- Tth DNA polymerase -Thermostable DNA
polymerase with reverse polymerase activity.
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- Description
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- Tth DNA polymerase (EC 2.7.7.7) is a thermostable DNA-dependent DNA
polymerase derived from Thermus thermophilus. The enzyme is also capable
of catalyzing the polymerization of DNA in the range of 1-2 kb using
an RNA template in the presence of MnCl2. The ability of
Tth DNA polymerase to reverse transcribe at elevated temperatures minimizes
the problems encountered with strong secondary structures in RNA since
they are unstable at higher reaction temperatures. Higher temperatures
also result in increased specificity of primer hybridization and extension.
Optimal temperature is 75°C, half-life at 95°C is 60 min. The
enzyme will incorporate modified nucleotides (e.g., digoxigenin-11-dUTP
and fluorescein-12-dUTP but not biotin-16-dUTP) at high rates. The enzyme
has 3´ terminal deoxynucleotidyl transferase actifity which usualy
results in addition of single dATP to the duplex DNA.
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- Unit definition
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One unit of the enzyme will incorporate 10 nmol of dNTPs into an acid-insoluble
form in 30 min at 72°C under standard assay conditions using a DNA
template.
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- Reaction buffer
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Reverse transcription buffer B6: (x10) : 670 mM Tris-HCl, pH 8.8, 166
mM (NH4)2SO4,
0.1% Tween 20. Supplied with 10 mM MnCl2. Optimal concentration
of MnCl2 is 1-1,5 mM. DMSO should be added at a final conc.
of 5%.
Amplification buffer B9: (x5): 125 mM Tris-HCl pH 8.9, 500 mM KCl, 0,25%
Tween 20, 12,5 mM MgCl2, 3,75 mM EGTA, 0,05% gelatine, 25%
glycerol.
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- Storage
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- Store at -20°C.
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- Applications
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- RT-PCR.
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Quality control
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Activity, SDS-PAGE purity, absence of endonucleases and exonucleases,
specific performance tests.
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References
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1. Rüttimann, C. et al. (1985) Eur. J. Biochem. 149, 41.
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- Catalog #
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- T12.3
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T12.4
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Pack size
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500 U
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1000 U
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