Introduction

Description

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 I2 × 1.25 ml12.5 ml


Components

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.
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Ordering Information

Sku Name Size Price Qty
12342010 AccuPrime™ SuperMix I 200 reactions USD 313.00
12342028 AccuPrime™ SuperMix I 1,000 reactions USD 1,482.00

Basic PCR Protocol

The following general procedure is suggested as a guideline and starting point when using AccuPrime™ SuperMix I in any PCR amplification. The reaction size may be scaled as needed.
  1. 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


  2. Add the following components in any order to each reaction tube/plate well. A final primer concentration of 200 nM each is recommended.

    Component10-μl Rxn
    25-μl Rxn
    50-μl Rxn
    AccuPrime™ SuperMix I5 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 H2OUp to 10 μl
    Up to 25 μl
    Up to 50 μl


  3. Cap or seal the tube/plate, tap gently to mix, and centrifuge briefly to collect the contents.

  4. 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.

  5. Analyze the amplification products by agarose gel electrophoresis. We recommend using E-Gel® 1.2% gels and TrackIt™ 100 bp DNA ladder.
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Additional Products

Product Amount Catalog no.
E-Gel® 1.2% Starter Pak 6 gels plus PowerBase™ G6000-01
E-Gel® 1.2% 18-Pak18 gelsG5018-01
TrackIt™ 100 bp DNA Ladder100 applications 10488-058

References

  1. 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

  2. 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

  3. 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
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 MAN0001078         Rev. date: 11-Jun-2010