Optimized for cycle sequencing    Uniform intensities    Low backgorund

DESCRIPTION
KlenThermase™ DNA polymerase is an optimised version of KlenTherm™ DNA polymerase designed for cycle sequencing with dideoxynucleotides. This enzyme is recommended both for manual DNA sequencing with 35S label and for automated fluorescent DNA sequencing. Mutations have been introduced into the KlenTherm™ DNA polymerase that confer on this enzyme enhanced properties for cycle sequencing of double-stranded PCR products. KlenThermase™ is similar to, yet distinct from, USB ThermoSequenase. Occasional weak bands occur with prolonged reaction or cycle times caused by sequence-specific pyrophosphorolysis which is catalyzed by the polymerase. We recommend to use KlenThermase™ with our thermostable Tth inorganic pyrophosphatase (1 unit of Tth inorganic pyrophosphatase added to 10 units of KlenThermase™) for further improvement of uniformity of band intensities.

APPLICATION
Fidelity: The relative mutation rate during polymerisation is two-fold lower for KlenThermase™ as compared to the full-length Taq DNA polymerase.
Cycle sequencing: The absence of the 5'-3' exonuclease activity makes KlenThermase™ especially suitable for cycle sequencing. It gives higher sequence intensity and very low backgrounds. The mutational optimization improves the uniformity of band intensities. A combination of KlenThermase™ with Tth inorganic pyrophospatase generates uniform bands that improve sequencing accuracy and give long read lengths.

CONCENTRATION
25 units/µl

UNIT DEFINITION
One unit is defined as the amount of enzyme that incorporates 10 nmoles of dNTPs into acid-insoluble form in 30 minutes at 72°C under the assay conditions (25 mM TAPS (tris-(hydroxymethyl)-methyl-aminopropane-sulfonic acid, sodium salt) pH 9.3 (at 25°C), 50 mM KCI, 2 mM MgCl₂, 1 mM β-mercaptoethanol and activated calf thymus DNA as substrate.

STORAGE BUFFER
10 mM K-phosphate buffer pH 7.0, 100 mM NaCI, 0.5 mM EDTA, 1 mM DTT, 0.01% Tween 20; 50% glycerol (v/v)

STORAGE TEMPERATURE
Store KlenThermase™ DNA polymerase below 0°C, preferably at -20°C, in a constant temperature freezer.

REFERENCES
Tabor, S. & Richardson, C. C. (1995), Proc Natl Acad Sci U S A 92(14), 6339--6343.

PRODUCT CITATIONS
Gudnason, H.; Dufva, M.; Bang, D. D. & Wolff, A. (2008), Biotechniques 45(3), 261--271.
Milani, L.; Gupta, M.; Andersen, M.; Dhar, S.; Fryknäs, M.; Isaksson, A.; Larsson, R. & Syvänen, A.-C. (2007), Nucleic Acids Res 35(5), e34.
Milani, L. & Syvänen, A.-C. (2009), Methods Mol Biol 529, 215--229.