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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
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. Go to www.invitrogen.com/support 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.
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. Go to www.invitrogen.com/support 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.
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PCR Protocol
The following general procedure is suggested as a starting point when using AccuPrime™ Pfx DNA Polymerase in any PCR amplification.
- Add the following components to an autoclaved microcentrifuge tube at either room temperature or on ice:
Component Volume 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 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.
References
- 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.
MAN0001079 Rev. date: 1-Jun-2010
