Phoenix™ Hot Start Taq DNA Polymerase

Product Description

Phoenix™ Hot Start Taq DNA Polymerase is a recombinant, thermostabile Taq DNA polymerase complexed with a thermolabile, neutralizing antibody that blocks the 5′→3′ polymerase activity prior to the initial DNA denaturation step of PCR (1,2). Such antibody-mediated Hot-Start capability enhances the overall specificity, sensitivity and yield of the PCR by reducing nonspecific amplification and primer-dimer formation prior to PCR cycling, and allows the convenience of reaction set up at room temperature. When the temperature of the PCR reaction mix reaches ≥94°C during the initial DNA denaturing step of PCR cycling, activity of the Taq DNA polymerase is fully restored. Phoenix Hot Start Taq DNA Polymerase, like standard Taq DNA polymerase, also has 5′→3′ exonuclease activity, but lacks any detectable 3′→5′ exonuclease activity.

Source of Protein
A recombinant E. coli strain carrying the Taq DNA polymerase gene from the thermophilic organism Thermus Aquaticus YT-1 complexed with a monoclonal antibody derived from murine cell culture.

Supplied in
20 mM Tris-HCl
100 mM NaCl
0.1 mM EDTA
Stabilizer
50% Glycerol
pH 7.5 @ 25°C

Supplied With:
5X Phoenix Hot Start Taq Reaction Buffer (B7590)
5X Phoenix Hot Start Taq GC Reaction Buffer (B7591)

Unit Definition
1 unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid-insoluble material in 30 minutes at 75°C.

Quality Control Analysis

Functional Assay
Phoenix Hot Start Taq DNA polymerase was tested for its ability to amplify DNA targets by PCR in reaction buffers B7590 and B7591 following a 24 hour incubation at room temperature. After heat activation and PCR amplification the samples resulted in visible single band amplicons as determined by agarose gel electrophoresis.

Taq Inhibition
This assay measured the residual activity of Phoenix Hot Start Taq DNA polymerase in the absence of a ≥ 94°C heat activation step. Phoenix Hot Start Taq DNA polymerase and Taq DNA Polymerase (Taq DNA Polymerase without Taq Antibody) were incubated in the presence of Calf Thymus DNA, 50 µM ³H-dTTP, 100 µM dNTPs and 1X reaction buffer B7590 or B7591. After a 24 hour incubation at room temperature the samples were analyzed for total 3H-dTTP counts incorporated using the method of Sambrook and Russell (Molecular Cloning, v3, 2001, pp. A8.25-A8.26).

Contamination Tests

Single-Stranded Exonuclease Activity
Radiolabeled single-stranded DNA substrate incubated with enzyme solution for 4 hours at 37°C resulted in less than 5% release of TCA-soluble counts.

Double-Stranded Exonuclease Activity
Radiolabeled double-stranded DNA substrate incubated with enzyme solution for 4 hours at 37°C resulted in less than 1% release of TCA-soluble counts.

Double-Stranded Endonuclease Activity
A reaction containing plasmid DNA incubated with enzyme solution for 4 hours at 37°C resulted in no visually discernible conversion to nicked circular DNA as determined by agarose gel electrophoresis.