BIO-HELIX - MB101-0500
Taq DNA Polymerase
Size: 500 units
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.
- A compatible PCR instrument
- Vortex or equivalent
- Plates and seals for your instruments
1. For each 50 μl reaction, assemble the following in a 0.5 ml PCR tube on ice just prior to use:
|add to 50μl||-||PCR Grade Water|
|1 μl||200 μM||dNTP Mix (10 mM each dATP, dCTP, dGTP|
|1 μl||0.1-0.2 μM||Forward primer, 5-10 μM|
|1 μl||0.1-0.2 μM||Reverse primer, 5-10 μM|
|5 μl||2 mM MgCl2||10X PCR Buﬀer|
|0.25 μl||1.25 units||Taq DNA Polymerase (5 units/μl)|
|X μl||10 ng||DNA template|
|50 μl||Total volume|
2. Mix gently. If necessary, centrifuge briefly. Cap tubes and place in thermal cycler.
3. Process in thermal cycler for 25-35 cycles as follows:
|Initial Denaturation||2-5 minutes at 94°C|
|Annealing||1 min at the proper annealing temperature|
|Extention||2 min at 72°C|
|Final extension||5 min at 72°C|
Optimal conditions for amplification will vary depending on the primers and thermal cycler used. It may be necessary to optimize the system for individual primers, template, and thermal cycler.
Purified high quality DNA is needed for a success PCR reaction. The final concentration of cDNA template please refer to “Reaction Setup”
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.