |
| Ready for TA cloning | Products carry a 3'-dA overhang for direct cloning into T vectors. | |
| High purity | Free of contaminating endonuclease, exonuclease and bacterial DNA. | |
| Complete with buffer | Supplied with 10× Taq Buffer containing Mg2+. |
Specifications
| Enzyme | Thermus aquaticus DNA polymerase, recombinant (expressed in E. coli) |
| Concentration | 5 U/μl |
| Activities | 5'→3' polymerase and 5'→3' exonuclease; no 3'→5' exonuclease |
| Product ends | 3'-dA overhang (TA cloning compatible) |
| Applications | DNA amplification from animal, plant and microbial samples |
| Unit definition | One unit incorporates 10 nmol of dNTPs into acid-insoluble material in 30 min at 74°C, using activated salmon sperm DNA as template/primer. |
| Shelf life | 2 years |
Components
| Components | 10,000 U |
| 10× Taq Buffer (Mg2+ plus) | 10 × (4 × 1 ml) |
| GenieTaq DNA Polymerase (5 U/μl) | 10 × 200 μl |
The 10,000 U pack contains ten 1,000 U sets, each with 4 × 1 ml buffer and 200 μl enzyme.
Storage
Store at -30 ~ -15°C.
- Prepare the reaction system
- Taq DNA polymerase is somewhat active at room temperature. Set up reactions on ice, then transfer to the thermal cycler, to reduce nonspecific amplification during setup.
- Set up a 50 μl reaction as follows:
| Components | Volume |
| ddH2O | To 50 μl |
| 10× Taq Buffer (Mg2+ plus) | 5 μl |
| dNTP Mix (10 mM each) | 1 μl |
| Primer 1 (10 μM) | 2 μl |
| Primer 2 (10 μM) | 2 μl |
| Template DNAa | x μl |
| GenieTaq DNA Polymerase (5 U/μl)b | 0.5 μl |
a Recommended template amount in a 50 μl reaction:
| Template type | Amount |
| Animal and plant genomic DNA | 0.1–1 μg |
| Escherichia coli genomic DNA | 10–100 ng |
| λDNA | 0.5–10 ng |
| cDNA | 1–5 μl (≤1/10 of the total PCR volume) |
| Plasmid DNA | 0.1–10 ng |
b The enzyme volume can be adjusted between 0.25 and 1 μl. More enzyme usually increases yield but may reduce specificity.
For fragments with GC content >60% that do not amplify under optimised conditions, use a PCR enhancer.
- Run the reaction program
| Step | Temperature | Time | Cycles |
| Initial denaturationa | 95°C | 3 min | 1 |
| Denaturation | 95°C | 15 sec | 30–35 |
| Annealingb | 60°C | 15 sec | 30–35 |
| Extension | 72°C | 60 sec/kb | 30–35 |
| Final extension | 72°C | 5 min | 1 |
a Suitable for most reactions. For templates with complex structure, extend initial denaturation to 5–10 min.
b Set the annealing temperature 3–5°C below the primer Tm. For complex templates, adjust the annealing temperature and extend the extension time.
Q1: Can I clone the PCR products directly into a T vector?
- Yes. The enzyme lacks 3'→5' exonuclease activity, so products carry a 3' adenine overhang for TA cloning. They are also compatible with seamless and TOPO cloning kits.
Q2: Why should I set up reactions on ice?
- Taq DNA polymerase has some activity at room temperature. Preparing reactions on ice and then transferring them to the thermal cycler reduces nonspecific amplification during setup.
Q3: How much enzyme should I use?
- Use 0.5 μl (5 U/μl) per 50 μl reaction, adjustable between 0.25 and 1 μl. Increasing the enzyme usually improves yield but may reduce specificity.
Q4: How should I handle GC-rich or complex templates?
- For complex templates, extend initial denaturation to 5–10 min, adjust the annealing temperature and extend the extension time. For fragments with GC content >60% that still fail to amplify, use a PCR enhancer.
