Why is the ROBUST DNA polymerase called "ROBUST"?
DNA polymerases are enzymes responsible for synthesizing new DNA strands during PCR. They do this by adding nucleotides to a primer attached to a template DNA strand. The ROBUST DNA polymerase is designed for challenging PCR conditions, featuring enhanced processivity, improved resistance to PCR inhibitors, thermal stability and hot start technology. It can amplify from lower-quality templates and allows for reduced cycle times.
What is the significance of the 5' adenine overhang produced by some of these DNA polymerases?
The 5' adenine (A) overhang produced by ROBUST and TAQ DNA polymerases is crucial for a cloning method called TA cloning. In TA cloning, the PCR product is directly ligated into a vector with a complementary 3' thymine (T) overhang. The matching 'A' and 'T' overhangs facilitate efficient ligation, creating recombinant plasmids with the PCR product inserted into the vector.tent
What is processivity, and how does the ROBUST and High Fidelity DNA polymerases compare to Taq DNA Polymerase in this aspect?
Processivity refers to the ability of a DNA polymerase to synthesize a long chain of DNA without detaching from the template. High processivity enables longer amplicons to be generated more quickly and with less opportunity for non-specific extension to occur. The ROBUST and High Fidelity polymerases have enhanced processivity compared to standard Taq polymerase, meaning they can synthesize longer DNA fragments more efficiently.
What is 'Hot Start' technology and how does it benefit PCR amplification with Hot Start Taq Polymerase?
Hot Start technology, in the context of Hot Start Taq Polymerase, uses an aptamer that has a 3’ cap to prevent amplification during PCR setup. The aptamer is a short single-stranded DNA or RNA molecule that binds specifically to the polymerase, blocking its activity until the high-temperature denaturation step. This prevents mis-priming and the formation of primer-dimers and other non-specific amplification products, resulting in more accurate and reliable PCR reactions. This means you don't need to initially incubate at 94 - 95 °C.
What is the purpose of GC Extender, and when should it be used?
GC Extender is an optional component intended to be used when amplifying difficult templates, such as those with high GC content (greater than 60%) or templates longer than 2.5 kbp. The precise volume needed (5µL – 15µL) should be optimized for each template and primer set, which helps the polymerase to efficiently amplify through complex regions and longer sequences of DNA.
What considerations should be made when optimizing a PCR reaction using these polymerases?
Optimization is crucial when using these polymerases, which include optimizing primer annealing temperature, the amount of template DNA, the concentrations of primers, the amount of DNA polymerase added, and magnesium sulfate concentration. The optimal number of cycles and the extension time also depend on your template. Using GC Extender can be optimized to a specific amount needed for difficult templates.
What is High Fidelity DNA Polymerase and what makes it different from standard DNA polymerases like Taq and ROBUST?
High Fidelity DNA Polymerase is a recombinant enzyme designed for accurate amplification of DNA. It's unique because it possesses a 5’ to 3’ DNA polymerase activity, a 3’ to 5’ exonuclease domain (which allows it to proofread and correct errors during DNA synthesis), and an Sso7D tethering domain that increases processivity. Compared to Taq DNA polymerase, it boasts 55-fold higher fidelity, generates blunt-end PCR products, can amplify larger DNA fragments (up to 10 kbp) with greater accuracy, and has two to three times the processivity.
What are blunt end PCR products?
Blunt end PCR products are double-stranded DNA fragments generated by PCR in which both strands end at the same nucleotide position, resulting in flush or blunt ends with no single-stranded overhangs. In other words, the 5′ and 3′ termini of each strand line up exactly, creating a perfectly straight (blunt) edge on both ends of the DNA fragment.
Are all Master Mixes formulated for the same performance?
No, the CSR 2X Taq Master Mix does not contain any special additives, making it suitable for standard PCR with minimal, if any, adjustments to your existing protocols. However, buffer and polymerase compositions vary between manufacturers and may perform differently under challenging PCR conditions, such as high-GC templates, impure DNA preparations, or the presence of low levels of PCR inhibitors. In such cases, optimal conditions for one master mix may not translate directly to another. If issues arise, it is recommended to perform independent optimization for the 2X Taq Master Mix.
Why doesn't CSR 2x Taq Master or CSR 2X Hot Start Taq Mix work when I use my existing PCR protocol?
There are three likely reasons.
- PCR Enhancer Reagent: If your PCR conditions require a PCR Enhancer Reagent to improve amplification efficiency, determine the optimal amount by titrating in 5 µL increments, up to a maximum of 20 µL per 50 µL reaction.
- Annealing Temperature: If no amplification product is visible, try reducing the annealing temperature by 3°C. If no band appears after a total adjustment of 7°C, determine the optimal annealing temperature by testing a range from the primer's calculated melting temperature (Tm) down to 7°C below it. For primers with different Tm values, use the lower Tm as the reference point.
- Cycle Number: If no band is visible, increase the number of cycles in increments of 3. 2X Taq Master Mixes work effectively between 25 and 40 cycles.
