Learn more Updated Shipping Rates and Promotions
Explore More eSpOT-ON Nuclease Protein Available Now
Explore More Order eSpOT-ON Nuclease mRNA Now
My Cart
Contact Sales
PRODUCT

Hot-Start DNA Polymerase, High Fidelity

High‑fidelity hot‑start enzyme for sensitive applications

Proofreading hot‑start polymerase delivers higher fidelity than Taq with robust amplification of complex targets. Produces blunt-end products for cloning and is well-suited for NGS library amplification.

  • Approximately 50x higher fidelity than Taq with proofreading
  • Robust amplification of 5–10 kb targets
  • Produces blunt ends ideal for blunt cloning
  • Reliable for NGS library amplification

Select a Size

Hot-Start DNA Polymerase, High Fidelity (100 Units)

#EM15HIFIPOL-Sm
Product image 1
Specifications Description Quality FAQ Resources

Product Specifications

Product Number EM15HIFIPOL
Function Proofreading polymerase generating blunt-end products
For Use In Cloning; High-Fidelity PCR; NGS library amplification
Molecular Weight 94 kDa
Concentration 1 U/µL
Delivery Format Tubes
Intended Use This product is intended for research use only
Volume 100 µL
Source Recombinant E. coli
Shipping Conditions Cold Pack
Storage Temperature -20°C
Stability ≥12 months

High-Fidelity Hot-Start Enzyme for Sensitive Applications

Hot-Start High-Fidelity DNA Polymerase is a recombinant, thermostable proofreading DNA polymerase engineered for exceptional accuracy and reliability. The enzyme features a ‘hot-start’ mechanism, where antibodies block polymerase activity at ambient temperatures and dissociate after the initial denaturation step at 94°C. This 'hot start' enhances sensitivity, specificity, and yield, while enabling reaction assembly at room temperature. With fidelity ~50 times higher than standard Taq polymerase, Hot-Start High-Fidelity Polymerase ensures precise DNA amplification.

Our Hot-Start High-Fidelity DNA Polymerase efficiently amplifies targets up to 5–10 kb in size, even from high-complexity templates. It demonstrates high resistance to PCR inhibitors, ensuring robust performance across a variety of challenging sample types. Our enzyme features 3'→5' exonuclease (proofreading) activity, which corrects nucleotide incorporation errors during DNA synthesis. Additionally, the enzyme lacks 5'→3' exonuclease activity and reverse transcriptase activity, ensuring the production of blunt-end PCR products suitable for downstream applications like blunt cloning.

Key Features

  • Exceptional Fidelity: Approximately 50 times higher accuracy than Taq polymerase for more precise DNA amplification.
  • Hot-Start Mechanism: Antibodies block polymerase activity at room temperature and dissociate at 94°C, improving sensitivity, specificity, and yield.
  • Blunt-End PCR Products: Ideal for cloning workflows requiring blunt-end products.
  • Amplification of Large Targets: Supports amplification of DNA targets up to 5–10 kb in size.
  • Robust Performance: High resistance to PCR inhibitors ensures reliable results across diverse sample types.
  • Efficient Amplification: Ideal for high-complexity DNA, like GC-rich templates.

Applications

  • High-Fidelity PCR: For applications requiring precise and accurate DNA amplification.
  • Blunt-End Cloning: Produces blunt-end PCR products for seamless cloning workflows.

Technical Notes

  • Optimization Tip: Recommended annealing temperature is 5°C above primer Tm; gradient PCR is suggested for optimization.

Custom Options

We offer flexible customization to meet your specific needs:

  • Volume: Optimize packaging sizes to match your application scale.
  • Concentration: Customize concentrations for optimal performance.
  • Formulation: Available in liquid and lyophilized formats to suit your protocols.
  • Master Mix Creation: Custom manufacturing of PCR master mixes based on your optimized formulations.

Quality

One unit is defined as the amount of enzyme required to catalyze the incorporation of 10 nmol of dNTP into acid-soluble form in 30 minutes at 72°C in the presence of the reaction buffer.

Functional assay
Human genomic DNA was amplified using the DNA Polymerase and specific primers to produce a distinct band of 1100 bp.

Self-priming activity
Standard PCR is carried out without primers, using the DNA Polymerase and human genomic DNA. No products were amplified.

Exonuclease assay
Linearized lambda/HindIII fragments are incubated with the DNA Polymerase in a 50 µl reaction mixture for 4 h at 37°C. No degradation of DNA was observed.

Endonuclease assay
lambda DNA is incubated with the DNA Polymerase in a 50 µl reaction mixture for 4 h at 37°C. No degradation of DNA was observed.

Nick Activity
Supercoiled plasmid DNA is incubated with the DNA Polymerase in a 50 µl reaction mixture for 4 h at 37°C. No conversion of covalently closed circular DNA to nicked DNA was detected.

Contamination with E. coli DNA
A sample of the denatured DNA Polymerase is analyzed with specific primers targeting the 16S rRNA gene in qPCR for the presence of contaminating E. coli DNA. No E. coli was detectable.

Frequently Asked Questions

Why is the recommended annealing temperature for the Hot-Start Taq DNA Polymerase, High-Fidelity different from the commonly seen "5°C below primer Tm"?

The enzyme and its buffer system are optimized for a slightly higher melting temperature for researchers to begin with aiming for a better specificity and sensitivity for assays in general. Since each assay is unique, we strongly recommend performing a gradient PCR as part of the optimization to identify the most suitable annealing temperature for your specific design.

What is the difference between High-Fidelity Hot Start DNA polymerase and standard Hot Start DNA polymerase?

Both DNA polymerases include antibodies that prevent DNA amplification before the initial denaturation step, reducing non-specific amplification and primer-dimer formation. However, the High-Fidelity Hot-Start DNA polymerase also includes 3′→5′ exonuclease (proofreading) activity, meaning that it can correct nucleotide mismatches during synthesis.

What does high fidelity mean in terms of DNA amplification?

Our High Fidelity, Hot Start DNA polymerase is approximately 50 times more accurate than standard Taq polymerase. This is due to the 3′→5′ exonuclease (proofreading) activity, which standard Taq polymerase lacks.

When do I need High-Fidelity DNA Polymerase?

In complex applications where single-base errors can create significant downstream issues, such as cloning, NGS library preparation, or long-range PCR, high-fidelity polymerases are a preferred pick to minimize amplification-induced errors. Learn how to select the right DNA polymerase for your experiments.

How much primer should I use in a PCR reaction?

The optimal primer concentration depends on the application, but 0.2–1 µM of each primer (forward and reverse) is commonly used in PCR reactions and is also the range recommended in the user guide. We highly recommend optimizing primer concentration based on your experimental design. Using too little primer can reduce amplification efficiency and yield, while excessive primer concentrations may increase nonspecific amplification and primer-dimer formation.

Ready to get started?

Get in touch