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Introduction
Platinum® Taq Antibody is a proprietary inhibitor which, when bound to Taq DNA polymerase, inactivates polymerase activity. This reagent provides an automatic “hot start” for Taq DNA polymerase in PCR (1,2,3). Hot starts are typically used in PCR to increase sensitivity, specificity, and yield while allowing assembly of reactions at ambient temperatures. The extra time, effort, and contamination risks associated with manual hot start procedures are addressed with the use of Platinum® Taq Antibody. Platinum® Taq Antibody blocks Taq DNA polymerase activity at ambient temperatures but releases active polymerase after the denaturation step in PCR cycling at 94°C. By increasing the effectiveness of Taq DNA polymerase through use of this product, it is possible to reduce the optimization and handling of reaction components and improve PCR results. Platinum® Taq Antibody is supplied in a concentration sufficient to eliminate non-specific priming during amplification. To prepare an antibody:polymerase complex, Platinum® Taq Antibody is mixed with an equal number of units of Taq DNA polymerase. The mixture is used at the diluted enzyme unit concentration with no other modifications to PCR reactions necessary. Other native, recombinant, or truncated forms (4,5) of Taq DNA polymerase, including Elongase® Enzyme (Cat. No. 10480-010/-028), are also effectively inhibited.
Unit Definition:
One unit of Platinum® Taq Antibody is the amount of product required to inhibit one unit of Taq DNA polymerase from incorporating deoxyribonucleotide into acid-precipitable material.
Storage Buffer:
20 mM Tris-HCl (pH 8.0), 40 mM NaCl, 2 mM Sodium Phosphate, 0.1 mM EDTA, 1 mM DTT, stabilizers, 50% (v/v) glycerol.
Quality Control:
The Certificate of Analysis (CofA) provides detailed quality control information for each product. The CofA is available on our website at www.invitrogen.com/cofa, and is searchable by product lot number, which is printed on each box.
PCR Precautions:
Since PCR is a powerful technique capable of amplifying trace amounts of DNA, all appropriate precautions should be taken to avoid cross-contamination. Ideally, amplification reactions should be assembled in a DNA-free environment. Use of aerosol-resistant barrier tips is recommended. Take care to avoid contamination with the primers or template DNA used in individual reactions. PCR products should be analyzed in an area separate from the reaction assembly area.
Unit Definition:
One unit of Platinum® Taq Antibody is the amount of product required to inhibit one unit of Taq DNA polymerase from incorporating deoxyribonucleotide into acid-precipitable material.
Storage Buffer:
20 mM Tris-HCl (pH 8.0), 40 mM NaCl, 2 mM Sodium Phosphate, 0.1 mM EDTA, 1 mM DTT, stabilizers, 50% (v/v) glycerol.
Quality Control:
The Certificate of Analysis (CofA) provides detailed quality control information for each product. The CofA is available on our website at www.invitrogen.com/cofa, and is searchable by product lot number, which is printed on each box.
PCR Precautions:
Since PCR is a powerful technique capable of amplifying trace amounts of DNA, all appropriate precautions should be taken to avoid cross-contamination. Ideally, amplification reactions should be assembled in a DNA-free environment. Use of aerosol-resistant barrier tips is recommended. Take care to avoid contamination with the primers or template DNA used in individual reactions. PCR products should be analyzed in an area separate from the reaction assembly area.
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Protocol
The following general procedure is suggested as a guideline and as a starting point when using Platinum® Taq Antibody in any PCR amplification. Optimal reaction conditions (incubation times and temperatures, concentration of Taq DNA polymerase, primers, MgCl2, and template DNA) vary and may need to be optimized. Final reaction volumes described below are approximately 50 μl. Reaction size may be altered to suit user preferences.
- Measure out the amount of Taq DNA polymerase needed. When using a reaction cocktail, determine the total number of units in the mix.
- Add an amount of “equivalent units” of Platinum® Taq Antibody to an equal amount of Taq DNA polymerase units. For Taq DNA polymerase at 5 unit per μl, prepare a 1:1 mixture. Mix well using a Vortex Mixer.
- Add the following components to a sterile 0.5-ml microcentrifuge tube:
Components Volume Final Concentration 10X PCR Buffer, Minus Mg 5 μl 1X 10 mM dNTP mixture 1 μl 0.2 mM each 50 mM MgCl2
1.5 μl 1.5 mM Primer mix (10 μM each) 1 μl 0.2 μM each Template DNA ≥1 μl (as required) Platinum® Taq Antibody:
Taq DNA polymerase1 μl 2.5 units (or as required) Autoclaved, distilled water to 50 μl Not applicable
If desired, a master mix can be prepared for multiple reactions to minimize reagent loss and to enable accurate pipetting. - Mix contents of the tubes and overlay with 50 μl of mineral or silicone oil, if necessary.
- Cap the tubes and centrifuge briefly to collect the contents
- Incubate tubes in a thermal cycler at 94°C for 30 s to 2 min to completely denature the template and activate the enzyme.
- Perform 25-35 cycles of PCR amplification as follows: Denature 94°C for 30 s Anneal 55°C for 30 s Extend 72°C for 1 min per kb
- Maintain the reaction at 4°C after cycling. The samples can be stored at -20°C until use.
- Analyze the amplification products by agarose gel electrophoresis and visualize by ethidium bromide staining. Use appropriate molecular weight standards
References
- Chou, Q., et al. (1992) Nucl. Acids Res., 20, 1717.
- Sharkey, D.J., et al. (1994) BioTechnology, 12, 506.
- Westfall, B.A., et al. (1997) Focus®, 19.3, 46.
- Barnes, W.M. (1992) Gene, 112, 29.
- Lawyer, F.C., et al. (1993) PCR Methods and Applications, 2, 275.
MAN0000923 28-Jun-2010
