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Taq DNA Polymerase 

Taq DNA Polymerase is a 94 kDa, thermostable, recombinant DNA polymerase obtained by expression of a modified form of the Thermus aquaticus (Taq) DNA polymerase gene in E. coli.
Cat. No. Size Price Quantity  
TB10001A 500 U USD 50
TB10001B 2 x 500 U USD 90
TB10001C 5000 U USD 450
Tel: +1 (973) 789-8318Email: [email protected]

Reagents Supplied

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

Description

Taq DNA Polymerase is a 94 kDa, thermostable, recombinant DNA polymerase obtained by expression of a modified form of the Thermus aquaticus (Taq) DNA polymerase gene in E. coli. It has both 5'→3' polymerase and 5'→3' exonuclease activity, but lacks 3'→5' exonuclease activity. As most PCR products amplified with Taq DNA Polymerase have one A added at 3'-termini, the obtained PCR product can be directly used for cloning into T-Vector.

Applications

  • General PCR
  • Colony PCR
  • Primer Extension

Source

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× Taq PCR Buffer

  • 500 mM Tris-HCl (pH 9.0 @ 25°C)
  • 200 mM KCl
  • 100 mM (NH4)2SO4

Storage Buffer

  • 20 mM Tris-HCl (pH 8.0 @ 25°C)
  • 100 mM KCl
  • 0.1 mM EDTA
  • 1 mM DTT
  • 0.5% Tween-20
  • 50% Glycerol

Reaction Setup

Typical PCR reaction setup

Set up the following reaction in a sterile, nuclease-free microcentrifuge tube on ice.

Component Volume Final Concentration
10× 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);
Colony
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 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 2 min
Denaturation 95°C 30 sec
Annealing (Tm-5) °C 30 sec
Extension 72°C 1 min/kb
Final Extension 72°C 5 min
 30 - 40 cycles
 
  • With colony PCR, an initial 5-minutes pre-denaturation at 95°C is recommended.
  • Generally, 25–35 cycles result in optimal amplification of desired products. Up to 40 cycles may be required to detect low-copy-number targets.

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.

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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