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|
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
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.
Add the following components to an autoclaved microcentrifuge tube at either room temperature or on ice:
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**
Autoclaved distilled water to 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).
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.)
Cap the tubes and centrifuge briefly to collect the contents.
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.
Maintain the reaction at 4°C after cycling. The samples can be stored at -20°C until use.
- Analyze the products by agarose gel electrophoresis and visualize by ethidium bromide staining.
- Takagi, M., Nishioka, M., Kakihara, H., Kitabayashi, M., Inoue, H., Kawakami, B., Oka, M., and Imanaka, T. (1997) Appli. Environ. Microbiol., 63, 4504-4510.
- Nishioka M, Mizuguchi H, Fujiwara S, Komatsubara S, Kitabayashi M, Uemura H, Takagi M, Imanaka T. (2001) J. Biotechnol., 88, 141–9.
- Cline, J., Braman., and Hogrefe, H. H. (1996) Nucleic Acid Res., 24, 3546.
- Sharkey, D.J., Scalice, E.R., Christy, K.G., Atwood, S.M., Daiss, J.L. (1994) BioTechnology, 12, 506.
- Rapley, R. (1994) Mol. Biotechnol., 2, 295–298.