What is KOD polymerase?
KOD is a high fidelity thermostable DNA polymerase that amplifies target DNA up to 6 kbp with superior accuracy and yield for PCR applications. The enzyme′s 3′→5′ exonuclease-dependent proofreading activity results in a lower PCR mutation frequency than any other commercially available DNA polymerase.
How do hot start polymerases work?
Hot Start Taq DNA Polymerase is a mixture of Taq DNA Polymerase and an aptamer-based inhibitor. The inhibitor binds reversibly to the enzyme, inhibiting polymerase activity at temperatures below 45°C, but releases the enzyme during normal cycling conditions, allowing reactions to be set up at room temperature.
What is the principle of hot start PCR?
The basic principle of Hot Start PCR methods and reagents is that they are designed to inhibit Hot Start Taq DNA polymerase activity, or the incorporation modified dNTPs during reaction set up until a heat activation step occurs.
Why is it called hot start PCR?
Denaturation is the first step in the PCR reaction. The thermocycler heats up to roughly 95 degrees Celsius, which causes the double-stranded DNA helix to melt open into two single-stranded DNA templates. Simultaneously, the heat from this step also activates the DNA polymerase – hence, hot start.
Does Taq polymerase denature DNA?
Taq polymerase has the important characteristic of being stable at temperatures up to 95°C2. That’s critical because this is the temperature at which DNA denatures – a required step at the beginning of the PCR reaction.
How does a colony PCR work?
Principle of Colony PCR: Two sets of primers amplify two different plasmid DNA, the insert-specific primers amplify the gene of interest whereas the vector-specific primer amplifies the flanking region along with the insert. Both primer sets amplify DNA in the same direction but have a different purpose.
What are the applications of hot-start PCR?
The results of this procedure has many applications both medically and industrially. For example, applications of PCR including forensics, paternity testing, biodefence, cloning, mutation detection, genetic testing and DNA sequencing.
What are the advantages of a thermal stable Hot-Start DNA Polymerase?
What are the benefits of hot-start technology?
- Prevents extension of primers binding to template sequences with low homology (mispriming)
- Prevents extension of primers binding to each other (primer-dimer formation) during reaction setup.
- Increases sensitivity and yield of the desired target fragments.
What are the advantages of a thermal stable Hot Start DNA polymerase?
Why does PCR need high temperature?
The bacteria’s DNA polymerase is very stable at high temperatures, which means it can withstand the temperatures needed to break the strands of DNA apart in the denaturing stage of PCR.
Why is a PCR cycle repeated 30 times?
At the end of a cycle of these three steps, each target region of DNA in the vial has been duplicated. This cycle is usually repeated 30 times. Each new DNA piece can act in the next cycle as a new template, so after 30 cycles, 1 million copies of a single fragment of DNA can be produced (Scheme – Diagram of PCR).
Can PCR occur without Taq polymerase?
Taq polymerase Like DNA replication in an organism, PCR requires a DNA polymerase enzyme that makes new strands of DNA, using existing strands as templates.
What is hot start DNA polymerase?
KOD Hot Start DNA Polymerase* is a premixed complex of KOD DNA Polymerase and two monoclonal antibodies that inhibit the DNA polymerase and 3’→5′ exonuclease activities at ambient temperatures (Mizuguchi 1999). KOD Hot Start amplifies genomic DNA templates up to 21 kb including GC-rich genes for PCR applications.
Why choose KOD enzymes for PCR?
Our analysis has shown that KOD enzymes are an easy choice for fast, accurate and high-yielding PCR. EMD Millipore’s molecular biologists work to develop and formulate polymerases offering the highest specificity, fidelity and yield during PCR amplification.
How much DNA polymerase is in a 50 µL reaction?
The final diluted reaction contains 1 U KOD Hot Start DNA Polymerase per 50 µl reaction. The smaller available size provides sufficient master mix for 100 (50 µl scale), or 250 (20 µl scale) reactions, while the larger size is adequate for 500 (50 µl scale) or 1250 (20 µl scale) reactions.
Which DNA polymerase is suitable for blunt-ended PCR products?
KOD Hot Start DNA Polymerase generates blunt-ended PCR products suitable for cloning with the Novagen Perfectly Blunt® and LIC Vector Kits. *Manufactured by Toyobo and distributed by EMD. Not available in Japan.