Antibody-Based Hot-Start DNA Polymerase Mix

Abbexa's Antibody-Based Hot-Start DNA Polymerase Mix is a convenient ready-to-use 2X reaction mix specifically designed for TA cloning, amplifying genomic DNA up to 10 kb and offering 3.5 times higher fidelity than native Taq. The enzyme's antibody-based hot-start property allows reaction set-up at room temperature, eliminating all non-specific priming and the formation of primer-dimers. It contains DNA Polymerase (abx461028), dNTPs, MgCl₂ and enhancers at optimal concentrations.. The mix is optimized and ready-to-use, simply add water, template and primers. Antibody-Based Hot-Start DNA Polymerase Mix (2X) dramatically reduces the time needed to set up reactions, thereby minimizing the risk of contamination. Greater reproducibility is ensured, by a reduction in the number of pipetting steps that can lead to pipetting errors. The 100 rxns size is provided as 2 × 1.25 ml 2X Antibody-Based Hot-Start DNA Polymerase Mix, and the 500 rxns size is provided as 10 × 1.25 ml 2X Antibody-Based Hot-Start DNA Polymerase Mix.

Target	Antibody-Based Hot-Start DNA Polymerase Mix
Tested Applications	PCR
Form	Liquid
Storage	Store all components at -20 °C. It is not recommended to store the enzyme at -80 °C as ice crystals may form on the active site, which can affect the enzyme activity. Avoid repeated freeze/thaw cycles.
Validity	Up to 12 months.
Buffer	Contains dNTPs, MgCl2, and enhancers.
Biological Activity	One unit is defined as the amount of enzyme that incorporates 10 nmol of dNTPs into acid-insoluble form in 30 minutes at 72 °C.
Concentration	2X
Availability	Shipped within 3-7 working days.
Note	This product is for research use only. Not for human consumption, cosmetic, therapeutic or diagnostic use.
Directions for use	Reaction Components: Component Volume Template 200 ng Primers (20 µM each) 1 µl Antibody-Based Hot-Start DNA Polymerase Mix (2X) 25 µl Water Variable, up to 50 µl Total Volume 50 µl Thermal Cycling Conditions: Step Number of Cycles Temperature Time per Cycle Initial Denaturation 1 cycle 95 °C 1 min Denaturation 25-35 cycles 95 °C 15 seconds Annealing ≥ 55 °C (primer dependent) 15 seconds Extension 72 °C 15 seconds Notes: Forward and reverse primers are generally used at a final concentration of 0.2-0.6 µM each. It is recommended to start with 0.4 µM as the final concentration (i.e. 10 pmol of each primer per 25 µl reaction volume). A primer concentration that is too high can reduce the specificity of priming, resulting in non-specific products. Primers should have a melting temperature (Tm) of approximately 60 °C. The amount of template in the reaction depends mainly on the type of DNA used. For templates with low structural complexity, such as plasmid DNA, it is recommended to use 25 pg - 5 ng DNA per 25 µl reaction volume. For eukaryotic genomic DNA, it is recommended to use a starting amount of 100 ng DNA per 25 µl reaction, this can be varied between 2.5 ng - 250 ng. It is important to avoid using templates re-suspended in EDTA-containing solutions (e.g. TE buffer) since EDTA chelates free Mg2+. The initial denaturation step at 95 °C for 1 minute is required to activate the enzyme and fully melt the template. For most PCR, this is sufficient to melt the DNA template, though complex templates such as eukaryotic genomic DNA may require up to 3 min. It is recommended to run the subsequent denaturation steps at 95 °C for 15 seconds each cycle, which is suitable for GC-rich templates. Increasing this step up to 30 seconds may improve problematic reactions. The optimal annealing temperature is primer dependent and is usually 2-5 °C below the lower Tm of the pair. It is recommended to start with an annealing temperature of 55 °C and, if necessary, run a temperature gradient to determine the optimal annealing temperature. 15 seconds per cycle is usually sufficient, though increasing this step up to 45 seconds may improve problematic reactions. The optimal extension time is dependent on the length of the amplicon and the complexity of the template. An extension time of 15 seconds is sufficient for amplicons under 1 kb. Longer extension times are recommended for fragments larger than 1 kb. The extension time may be increased up to 45 seconds/kb to find the fastest optimal condition. The optimal conditions will vary from reaction to reaction and are dependent on the system used. Each parameter needs to be adjusted by the end user and some optimization may be required.

Research Articles on Antibody-Based Hot-Start DNA Polymerase Mix

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