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Description:
Tth DNA polymerase is a thermostable 93 kDa single-subunit enzyme purified from the E. coli recombinant strain expressing Thermus thermophilus HB-8 DNA polymerase gene. Amplification of target DNA fragments from 100 bp to 3000 bp can be achieved with this enzyme. Tth DNA polymerase also exhibits reverse transcription activity in presence Mn²⁺ ions much higher than that demonstrated for Taq DNA polymerase.

Storage and dilution buffer:
10 mM K phosphate buffer, pH 7.0, 100 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 50 % glycerol, 0.1 mg/ml BSA.

Unit definition:
One unit is defined as the amount of enzyme that incorporates 10 nmol of total dNTP’s into acid-insoluble form in 30 minutes at 70 ^oC under standard assay conditions.

Amplification buffer 10x, RT-PCR buffer 10x:
NH₄-buffer: 166 mM (NH₄)₂SO₄, 670 mM Tris-HCl (pH 8.8 at 25 °C), 0.1 % Tween-20.

Chelating buffer 10x (for RT-PCR):
166 mM (NH₄)₂SO₄; 670 mM Tris-HCl (pH 8.8 at 25 °C); 0.25 % Tween-20; 7.5 mM EGTA; 50 % glycerol.

Applications:
Reverse transcription: synthesizes DNA in the 5′-3′ direction from RNA template.
Primer extension: polymerizes dNTP’s in the 5′-3′ direction on single-stranded DNA template.

Quality control:
Activity, SDS-PAGE purity; endonuclease/nickase and exonuclease activities were not detectable under standard assay condition.

Concentration:
5 units/µl

Storage:
-20 °C

Available from http://www.labsupplymall.com

Description:
Thermus thermophilus PPi – recombinant form of the enzyme purified from E. coli strain carrying PPi Thermus thermophilus (strain HB8) overproducing plasmid. This enzyme catalyzes the hydrolysis of inorganic pyrophosphate to form orthophosphate:
P₂O₇^-4 + H₂O –> 2HPO₄^-2.
Inhibition of PCR reaction by pyrophophatase can be prevented by addition of PPi into reaction mixture.

Storage and dilution buffer:
50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.1 mM EDTA, 2 mM DTT, 50 % glycerol.

Unit definition:
One unit of activity is the amount of enzyme that will generate 1 µmol of phosphate per minute from pyrophosphate under standard reaction conditions.

Reaction buffer 10x:
PCR buffer of choice or sequencing buffer.

Applications:
PCR, RT-PCR and sequencing.

Quality control:
Activity, SDS-PAGE purity, absence of endonucleases/nickases and exonucleases.

Concentration:
2-5 units/µl

Storage:
-20 °C

Available from http://www.labsupplymall.com

Thermostable dUTPase (Cloned)

Thermostable dUTPase (pyrococcus fruriosus) was prepared and purified from Escherichia coli expression system. dUTPase is specific for dUTP and is critical for the fidelity of DNA replication and repair. dUTPase hydrolyzes dUTP to dUMP and pyrophosphate, simultaneously reducing dUTP levels and providing the dUMP for dTTP biosynthesis.

Thermostable dUTPase used for PCR with Pfu DNA polymerase improves the efficiency and longer targets of PCR reaction.

Unit Definition

One unit of enzyme catalyzes hadrylazation of 10 nanomoles of dUTP to dUMP in one hour at 85 Centigrade

Activity

(A) Measured by its ability to hydrolyze dUTP to dUMP in reaction buffer 20mM Hepers pH7.5, 150mM KCI, 5mM MgCl₂, 10mM dUTP at 85 Centigrade for 1 hour. The PPi production was quantified by using the enzymatic determination kit from Sigma.

(B) Enhancing PCR amplication: 50ul of Pfu PCR reaction system with 1-3u of dUTPase to amplify genomic DNA target up to 15-19 kb in length.

Specific activity: 10³ u/ug

Formulation: dUTPase is supplied in 20mM Tris-HC1 (pH 8.2), 1mM DTT, 0.1mM EDTA, 100mM KC1, 0.1% Nonidet P40, 0.1% Tween 20 and 50% glycerol at concentration of 1u/ul of the enzyme.

Reference

1. Dabrowski S. and Ahring B.K. protein Exp. Pur. 2003,31,72-78

2. Hogrefe H. et al. Proc. Natl. Acad. Sci. USA 2002,99,596-601