KlenTaq1TM Data Sheet
KlenTaq1TM 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 98°C in reaction buffer PC2 does not seem to diminish the enzyme activity. Significant activity remains even after exposure to 99°C. The full length enzyme does not tolerate these treatments.
The KlenTaq1TM 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. KlenTaq1TM is therefore similar to, yet distinct from, Hoffman LaRoche's Stoffel Fragment.
Store enzyme at -20°C upon arrival.Store buffer at 4°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 O°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 or Triton X-100 or NP-40.
Concentration: 5 KlenTaq1TM 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 nmoles/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 the order). Note the absence of KCl and gelatin when compared to other buffers for thermostable DNA polymerases. The dNTP concentration can vary from 50 mM each to 1.2 mM each, but 200 mM is normally used.
You will need more KlenTaq1TM protein than Taq protein if the DNA incorporation is more than 500 bp.KlenTaq1TM is shipped at 25-30 u/ml 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. KlenTaq1TM 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 KlenTaq1TM needed to perform a reaction depends on the length of incorporation. For KlenTaq1TM, 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 2 kb template DNA. Since excess KlenTaq1TM is harmless, we conservatively recommend 0.50 microliter per reaction, just to be sure everything up to 2.5 kb will work. This is how we check and titre the enzyme.
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