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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).
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.
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 | ||
| Component | 200 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.
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.
- 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™ Buffer 5 μl Sterile Water to 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.
- Cap/seal the reaction vessels and flick with your finger for several seconds to mix. Place reaction(s) on ice until ready to cycle.
- 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 Denaturation 98°C 95°C 3 min 1 Denaturation 98°C 95°C 30 sec 25–30 Annealing 65°C (5°C < Tm) 55°C (5°C < Tm) 30 sec 25–30 Extension 72°C 74°C 1 min/kb 25–30 Final Extension 72°C 74°C 10 min 1
- 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.
Reference
- Barnes, W. M. (1992). “The Fidelity of Taq Polymerase Catalyzing PCR is Improved by an N-terminal Deletion.” Gene 112: 29–35.
- 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.
MAN000142 18-Jun-2010
