Taq DNA Polymerase

Taq DNA Polymerase is purified from E. coli. expressing a Thermus aquaticus DNA polymerase gene. This enzyme has a 5′ → 3′ DNA polymerase and a 5′ → 3′ exonuclease activity but lacks a 3′ → 5′ exonuclease activity. The enzyme consists of a single polypeptide with a molecular weight of approximately 94 kDa. Taq DNA polymerase is heat-stable and will synthesize DNA at elevated temperatures from single-stranded templates in the presence of a primer.

Taq Polymerase is recommended for use in routine PCR reactions. The buffer system is optimized for high specificity and guarantees minimal by-product formation. We supplied Taq Polymerase with appropriate buffers. Usually 1-1.5 u of Taq DNA Polymerase are used in 50 µl of reaction mix. Higher Taq DNA Polymerase concentrations may cause synthesis of nonspecific products. However, if inhibitors are present in the reaction mix (e.g., if the template DNA used is not highly purified), higher amounts of Taq DNA Polymerase (2-3 u) may be necessary to obtain a better yield of amplification products.

• For each 50 μl reaction, assemble the following in a 0.5 ml PCR tube on ice just prior to use:

• Mix gently. If necessary, centrifuge briefly. Cap tubes and place in thermal cycler.
• Process in thermal cycler for 25-35 cycles as follows:

Initial Denaturation	2-5 minutes at 94°C
Denaturation	20-40 seconds
Annealing	1 min at the proper annealing temperature
Extension	2 min at 72°C
Final extension	5 min at 72°C

Template

Purified high quality DNA is needed for a success PCR reaction.

Storage Buffer

The enzyme is supplied in a storage buffer consisting of 20 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 1 mM DTT, 50% (v/v) glycerol, and 1% Triton X-100.

One unit is defined as the amount of enzyme that will catalyze the incorporation of 10 nmol of dNTP into acid- insoluble form in 30 min at 74°C in a reaction containing 25 mM TAPS (tris-[hydroxymethyl]-methyl-amino- propane-sulfonic acid, sodium salt), pH 9.3 at 25°C, 50 mM KCl, 2 mM MgCl₂, 1 mM β-mercaptoethanol, 0.2 mM dATP, dGTP, and dTTP, 0.1 μM [α-32P] dCTP, and activated salmon sperm DNA.

• A compatible PCR instrument
• Vortex or equivalent
• Microcentrifuge
• Plates and seals for your instruments