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  • Taq DNA Polymerase
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Introduction

ThermalAce™ DNA Polymerase is an extremely thermostable enzyme from a proprietary archaebacterium that is specifically designed for high-yield PCR amplification of GC-rich templates (>65% GC content). The enzyme
retains full activity after incubation at 95°C for 4 hours and has five-fold better processivity than Taq DNA polymerase. An optimized buffer reduces the need for additional optimization in many cases, making ThermalAce™ the enzyme of choice for a wide variety of applications. Sufficient reagents are provided for 100 or 500 amplification reactions of 50 μl each (at 2 units of ThermalAce™ DNA Polymerase per reaction).


Kit Size

Component200 Units
1000 Units
ThermalAce™ DNA Polymerase (2 U/μl)
100 μl
500 μl
10X ThermalAce™ Buffer
1 ml
5 ml
50X dNTP Mix (10 mM each of dATP,
dCTP, dGTP, dTTP, at pH 8.0) 200 μl 1 ml

200 μl

1 ml

 
ThermalAce™ DNA Polymerase

2 U/μl in 50 mM Tris-HCl (pH 8.0), 100 mM KCl, 1 mM Dithiothreitol (DTT), 0.1 mM EDTA, 50% Glycerol, 0.1% Triton® X-100

10X ThermalAce™ Buffer

600 mM Tris-HCl (pH 9.25), 15 mM MgSO4, 300 mM NaCl, 0.1 mg/ml bovine serum albumin (BSA), 0.1% Triton® X-100, and proprietary components

Unit Definition

One unit of enzyme is the amount of enzyme required to incorporate 10 nmoles of dNTPs into acid insoluble material in 30 minutes at 74°C. This is the standard unit definition for all thermostable polymerases used for PCR.
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Ordering Information

Catalog # Name Size List Price (USD) Qty
18038240 Taq DNA Polymerase, native 5,000 units 1,222.00
18038018 Taq DNA Polymerase, native 100 units 45.00

Basic PCR Protocol

The following protocol is recommended as a starting point. Optimal reaction conditions (incubation times and temperatures; concentrations of enzyme, primers, and template) may vary, and may be adjusted as needed. Recommended cycling temperatures are provided for both GC-rich templates (>65% GC content) and standard templates.
  1. Add components in the following order to each reaction vessel on ice.

    DNA template x μl
    Primers (100 ng each)1 μl
    50X dNTP Mix (10 mM each dNTP) 1 μl
    10X ThermalAce™ Buffer5 μl
    Sterile Waterto 49 μl
    ThermalAce™ (2 U/μl)*
    1 μl
    Final volume
    50 μ

    Note: Up to 3 U of enzyme (1.5 μl) may be added for difficult templates. A master mix can be prepared for multiple reactions to enable accurate pipetting.

  2. Cap/seal the reaction vessels and flick with your finger for several seconds to mix. Place reaction(s) on ice until ready to cycle.

  3. Program the thermal cycler as follows. Note that the annealing temperature may vary depending on the Tm of your primers. The optimal annealing temperature is typically 5°C below the Tm of the primers.

    Step 
    Temp (GC-rich
    template)
    Temp (standard
    template)
    Time
    Cycle
    Denaturation98°C95°C
    3 min
    1
    Denaturation 98°C 95°C
    30 sec
    25–30
    Annealing65°C (5°C < Tm)55°C (5°C < Tm)
    30 sec
    25–30
    Extension72°C 74°C
    1 min/kb
    25–30
    Final Extension
    72°C
    74°C
    10 min
    1


  4. Maintain the reaction at 4°C after cycling. The samples can be stored at -20°C until use. Analyze 5–10 μl of sample by agarose gel electrophoresis.
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Reference

  1. Barnes, W. M. (1992). “The Fidelity of Taq Polymerase Catalyzing PCR is Improved by an N-terminal Deletion.” Gene 112: 29–35.

  2. Barnes, W. M. (1994). “PCR Amplification of Up to 35-kb DNA with High Fidelity and High Yield from Lambda Bacteriophage Templates.” Proc. Natl. Acad. Sci. USA 91: 2216–2220.
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MAN000142      18-Jun-2010