AccuPrime™ SuperMix I provides qualified reagents for the amplification of nucleic acid templates by polymerase chain reaction (PCR). The mixture contains anti-Taq DNA polymerase antibodies, thermostable AccuPrime™ protein, Mg++, deoxyribonucleotide triphosphates (dNTPs), and recombinant Taq DNA polymerase at concentrations sufficient to allow amplification during PCR. Anti-Taq DNA polymerase antibodies inhibit polymerase activity at room temperature, providing an automatic “hot start” in PCR (Chou et al., 1992; Sharkey et al., 1994). The thermostable AccuPrime™ protein enhances specific primer-template hybridization during every cycle of PCR.
Antibody/AccuPrime™ protein-mediated amplification dramatically improves PCR specificity. It also improves the fidelity of Taq by 2-fold, and provides the most robust PCR for multiplex PCR and sub-optimal primer sets. AccuPrime™ SuperMix I may be stored at either -20°C or 4°C. Storage at 4°C avoids the necessity of thawing the mix before assembling the PCR. No detectable reduction of PCR performance or enzyme activity is observed after storage of AccuPrime™ SuperMix I for twelve months at 4°C. Repeated freeze-thaw cycles can reduce performance or activity. Reagents are provided for 200 or 1,000 amplification reactions of 25 μl each.
| Component ||200-rxns||1,000-rxns|
|AccuPrime™ SuperMix I||2 × 1.25 ml||12.5 ml|
40 mM Tris-HCl (pH 8.4); 100 mM KCl; 3 mM MgCl2, 400 μM each dGTP, dATP, dTTP, dCTP; AccuPrime™ Taq DNA Polymerase; thermostable AccuPrime™ protein; stabilizers
Guidelines for PCR
- AccuPrime™ SuperMix I is designed for the amplification of genomic DNA amplicons (≤200 bp), plasmid DNA, or cDNA templates. AccuPrime™ SuperMix I is not recommended for amplification of genomic DNA templates greater than 200 bp.
- General PCR parameters and troubleshooting information are documented in Innis, et al (Innis et al., 1990). PCR reactions should be assembled in a DNA-free environment using clean, dedicated automatic pipettors and aerosol resistant barrier tips. Always keep the control DNA and other templates to be amplified isolated from the other components.
- Optimal reaction conditions (incubation times and temperatures, primers, and template DNA) will vary. Adjust as needed.
- Program the thermal cycler as follows (note that the annealing temperature will vary depending on the Tm of your primers): Initial denaturation: 94ºC for 2 minutes 25–35 cycles of:
- Denaturation: 94ºC for 15–30 seconds
- Annealing: Tm of primers minus 5ºC for 15–30 seconds
- Extension: 68ºC for 1 minute per kb of PCR product
- Add the following components in any order to each reaction tube/plate well. A final primer concentration of 200 nM each is recommended.
Component 10-μl Rxn 25-μl Rxn 50-μl Rxn AccuPrime™ SuperMix I 5 μl 12.5 μl 25 μl Primer mix (10 μM each) 0.2 μl 0.5 μl 1 μl Template DNA 1–200 ng 1–200 ng 1–200 ng DNase-free H2O Up to 10 μl Up to 25 μl Up to 50 μl
- Cap or seal the tube/plate, tap gently to mix, and centrifuge briefly to collect the contents.
- Place the tube in the thermal cycler and run the program from Step 1. After cycling, maintain the reaction at 4ºC. Samples can be stored at –20ºC until use.
- Analyze the amplification products by agarose gel electrophoresis. We recommend using E-Gel® 1.2% gels and TrackIt™ 100 bp DNA ladder.
|E-Gel® 1.2% Starter Pak||6 gels plus PowerBase™||G6000-01|
|E-Gel® 1.2% 18-Pak||18 gels||G5018-01|
|TrackIt™ 100 bp DNA Ladder||100 applications||10488-058|
- Chou, Q., Russell, M., Birch, D., Raymond, J., and Bloch, W. (1992) Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications. Nucl. Acids Res., 20, 1717-1723
- Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. S. (eds) (1990) PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego, CA
- Sharkey, D. J., Scalice, E. R., Christy, K. G., Atwood, S. M., and Daiss, J. L. (1994) Antibodies as thermolabile switches: high temperature triggering for the polymerase chain reaction. Biotechnology, 12, 506-509