AccuTherm™ is a thermostable enzyme possessing 5'-3' DNA polymerase and 3'-5' proof reading exonuclea-se activities. It is isolated from the hyperthermophilic marine archae Pyrococcus furiosis (Pfu). AccuTherm™ provides extremely high fidelity. Whereas the enzyme is not able to amplify long fragments as efficiently as BioTherm™ or KlenTherm™ because of its very high exonuclease activity, a mixture of either KlenTherm™ or BioTherm™ with AccuTherm™ provides more robust synthesis of longer amplification products (Barnes, 1994. Proc. Natl. Acad. Sci. USA 91:2216-2220).
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-amino-propane-sulphonic acid, sodium salt) pH 9.3 (at 25°C), 50 mM KCI, 2 mM MgCl2, 1 mM .-mercaptoethanol) and activated 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 AccuTherm™ polymerase below 0°C, preferably at -20°C, in a constant temperature freezer
Synergy™ DNA polymerase, SynergyN™ DNA polymerase
5X REACTION BUFFER
83 mM (NH 4) 2 S0 4 , 350 mM Tris-HCI pH 8.8 (at 25°C), 22 mM MgCl2 , 0,75% Triton X100, 100 mM KCl