Taq DNA Polymerase, recombinant

Beschreibung

The Simplebiotech Taq Polymerase is purified from an E. coli strain carrying a plasmid with Taq DNA polymerase gene from Thermus aquaticus YT-1.

Description: Taq DNA Polymerase is a thermostable enzyme that catalyses 5’→3′ synthesis of DNA. The enzyme has no 3’→5’ proofreading exonuclease activity but possesses low 5’→3′ exonuclease activity.

Unit definition: One unit is defined as the amount of enzyme required to catalyse the incorporation of 10 nmoles of dNTPs into acid insoluble material in 30 minutes at 72 °C.

Assay conditions: The polymerase activity was assayed in 10 mM Tris-HCl (pH 8.5 at 25 °C), 50mM KCl, 1.5 mM MgCl2, 200 µM each of dATP, dGTP, dCTP, dTTP (a mix of unlabelled and [3H] dTTP) and 12.5 µg activated calf thymus DNA, in a final volume of 50 µL.

Storage buffer: 100 mM NaCl, 50 mM Tris-HCl (pH 8.0 at 25 °C), 1mM DTT, 0.1 mM EDTA, 1% Triton X-100 and 50% Glycerol.

Taq DNA polymerase buffer: 50 mM KCl, 10 mM Tris-HCl (pH 8.5 at 25 °C) 1.5 mM MgCl₂, 0.1 % Triton X-100.

Quality control:

Our Taq DNA Polymerase has been extensively functionally tested and carefully tested for absence of endo- and exodeoxyribonucleases.

Recommended PCR set-up:

Thaw buffer, dNTPs and primers and put them back on ice. Keep all components including the reaction mix on ice during the hole PCR set-up. Always include a “no template control” including all components except template DNA and if possible, a positive control. The 10x Taq Polymerase buffer can be replaced for the “5x Magic orange” direct load buffer.

Vortex gently and spin down.

Manual hot start can be simply achieved by keeping PCR mix on ice, until the thermal cycler has reached 94 °C. Then place the PCR tubes into the thermal cycler.

                                         
1) The recommended annealing temperature is 5 °C below the calculated melting temperature of the primer with the lower melting temperature.

2) Use 1min per kb of PCR product.

Calculating melting Temperature of Primers:

For primers shorter than 20 nucleotides melting temperature (Tm) can be approximated by the following equation:

Tm= 4 (G + C) + 2 (A + T),

G, C, A, T represent the number of the respective nucleotides in the primer sequence.

For longer primers thermodynamic calculations taking into account base stacking energy for calculation are recommended. This can be done easily using free online oligo calculation programs (e. g. https://www.biophp.org/minitools/melting_temperature/demo.php?formula=basic

http://biotools.nubic.northwestern.edu/OligoCalc.html).

Cloning of PCR Products: Taq Polymerase generates 3’ single-base dA overhangs that can be used for direct cloning into TA cloning vectors. For direct cloning into blunt vectors blunting of the purified PCR fragment (e. g. using the Klenow enzyme) is strongly recommended.

Licenses/Patents/Disclaimers:
The PCR process is disclosed and claimed in European patents EP0201184B1 and EP0200362B1 or United States patent US4965188A, which have expired along with all family members/equivalents in all territories. However, some specific product applications may fall within the scope of protection of granted patents, which may be in force in the United States, European Union and certain countries. The purchase of this product does not include a license to perform any application subject to patent protection, it is the sole responsibility of the buyer to ensure that use of the product does not infringe the patent rights of third parties.
For Research Use Only. Not for use in diagnostic procedures.