Description
Phoenix™ Hot Start Taq DNA Polymerase provides antibody-based hot start for robust PCR performance with exceptional pre-PCR cycling room-temperature stability. Supplied in: 20 mM Tris-HCl, 100 mM NaCl, 0.1 mM EDTA, Stabilizer and 50% glycerol; pH 7.5 at 25°C. Supplied with: 5X Phoenix Hot Start Taq Reaction Buffer (B7590) and 5X Phoenix Hot Start Taq GC Reaction Buffer (B7591) OEM by QIAGEN offers bulk manufacturing of Phoenix Hot Start Taq DNA Polymerase in custom formulations, including Low glycerol and glycerol-free formulations.
Phoenix Hot Start Taq DNA Polymerase is a recombinant, thermostable Taq DNA polymerase complexed with a thermostable, neutralizing antibody that blocks the 5ʹ→3ʹ 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 mixture 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 5ʹ→3ʹ exonuclease activity, but lacks any detectable 3ʹ→5ʹ exonuclease activity. Increased amplification specificity compared to regular Taq DNA polymerase Increased amplification yield and overall success rate Rapid reactivation and shorter PCR cycle time with antibody method High sensitivity amplification with 300 pg human genomic DNA Multiplexing capability with minimal optimization High success rates with GC-rich targets Polymerase properties Storage temperature: –25°C to –15°C Test Units Tested Specification Purity N/A Specific activity N/A 74,625 U/mg Single-stranded exonuclease 50 U <10% Double-stranded exonuclease 50 U <1% Double-stranded endonuclease 50 U No conversion Taq inhibition N/A Pass Functional assay N/A Functional with buffers ordered with cat. nos. B7590 and B759
Source of recombinant enzyme 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. Unit definition: One 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. Molecular weight: 94kDa Optimum extension temperature: 66–72°C Extension rate: 60 seconds per kilobase at 72˚C Proofreading (3'-5' exo): No Nick-translation (5'-3' exo): Yes Strand displacement: No Extends from a nick: Yes
General precautions should be taken when setting up PCR, including steps to avoid cross-contamination and ensure gentle pipetting, thorough mixing and brief centrifugation after all components are added to the reaction. The following procedure can be used as a guideline. Reactions may need to be optimized individually depending on the desired result. The tables below provide information for 50 µl reactions and typical cycling conditions. The total reaction volume can be adjusted as needed. Cycling conditions may need to be optimized, depending on the amplicon of interest. Component Volume (µl) Final concentration Sterile H 2 O Variable n/a 5x Phoenix Hot Start Taq Reaction Buffer or 5x Phoenix Hot Start Taq GC Reaction Buffer 10 1X 10 mM dNTP mix 1 200 µM each Primer 1 Variable 0.2 µM Primer 2 Variable 0.2 µM DNA template Variable See note 3, below Phoenix Hot Start Taq DNA Polymerase 0.2 0.02 U/µl (or 1U) Step Temperature Time Cycles Initial denaturation* 94°C 30 seconds to 3 minutes 1 Denaturation Annealing Extension 94°C Varies 72°C 30 seconds 30 seconds 60 seconds/kb 25–40 Final extension 72°C 4°C 5 min hold 1 * Required for template denaturation and activation of Phoenix Hot Start Taq Polymerase. Usage Notes 1. 5x Phoenix Hot Start Taq Buffer should be used as the default buffer. For GC-rich and difficult templates, use 5x Phoenix Hot Start Taq GC Buffer. 2. A final concentration of 0.2 µM is recommended for each primer, but can be varied in the range of 0.2–1 µM. 3. The following table give the recommended template quantities. Complexity Source (example) Guideline Low Plasmid, virus, BAC 1 pg – 10 ng High Genomic DNA 50–250 ng 4. One unit of enzyme is usually sufficient for amplifying most targets, but more may be required (up to 2.5 units) in multiplex PCR or to increase yields of difficult or long targets. 5. Both 5x Phoenix Hot Start Taq Buffer and the GC buffer are formulated so that they will provide 2 mM Mg 2+ in the final reaction (i.e., when diluted to 1x). In cases where additional Mg 2+ optimization is required, adjust the final Mg 2+ concentration in 0.2 mM steps. Quality control analysis Functionality of Phoenix Hot Start Taq DNA polymerase is evaluated by 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 is measured by the residual activity of Phoenix Hot Start Taq DNA polymerase in the absence of a ≥ 94⁰C 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). Single-Stranded Exonuclease is determined in a 50 µl reaction containing a radiolabeled single-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C. Double-stranded exonuclease activity is determined in a 50 µl reaction containing a radiolabeled double-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C. Double-stranded endonuclease activity is determined in a 50 µl reaction containing 0.5 µg of plasmid DNA and 10 µl of enzyme solution incubated for 4 hours at 37°C.
