Substitution of Asn for the conserved Ser543 in the thumb subdomain of the Taq DNA polymerase large fragment (KlenTherm™ DNA polymerase) prevents pausing during DNA synthesis and allows the enzyme to overcome template regions with a complex secondary structure. The mutant enzyme, KlenThermN™ DNA poly-merase (patent pending), provides specific PCR amplification and sequencing of difficult templates, e.g. those with a high GC content or complex secondary structure. Furthermore this substitution increases several times the efficiency of synthesis of long (over 2 kb) DNA molecules. The difference in the DNA synthesis efficiencies by the mutant and native enzymes increases with the increase in the DNA fragment length.CONCENTRATION
10 units/µlUNIT DEFINITION
One unit is defined as the amount of enzyme that incorporates 10 nmoles of dNTPs into acid-insoluble form in 30 min at 73°C under the assay conditions 25 mM TAPS (tris-(hydroxy-methyl)-methyl-amino-propanesul-fonic acid, sodium salt) pH 9.3 (at 25°C), 50 mM KCl, 3.5 mM MgCl2 , 1 mM .-mercaptoethanol) and activa-ted salmon sperm DNA as substrate.STORAGE BUFFER
10 mM K-phosphate buffer pH 7.0, 100 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 0.01% Tween 20; 50% glycerol (v/v)STORAGE TEMPERATURE
Store KlenThermN™ DNA polymerase, preferably at -20°C, in a constant temperature freezer.10X REACTION BUFER
500 mM KCl, 100 mM Tris-HCl (pH 9 at 25°C), 1% Triton X100COMPANION PRODUCTS
Extra solution: 50 mM MgCl 2 , add MgCl 2 to a final concentration of 3.5 mM.
Please note the difference between KlenThermTM and BioThermTM reaction buffers!
1.5 ml 10x reaction buffer Cat. No GC-001-006
KlenTherm™ DNA polymerase, Synergy™ DNA polymerase, SynergyN™ DNA polymerase, dNTPsREFERENCES
- Ignatov, K.B., Miroshnikov, A.I., Kramarov, V.M. (1998) FEBS Lett. 425, 249-250. Substitution of Asn for Ser543 in the large fragment of Taq DNA polymerase increases the efficiency of synthesis of long DNA molecules.