Introduction

Description

AccuPrime™ Pfx DNA Polymerase is a proprietary enzyme preparation containing recombinant DNA polymerase from Thermococcus species strain KOD (1,2). This polymerase possesses a proofreading 3’ to 5’ exonuclease activity that provides higher fidelity than Pfu DNA polymerase (3). AccuPrime™ Pfx DNA Polymerase is a highly processive enzyme and possesses a fast chain extension capability. It is provided in an antibody bound form that is inactive at ambient temperatures. The enzyme regains activity after the initial denaturation step at 94°C in PCR cycling, providing an automatic “hot start” that increases specificity, sensitivity, and yield, while allowing room temperature assembly (4). 10X AccuPrime™ Pfx Reaction Mix contains thermostable AccuPrime™ proteins, MgSO4, and dNTPs. Thermostable AccuPrime™ proteins enhance specific primer-template hybridization during every cycle of PCR (5). The high specificity, fidelity, and yield offered by AccuPrime™ Pfx DNA Polymerase make it ideal for demanding PCR applications


 Component 200-rxn kit 1,000-rxn kit
AccuPrime™ Pfx DNA Polymerase (2.5 U/μl) 100 μl 500 μl
50-mM Magnesium Sulfate 1 ml 2 × 1 ml
10X AccuPrime™ Pfx Reaction Mix 1 ml 5 × 1 ml

Unit Definition

One unit of AccuPrime™ Pfx DNA Polymerase incorporates 10 nmol of deoxyribonucleotide into acid-insoluble material in 30 min at 74°C.

AccuPrime™ Pfx DNA Polymerase Storage Buffer

50-mM Tris-HCl (pH 8.0), 50-mM KCl, 1-mM DTT, 0.1-mM EDTA, stabilizers, and 50% (v/v) glycerol

Quality Control

The Certificate of Analysis provides detailed quality control and product qualification information for each product. Certificates of Analysis are available on our website. Visit the support page and search for the Certificate of Analysis by product lot number, which is printed on the box.

General Recommendations and Guidelines for PCR

PCR is a powerful technique capable of amplifying trace amounts of DNA. All appropriate precautions should be taken to avoid cross-contamination.

MgSO4  MgSO4 is included in the 10X AccuPrime™ Pfx Reaction Mix at a final concentration of 1 mM, which is sufficient for most templates. For further optimization, add 0.1 μl to 1.0 μl of 50-mM MgSO4 (included in the kit) to the reaction.

dNTPs:   dNTPs are included in the 10X AccuPrime™ Pfx Reaction Mix at a final concentration of 0.3 mM.

Annealing Temperature  The optimal annealing temperature should be 5–10°C lower than the Tm of the primers used; if necessary, gradually increase the annealing temperature by 2–3°C for higher specificity.

KCl:   For difficult primer sets, prepare titrations of KCl (not included) at final concentrations of 20–50 mM for further optimization.

Ordering Information

Sku Name Size Price Qty
12344024 AccuPrime™ Pfx DNA Polymerase 200 reactions USD 334.00
12344032 AccuPrime™ Pfx DNA Polymerase 1,000 reactions USD 1,498.00

PCR Protocol

The following general procedure is suggested as a starting point when using AccuPrime™ Pfx DNA Polymerase in any PCR amplification.

  1. Add the following components to an autoclaved microcentrifuge tube at either room temperature or on ice:

    ComponentVolume
    Final Concentration
    10X AccuPrime™ Pfx Reaction mix*5 μl
    1X
    Primer mix (10 μM each)*1.5 μl
    0.3 μM each
    Template DNA (10 pg–200 ng)
    ≥ 1 μl
    as required
    AccuPrime™ Pfx DNA Polymerase**
    0.4–1 μl
    1.0–2.5 units
    Autoclaved distilled waterto 50 μl



    *AccuPrime™ Pfx DNA Polymerase will not function in reactions that contain dUTP either in the primers or in the dNTP mix.
    **For most targets, 1 unit is optimal. Higher concentrations may be inhibitory. More enzyme may be required for longer targets (>3 kb).

  2. Mix contents of the tubes and overlay with mineral or silicone oil, if necessary. (Note: The oil overlay is unnecessary in thermal cyclers equipped with a heated lid.)

  3. Cap the tubes and centrifuge briefly to collect the contents.

  4. Denature the template for 2 min at 95°C. Perform 25–35 cycles of PCR amplification as follows:

    Three-step cycling                                                                      Two-step cycling
    Denature: 95°C for 15 s                                                              Denature: 95°C for 15 s
    Anneal: 55–64°C for 30 s                                                           Extend: 68°C for 1 min per kb
    Extend: 68°C for 1 min per kb

    Note:  Two-step cycling can be used for long primers with high Tm.

  5. Maintain the reaction at 4°C after cycling. The samples can be stored at -20°C until use.

  6. Analyze the products by agarose gel electrophoresis and visualize by ethidium bromide staining.

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References

  1. Takagi, M., Nishioka, M., Kakihara, H., Kitabayashi, M., Inoue, H., Kawakami, B., Oka, M., and Imanaka, T. (1997) Appli. Environ. Microbiol., 63, 4504-4510.

  2. Nishioka M, Mizuguchi H, Fujiwara S, Komatsubara S, Kitabayashi M, Uemura H, Takagi M, Imanaka T. (2001) J. Biotechnol., 88, 141–9.

  3. Cline, J., Braman., and Hogrefe, H. H. (1996) Nucleic Acid Res., 24, 3546.

  4. Sharkey, D.J., Scalice, E.R., Christy, K.G., Atwood, S.M., Daiss, J.L. (1994) BioTechnology, 12, 506.

  5. Rapley, R. (1994) Mol. Biotechnol., 2, 295–298.
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MAN0001079          Rev. date: 1-Jun-2010