Phoenix Hot Start Taq DNA Polymerase

OEM by QIAGEN offers customized bulk manufacturing of this enzyme.

Products

The Phoenix Hot Start Taq DNA Polymerase is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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Phoenix Hot Start Taq DNA Polymerase (5000 U)

Cat. No. / ID: P7590L

5,000 U (evaluation pack) Phoenix Hot Start Taq DNA Polymerase (0.10mL at 5000 U/mL), 5x Phoenix Hot Start Taq Reaction Buffer (4 x 1.5 mL), and 5x Phoenix Hot Start Taq GC Reaction Buffer (2 x 1.5 mL)

Features

  • Thermostable 5ʹ→3ʹ DNA polymerase with antibody mediated hot start
  • Lacks a 3ʹ→5ʹ proofreading function
  • 72-hour stability at room temperature for simple setup and automated workflows
  • Tolerance to wide ranges of Mg2+ and annealing temperatures

Product Details

Phoenix Hot Start Taq DNA Polymerase provides antibody-based hot start for robust PCR performance with exceptional pre-PCR cycling room-temperature stability.

The enzyme is supplied in 20 mM Tris-HCl, 100 mM NaCl, 0.1 mM EDTA, Stabilizer and 50% glycerol; pH 7.5 at 25°C.

Low glycerol and glyocerol-free formulations also available.

OEM by QIAGEN offers customized bulk manufacturing of this enzyme.

Performance

OEM by QIAGEN offers bulk manufacturing of Phoenix Hot Start Taq DNA Polymerase in custom 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
SDS 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 B7590 and B7591

References
1. Chou, Q., et al. (1992) Nucleic Acids Res. 20: 1717.
2. Sharkey, D., et al. (1994) Nature Biotechnology 12: 506.

Principle

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

Procedure

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 H2O 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.

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 Mg2+ in the final reaction (i.e., when diluted to 1X). In cases where additional Mg2+ optimization is required, adjust the final Mg2+ concentration in 0.2 mM steps.

Quality control analysis

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.

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).

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.

Applications

This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.

  • Routine PCR amplification up to 5 kb
  • High-throuput PCR
  • Primer extension
  • Multiplex PCR and RT-PCR
  • Probe or dye-based real-time qPCR and RT-qPCR

Resources

FAQ

How can I optimize Mg2+ conditions for a specific amplicon when using Phoenix Hot Start Taq and the supplied reaction buffer?
Supplied Phoenix Hot Start Taq buffers contain 2 mM Mg2+ in the final reaction. Therefore, final Mg2+ reaction concentration may be increased according to user preference with a concentrated solution containing Mg2+.

Note: Increasing final Mg2+ concentration may lower fidelity of the PCR (1).     
FAQ ID - 3902
Why is the Phoenix Hot Start Taq sometimes cloudy upon removing from -20°C storage?
Phoenix Hot Start Taq enzyme solution may appear cloudy immediately upon removing from –20°C degree storage due the presence of a stabilizer in the storage buffer.
FAQ ID - 3906
What is the fidelity/error rate of Phoenix Hot Start Taq?
Phoenix Hot Start Taq has the same fidelity as standard Taq. The fidelity is approximately 2.7 x 10-5 in Phoenix Hot Start Taq reaction buffer when measured using a LacI-based assay (2).
FAQ ID - 3905
Is Phoenix Hot Start Taq capable of multiplex PCR?
Yes.  Phoenix Hot Start Taq is capable of amplifying multiple amplicons simultaneously in a single 50 µl PCR. Increasing the amount of polymerase added (up to 2.5 U) may improve results.
FAQ ID - 3901
Can Phoenix Hot Start Taq utilize cDNA as template for PCR?
Yes.
FAQ ID - 3900
How can PCR cycling conditions be optimized for Phoenix Hot Start Taq?
General Taq protocols for PCR cycling (see recommended cycling conditions from Phoenix Hot Start Taq on Product Information Sheet) are a good starting point.  For determining the optimal temperature for the primer annealing step, vary the temperature by 2 degree increments starting at about 10 degrees below the Tm of the primer with the lower Tm up to about 5 degrees above Tm of primer with the higher Tm.

Phoenix Hot Start Taq is also compatible with Touchdown PCR cycling.  
FAQ ID - 3899
What is the amplification length limit of Phoenix Hot Start Taq?
Phoenix Hot Start Taq has been demonstrated to amplify up to 4 kb human genomic DNA targets, and up to 8 kb lambda DNA targets.  
FAQ ID - 3904
How can yield for long targets be increased when using Phoenix Hot Start Taq?
Use of a PCR enhancer containing Betaine, DTT, BSA, and DMSO (3) may be used to improve yield of complex targets ≥ 2 kb.
FAQ ID - 3907
How stable is Phoenix Hot Start Taq when incubated in a PCR reaction mix at room temperature?
Phoenix Hot Start Taq is stable at room temperature for at least 72 hours when incubated in a PCR reaction mix containing 1X of the supplied, optimized Phoenix Hot Start Taq reaction buffer (standard or GC).  Similar testing on a smaller number of amplicons demonstrated functional stability by endpoint PCR following 20 days room temperature incubation.
FAQ ID - 3898
When should I use Phoenix Hot Start Taq GC reaction Buffer?
Phoenix Hot Start GC reaction buffer is recommended for use with difficult or GC-rich amplicons (≥ 55%). 
FAQ ID - 3903