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Home > Gene Recombination / Cloning > Golden Taq DNA Polymerase

Golden Taq DNA Polymerase 

Taq DNA Polymerase is a thermostable DNA polymerase that possesses a 5´→3´ polymerase activity and a 5´ flap endonuclease activity.
Cat. No. Size Price Quantity  
TB10002A 200 U USD 65
TB10002B 500 U USD 130
TB10002C 5000 U USD 650
Tel: +1 (973) 789-8318Email: [email protected]

Reagents Supplied

  • Golden Taq DNA Polymerase 5 U/μl
  • 10× Golden Taq PCR Buffer
  • 25 mM MgCl2

Description

Taq DNA Polymerase is a thermostable DNA polymerase that possesses a 5´→3´ polymerase activity and a 5´ flap endonuclease activity. Golden Taq DNA Polymerase is a chemically modified form of Taq DNA Polymerase requiring thermal activation. Golden Taq DNA Polymerase is provided in an inactive state and is activated by heat, allowing hot-start PCR for convenient, room-temperature reaction setup. Hot-start PCR is advantageous for most amplification targets by eliminating or minimizing primer-dimer and lowering non-specific products.

Applications

  • PCR
  • RT-PCR
  • Primer extension

Source

Golden Taq DNA Polymerase is prepared from E. coli carrying a plasmid that encodes the Thermus aquaticus DNA polymerase gene.

Activity Determination

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

10× Golden Taq PCR Buffer

200 mM Tris-HCl (pH 8.5 @ 25°C)
100 mM KCl
100 mM (NH4)2SO4

Storage Buffer

20 mM Tris-HCl (pH 9.0 @ 25°C)
100 mM KCl
0.1 mM EDTA
1 mM DTT
0.5% Tween-20
50% Glycerol

Typical PCR reaction setup

In a sterile, nuclease-free microcentrifuge tube, combine the following components at room temperature.

Component Volume Final Concentration
10× Golden Taq PCR Buffer 5 μl
10 mM dNTP 1 μl 0.2 μM
25 mM MgCl2 4 μl 2 mM
10 μM Forward Primer Variable 0.1−1.0 μM
10 μM Reverse Primer Variable 0.1−1.0 μM
Template DNA Variable Plasmid DNA: 1 ng (100 pg−50 ng);
Genomic DNA: 50 ng (10−200 ng);
cDNA: 50 ng (10−200 ng)
Golden Taq DNA Polymerase 0.5 μl 2.5 U
Nuclease-free water To 50 μl  
  • Mg2+ concentration of 1.5–2.0 mM is optimal for most PCR products generated with Golden Taq DNA Polymerase.
    If optimization is desired, Mg2+ can be further optimized in 0.5 or 1.0 mM increments using MgCl2.
  • The addition of DMSO (final conc. 2–5%) is beneficial for the amplification of GC-rich targets.

PCR cycle conditions

3-Step cycle
STEP TEMP. TIME
Pre-denaturation 95°C 10 min
Denaturation 95°C 30 sec
Annealing (Tm-5) °C 30 sec
Extension 72°C 1 min/kb
Final Extension 72°C 5 min
25 - 35 cycles
 
  • The 10 min, 95°C step is required to activate the Golden Taq DNA Polymerase.

Quality Control

Absence of Exonuclease Activity: Incubation of 50 U of the enzyme with 1 µg λ-HindIII DNA for 16 hours at 75ºC and 65ºC resulted in no smearing of bands on agarose gels.

Absence of Endonuclease Activity: Incubation of 50 U of enzyme with 1 µg of supercoiled pBR322 DNA for 16 hours at 75ºC resulted in no detectable conversion to relaxed or linear forms by agarose gel electrophoresis.

No detectable DNA contamination: 50 U of the enzyme was tested for E. coli genomic DNA contamination by PCR amplifying with the E. coli 16S ribosomal primers.

Functional Assay: Performance of DNA amplification by PCR was confirmed by using human genomic DNA as the template (amplified fragment: approx. 1.9 kb). The resulting PCR products are visualized as single bands on agarose gel.

Physical Purity: >99% pure by SDS-PAGE.

Notes

  • As most PCR products amplified with Golden Taq DNA Polymerase have one A added at 3'-termini, the obtained PCR product can be directly used for cloning into T-Vector. Also it is possible to clone the product in blunt-end vectors after blunting and phosphorylation of the end.

References

  • Chien, A., Edgar, D.B. and Trela, J.M. (1976) J. Bact., 127, 1550-1557.
  • Kaledin, A.S., Sliusarenko, A.G. and Gorodetskii, S.I. (1980) Biokhimiya., 45, 644-651.
  • Lawyer, F.C. et al. (1993). PCR Methods and Appl., 2, 275-287.
  • Longley, M.J., Bennett, S.E. and Mosbaugh D.W. (1990) Nucleic Acids Res., 18, 7317-7322.
  • Lyamichev, V., Brow, M.A. and Dahlberg, J.E. (1993) Science, 260, 778-783.
  • Chou, Q., Russell, M., Birch, D.E., et al. (1992) Nucleic Acids Res. 20, 1717-1723.

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