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.
|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
50-μl Rxn AccuPrime™ SuperMix I 5 μl
25 μl Primer mix (10 μM each) 0.2 μl
1 μl Template DNA
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