What differentiates StellarTaq™ DNA Polymerase (DNAP) from wild type Taq DNAP?
StellarTaq DNAP is an engineered thermostable DNA polymerase with extreme polymerization speed and increased tolerance to common PCR inhibitors that may be introduced from sample types or sample preparation.
What are relevant applications for StellarTaq DNAP?
- Pathogen detection, including infectious diseases
- PCR amplification of DNA fragments (≤5 kb)
- Probe and intercalating dye-based qPCR
- RT-qPCR (also explore StellarScript HT+ Reverse Transcriptase and RNase Inhibitor)
- PCR applications where inhibitors are present
- Fast PCR
- PCR applications where specificity is important
Can StellarTaq DNAP be used for 1-step RT-qPCR?
Yes. Note that because reverse transcriptases can inhibit polymerase activity, 1-step RT-qPCR assays may benefit from higher polymerase concentrations. Our RNase Inhibitor and StellarScript HT+ Reverse Transcriptase are excellent choices for one and two-step RT-qPCR.
What polymerase and exonuclease activity does StellarTaq DNAP possess?
StellarTaq DNAP catalyzes 5’→3′ DNA synthesis and has 5’→3′ exonuclease activity. It is deficient in 3’→5′ exonuclease activity.
Does StellarTaq DNAP have hot start functionality?
StellarTaq DNAP is supplied in both hot start and non-hot start formats.
What method of hot start is used with StellarTaq DNAP?
StellarTaq DNAP uses an antibody to enable hot start functionality.
Does the hot start functionality inhibit the 5’ → 3’ exonuclease activity as well as the 5’ → 3’ polymerase activity?
The hot start functionality inhibits the 5’ → 3’ exonuclease activity significantly but not completely.
What ends will my PCR products have?
StellarTaq DNAP generates A-tailed PCR products. This A-overhang can be used to ligate adapters with a T-overhang.
Can StellarTaq DNAP amplify uracil-containing templates?
Yes!
Can StellarTaq DNAP incorporate modified or alternative bases?
StellarTaq does not have 3’ to 5’ exonuclease ‘’proof-reading’’ activity which should make it tolerant to modified or alternative bases.
Does StellarTaq DNAP exhibit strand displacement activity?
No. Any strand displacement activity is likely to be far exceeded by its 5′-3′ exonuclease activity that will digest any downstream strand encountered during polymerization.
Is StellarTaq DNAP provided in a glycerol-free format?
Yes, StellarTaq DNA Polymerase is provided in a glycerol-free hot start and non-hot start format.
How many freeze/thaw cycles can glycerol-free StellarTaq DNAP tolerate?
StellarTaq DNAP is guaranteed to be stable up to 5 freeze/thaw cycles. However, repeated freeze-thaw cycles should be avoided if possible.
What is the storage buffer for StellarTaq DNAP?
- Glycerol-containing: 50 mM Tris-HCl, pH 7.5, 100 mM KCl, 0.1 mM EDTA, 50% Glycerol, 0.05% Tween 20
- Glycerol-free: 50 mM Tris-HCl, pH 7.5, 300 mM KCl, 0.05% Tween 20
What is the recommended reaction buffer for StellarTaq DNAP?
A standard 10X reaction buffer (not supplied with kit) is recommended:
- 200 mM Tris-HCl, pH 8.3, 1000 mM KCl, 0.04% Tween 20
- Additional optimization is likely required. See technical guide for more information
Can I use the same reaction buffer as for wild type Taq DNAP?
No. StellarTaq DNAP performs well in a range of buffers, but requires higher salt concentrations than wild type Taq DNAP to perform optimally. The composition of the recommended 10X buffer (not supplied with kit) is a standard PCR buffer, tailored for the optimal salt concentration of StellarTaq DNAP(1000 mM KCl).
This buffer will need to be optimized for specific applications. Please reference our technical guide for optimization instructions.
What StellarTaq DNAP concentration should I use?
A good general purpose starting point is 0.012 U/μL of polymerase in the reaction. We recommend using higher concentrations (up to 0.12 U/μL) in reactions with high concentrations of inhibitors, for fast PCR, or when no amplification is observed at 0.012 U/μL.
We recommend using lower concentrations of StellarTaq DNAP if non-specific amplification is observed.
What final MgCl₂ concentration should I use?
We suggest using 2 mM MgCl₂ in the reaction. Mg²+ concentrations can alter reaction performance and may need to be optimized. Probe-based qPCR applications will benefit from increased MgCl₂ concentration. A scouting range between 1.5 – 6 mM MgCl₂ is recommended.
Are protected primers required for StellarTaq-based PCR amplification?
No. StellarTaq DNA Polymerase 5’→3′ exonuclease activity targets double-stranded DNA and will not degrade primers.
How can I maximize the yield from StellarTaq DNAP?
- Set up an initial salt concentration vs enzyme concentration matrix experiment to identify the optimal salt and enzyme concentration.
- Find the optimal magnesium concentration by performing a magnesium titration between 1.5 mM – 6 mM MgCl₂.
- Use templates shorter than 5 kb.
- Optimize annealing temperature of the primers.
- Optimize combined annealing and extension time to minimize non-specific amplification. Note that longer annealing and extension times can result in increased non-specific product formation.
Please reference our technical guide for additional details.
What is the amplification speed of StellarTaq DNAP?
StellarTaq DNA Polymerase can amplify up to 2 kb DNA in 10 seconds.
Do you offer custom formats?
Yes! Watchmaker offers customer fills, packaging, concentrations, and labeling – including private label – designed to meet your unique needs. We offer flexible terms to serve organizations of any size, and our right-sized processes enable rapid turnaround time on customization. Please contact sales@watchmakergenomics.com to learn more about our capabilities.
Are you ISO 13485-certified?
Yes! Our Quality Management System has achieved ISO 13485:2016 Certification. Our certificate has been awarded for the design, development, manufacture, contract manufacture, and support of high performing reagents for genomics applications in medical research. Download the certificate here.