KlenTaq1, the thermostable DNA polymerase, is expressed from a 5’ deletion of the gene, encoding Thermus aquaticus DNA polymerase, leaving a highly active and even more heat-stable DNA polymerase activity. Repeated exposure to 980 C in reaction buffer PC2 does not seem to diminish the enzyme activity. Significant activity remains even after exposure to 990 C. The full length enzyme does not tolerate these treatments. The KlenTaq1™ enzyme is missing the N-terminal portion of the wild-type full length Taq DNA polymerase. This part of the protein is homologous to the 5’ – exonuclease region of E.coli DNA polymerase 1. KlenTaq1™ is therefore similar to, yet distinct from, Hoffman LaRoche’s Stoffel Fragment. For KlenTaq1 without glycerol (Cat# 1001 NGKT): Store the enzyme and the buffer at 4°C. The storage buffer is 50 mM ammonium sulfate, 20 mM Tris-HCl pH 8.55, 0.1 mM EDTA, 10mM mercaptoethanol, no gelatin, and 0.5% Thesit. For KlenTaq1 in 50% glycerol (Cat# 1001): Store the enzyme at -200 C and the buffer at 40 C. The glycerol in the storage buffer prevents freezing at -20°C but the Thesit causes some thickening at this temperature. It is optional, but advisable to warm up to 0°C, on ice, before dispensing. For long term storage, -70°C or -80°C may be employed, but we recommend that the enzyme not be frozen more than once. Re-freezing and thawing will cause degradation to the activity of the enzyme. The storage buffer is 50% glycerol (v/v; 63% w/v), 50 mM ammonium sulfate, 20 mM Tris-HCl pH 8.55, 0.1 mM EDTA, 10 mM mercaptoethanol, no gelatin, and 0.5% Thesit. Concentration: 5 KlenTaq1™ units (60 nmole incorporation in 30 minutes at 72°C). Activated salmon sperm DNA is the template; PC2 (see below) is the buffer. N.B. these units are 6 times larger than the standard units. In standard units (10 nmole/30min.), the enzyme concentration here is approximately 30 units/ml. Although the enzyme will function well under a broad range of conditions, the recommended reaction buffer, PC2, is: 50 mM Tris-HCl pH 9.1, 16 mM ammonium sulfate, 3.5 mM MgCl2, and 150 mg/ml BSA (supplied with order). Note the absence of KC1 and gelatin when compared to other buffers for thermostable DNA polymerases. The dNTP concentration can vary from 50 µM each to 1.2 mM each, but 200 µM is normally used.You will need more KlenTaq1™ protein than Taq protein if the DNA incorporation is more than 500 bp. KlenTaq1™ is shipped at 25-30 u/µl concentration so that it can easily incorporate 2000 bp, if the same quantity (recommended by Roche) is used as for full-length Taq DNA polymerase.
1 DNA incorporation unit = 10 nmoles / 30 min=’standard units’. Roche’s enzyme comes 5 ‘standard units’ per microliter. KlenTaq1™ comes with 25-30 ‘standard units’ per microliter. Roche recommends 0.5 microliter per 100 microliter reaction; that is, 1 microliter will catalyze 2 reactions. The amount of KlenTaq1™ needed to perform a reaction depends on the length of incorporation. For KlenTaq1™, in a 100 microliter reaction, one microliter will catalyze 15-20 reactions of 500 bp and 8-10 reactions of 1 kb and 3-5 reactions of 2kb template DNA. Since excess KlenTaq1™ is harmless, we conservatively recommend 0.50 microliters per reaction, just to be sure everything up to 2.5 kb will work. This is how we check and titre the enzyme.
Ref.Barnes, W.M., Gene, Vol. 112, pp. 29-35, 1992.Barnes, W. M., PNAS, Vol. 19, pp. 2216-2220, March 1994.Baskaran, N., et al, Genome Research, Vol. 6, pp. 633-638, 1996.
N.B. For KlenTaq1 without glycerol (Cat# 1001 NGKT): Store the enzyme and the buffer at 4°C. For KlenTaq1 in 50% glycerol (Cat# 1001): Store the enzyme at -200 C and the buffer at 40 C.