Description

Tth DNA Polymerase is a thermostable enzyme that replicates DNA at 74 °C and exhibits a half-life of 20 minutes at 95 °C isolated from eubacterium Thermus thermophilus strain HB8. Tth catalyzes the polymerization of nucleotides into duplex DNA in the 5´→3´ direction in the presence of magnesium and the polymerization of nucleotides into DNA using an RNA template in the 5´→3´ direction in the presence of manganese. The enzyme has a molecular weight of 94 000 daltons as estimated from the predicted amino acid sequence and exhibits 5´→3´ exonuclease activity. Tth is recommended for use in PCR, RT-PCR, reverse transcription and primer extension reactions at elevated temperature.

Recombinant enzyme with both intrinsic transcriptase and thermostable DNA polymerase activity, a convenient solution for single tube RT-PCR. The enzyme tolerates temperatures up to +50°C –in range of +50 to +70°C for the RT reaction, and up to +95°C for the PCR, overcoming problems caused by RNA secondary structures. Carryover prevention via the incorporation of dUTP and subsequent treatment with UNG is also possible.

Features

The thermostability and the reverse transcriptase (RT) activity of Tth DNA polymerase is useful in amplifying DNA from RNA templates that contain G-C-rich sequences or secondary structures since the elevated temperatures serve to denature the template RNA. Higher temperatures (in contrast to other enzymes for RT-PCR) also result in increased specificity of primer hybridization and extension. The concentration of RNA template for effective reverse transcription with Tth DNA polymerase should be higher if to compare with reverse transcription directed by Reverse Transcriptase (M-MuLV, AMV).

Applications

• PCR and RT-PCR - cDNA synthesis

Concentration: 5 u/µl

Storage Buffer

10 mM Tris-HCl, 1 mM dithiothreitol, 0.1 mM EDTA, 300 mM KCl, 0.1% Triton X-100 (v/v)*, 50% glycerol (v/v), pH 7.5 (25°C)

Reaction Buffer

5X RT/PCR reaction buffer (One Step-buffer): 250 mM bicine (pH 8.2, by KOH, at 25°C), 580mM KOAC, 40% Glycerol
10X PCR buffer: 100 mM Tris-HCl, (pH 8.8 at 25°C), 15 mM MgSO₄, 800mM (NH4)₂SO₄, 0.5 mg/ml BSA, 0.5% Tween 20

Usage

1.) One step RT PCR: - Reverse transcription and amplification in one Tube - Advantage: The one step One step reaction eliminates the risk of cross contaminations associated with two step RT-PCR.
2.) Two step RT PCR
3.) Standard PCR

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