Tth DNA Polymerase is the recombinant form of the enzyme obtained from the thermophilic eubacterium Thermus thermophilus expressed in E.coli. It is a highly processive 5' to 3´ DNA polymerase lacking 3' to 5´ exonuclease activity. Tth DNA Polymerase catalyzes the polymerization of nucleotides into duplex DNA in the 5' to 3´ direction in the presence of Mg²⁺. Tth DNA Polymerase has a very efficient intrinsic reverse transcriptase (RT) activity in the presence of Mn²⁺ ions. This RT activity is not associated with RNase H activity. The ability of Tth DNA Polymerase to catalyze the polymerization of DNA, using a RNA template at high temperature, minimizes the problems encountered with strong secondary structures in RNA, since they are unstable at higher reaction temperatures. Tth DNA Polymerase is resistant to prolonged incubation at 95℃.
- 5 unit/㎕ in 20 mM Tris-HCl, pH8.0, 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20, and 50% Glycerol.
- PCR, one-step RT-PCR, genomic PCR, and primer extension.
- One unit is defined as the amount of enzyme required to catalyze the incorporation of 10 nmole of dNTP into an acid-insoluble form in 30 min at 72℃. The reaction conditions are : 25 mM TAPS, pH9.3, 50 mM KCl, 2 mM MgCl₂, 1 mM β-mercaptoethanol, 200 μM each dNTPs, 100 μM [α-³²P]dCTP, 12.5 ㎍ activated calf thymus DNA in a total volume of 50 ㎕.
10x Reaction Buffer w/ MgCl₂
- 500 mM Tris-HCl, pH9.0, 160 mM (NH₄)₂SO₄, 25 mM MgCl₂, 1% Triton X-100, 1 mg/ml BSA.
- Optimization of [Mg²⁺] and [Mn²⁺] : Magnesium and manganese ion concentrations should be optimized for a cDNA synthesis. The optimal concentration of each metal cation is highly dependent on the dNTP concentration and on the template, primers, and protocol used.
For many templates, the optimal concentration of magnesium ions using Tth DNA Polymerase is approximately 1.5 mM if the concentration of each dNTP in the reaction is 0.2 mM. If used with 1.5 mM Mg²⁺, the optimal concentration of Mn²⁺ for RNA-dependent DNA synthesis is approximately 0.5 mM for many templates.
For an RT-PCR reaction, we recommend 2.5 mM Mg²⁺ and 0.5 mM Mn²⁺ for most templates.
- Activity, SDS-PAGE purity, performance tests.
- Store at -20℃.