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. 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.
- Fidelity The relative mutation rate during polymerisation is twofold 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 inten-sities. Combination of KlenThermase™ with Tth inorganic pyrophospatase generates uniform bands that improve sequencing accuracy and give long read lengths.
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 .-mercaptoethanol and activa-ted calf thymus DNA as substrate.
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)
Store KlenThermase™ DNA polymerase below 0°C, preferably at -20°C, in a constant temperature freezer.
5X ANNEALING BUFFER
260 mM Tris-HCl (pH 9.5), 65 mM MgCl2
KlenThermaseN™, KlenTherm™ DNA polymerase, KlenThermN™ DNA polymerase, Tth pyrophosphatase
- Tabor, S. and Richardson, C.C. I995. A single residue in DNA polymerase of the Escherichia coli DNA polymerase 1 family is critical for distinguishing between deoxy- and dideoxyribonucleotides. Proc. Natl. Acad. Scf. USA 92: 6339-6343