Taq-B DNA Polymerase

• OEM by QIAGEN offers customized bulk manufacturing of this enzyme
• Lacks 3ʹ→5ʹ proofreading function

Taq-B DNA Polymerase is a thermally stable, processive 5ʹ→3ʹ DNA polymerase. The 94 kDa protein possesses an inherent 5ʹ→3ʹ nick-translation moiety and lacks a 3ʹ→5ʹ proofreading function. Taq-B DNA Polymerase is the industry standard for routine PCR.

The enzyme is supplied in 20 mM Tris-HCl, 100 mM NaCl, 1.0 mM DTT, 0.1 mM EDTA, Stabilizer and 50% glycerol; pH 7.5 at 25°C.

10x PCR Buffer l (cat. no. B7030) contains 100 mM Tris-HCl, 500 mM KCl and 5 mM MgCl₂; pH 8.3 at 25°C.

Low-glycerol formulations available.

Properties

• Storage temperature: –25°C to –15°C
• Proofreading (3'→5' exo): No
• Nick-translation (5'→3' exo): Yes
• Strand displacement: No
• Molecular weight: 94 kDa

Source of recombinant enzyme protein

A recombinant E. coli strain carrying the Taq DNA polymerase gene from the thermophilic organism Thermus aquaticus YT-1.

Unit definition:
One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid-insoluble material in 30 minutes at 75°C.

Molecular weight: 94 kDa

Instructions

2x DNA/oligonucleotide master mix:
1.0 µL 10 mM dNTPs
1.0 µL 10 µM Forward Primer
1.0 µL 10 µM Reverse Primer
1.0 µL 500 ng/µL genomic DNA
21 µL Type I Water

2x Enzyme/buffer master mix:
5.0 µL 10x PCR Buffer I
0.2 µL 5 U/µL Taq-B DNA Polymerase
19.8 µL Type I Water

Notes
Taq DNA Polymerase is the original and most commonly used PCR enzyme. Taq excels at amplifying short (<5 kb) sequences from low-complexity template sources and produces robust yields with little or no optimization of reaction conditions.

Consider the following guidelines when designing PCR strategies using Taq-B DNA Polymerase.

• DNA template: Although extensive purification of PCR templates is typically not necessary, care should be taken with crude or partially purified DNA sources as handling and chemical agents can adversely affect the PCR process. Exposure to short-wave UV light or other DNA damaging agents should be avoided, as should exposure to high ionic strength, detergents, such as SDS, loading dyes and phenol. To prevent contamination from previous PCR, consider setting up reactions in a positive-pressure hood and with aerosol barrier pipet tips. In a typical 25 cycle PCR, 104 copies of target sequence will yield reproducible amplification product. This corresponds to roughly 0.1–1 ng/mL (final concentration) of plasmid DNA, and 1–10 µg/mL of genomic DNA. The use of lower DNA concentrations typically produces less non-specific product, while higher concentrations can allow for fewer cycles and lower mutation rates.
• Primer design: Ideally, oligonucleotide primers are 15–30 bases in length, nearly 50% G+C, and have equal (+/– 3°C) annealing temperatures. The use of software to detect self-complementary or hairpin-prone regions is advised and is offered as a service by some synthesis providers. Note that although the 5ʹ-terminus of the primer may contain untemplated sequence, the 3ʹ end must match perfectly. Typical oligonucleotide concentration in the reaction is 0.1–0.5 µM.
• Magnesium: Magnesium is a critical component of the PCR though its concentration can be modulated to promote various effects. Generally, 1.5–2.0 mM Mg2+ is targeted, but higher concentrations (up to 5 mM) may be used to stimulate the yield of reactions at the expense of fidelity. The converse is also true: lower magnesium concentrations will promote higher fidelity products with a lower overall amplification yield. Note that certain reaction components, in particular template DNA and oligonucleotides, may contribute chelating agents to the reaction which could lower the effective magnesium concentration and starve the reaction.
• dNTPs: Generally, a final concentration of 100–200 µM dNTPs is employed, though higher concentrations may stimulate yields (particularly with longer targets) and lower may offer increases in fidelity. Taq DNA Polymerase can also incorporate and read through deoxyuridine and inosine, two analogs used in certain applications.
• Taq polymerase: 1 unit/50 µL reaction (20 U/mL) is typical, though additional enzyme may be added to stimulate yields. Taq DNA Polymerase extends a DNA template at approximately 2000 nucleotides/minute, so it is recommended that 45–60 seconds of extension time be provided per cycle. Appropriate extension temperatures range from 66–72°C.

Quality control analysis
Unit characterization assay
Specific activity was measured using a twofold serial dilution method. Dilutions of enzyme were made in 1x reaction buffer and added to 50 µL reactions containing calf thymus DNA, 25 mM TAPS (pH 9.3), 50 mM KCl, 2.0mM MgCl₂, 1 mM DTT, 3H-dTTP and 100 µM dNTPs. Reactions were incubated 10 minutes at 75°C, placed on ice, and analyzed using the method of Sambrook and Russell (Molecular Cloning, v3, 2001, pp. A8.25-A8.26)

Protein concentration is determined by OD₂₈₀ absorbance.

Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the band corresponding to the protein of interest in the diluted sample.

Single-stranded exonuclease is determined in a 50 µL reaction containing a radiolabeled single-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C.

Double-stranded exonuclease activity is determined in a 50 µL reaction containing a radiolabeled double-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C.

Double-stranded endonuclease activity is determined in a 50 µL reaction containing 0.5 µg of plasmid DNA and 10 µL of enzyme solution incubated for 4 hours at 37°C.

E. coli contamination is evaluated using 5 µL replicate samples of enzyme solution that are denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.

This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.

• Routine PCR