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Introduction
TaqPCRx DNA Polymerase, Recombinant is similar to Taq DNA Polymerase, Recombinant (Cat No. 10342-020). However, in addition to 50 mM Magnesium Chloride and the standard 10X PCR Buffer, it is supplied with an optimized buffer system including 10X PCRx Amplification Buffer, 50 mM Magnesium Sulfate, and 10X PCRx Enhancer Solution (the cosolvent) that simplifies Polymerase Chain Reaction (PCR) amplification of problematic and/or GC-rich templates using standard dNTPs and thermocycling protocols.
PCR optimization (1) is often a difficult and time consuming process requiring adjustment of incubation times and temperature, magnesium, primer, dNTP, Taq DNA polymerase and DNA template concentration, and potentially, the design of multiple primer sets. DNA sequences containing stable secondary structure(s) are frequently refractory to conventional
PCR optimization strategies as these sequences are resistant to denaturation and pose barriers to primer annealing or procession of DNA polymerase.
10X PCRx Enhancer Solution is a novel PCR cosolvent that facilitates efficient amplification of GC-rich sequences and remedies difficulties associated with PCR amplification of problematic templates. For problematic and/or GC-rich templates, the combination of 10X PCRx Enhancer Solution and 10X PCRx Amplification Buffer offers higher primer specificity, broader magnesium concentration optima, broader annealing temperature optima, and improved thermostabilization of recombinant Taq DNA polymerase.
Storage Buffer
20 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 1 mM DTT, stabilizers, 50% (v/v) glycerol
10X PCR Buffer
200 mM Tris-HCl (pH 8.4), 500 mM KCl
Unit Definition
One unit incorporates 10 nmol of deoxyribonucleotide into acid-precipitable material in 30 min at 74°C.
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 optimization (1) is often a difficult and time consuming process requiring adjustment of incubation times and temperature, magnesium, primer, dNTP, Taq DNA polymerase and DNA template concentration, and potentially, the design of multiple primer sets. DNA sequences containing stable secondary structure(s) are frequently refractory to conventional
PCR optimization strategies as these sequences are resistant to denaturation and pose barriers to primer annealing or procession of DNA polymerase.
10X PCRx Enhancer Solution is a novel PCR cosolvent that facilitates efficient amplification of GC-rich sequences and remedies difficulties associated with PCR amplification of problematic templates. For problematic and/or GC-rich templates, the combination of 10X PCRx Enhancer Solution and 10X PCRx Amplification Buffer offers higher primer specificity, broader magnesium concentration optima, broader annealing temperature optima, and improved thermostabilization of recombinant Taq DNA polymerase.
| Component | Part No. | Amount |
| Taq DNA polymerase (5 U/μl) | 11508-017 | 500 U |
| 10X PCRx Enhancer Solution | 52391 | 2 × 1 ml |
| 50 mM Magnesium Sulfate | 52044 | 1 ml |
| 10X PCRx Amplification Buffer | 52395 | 2 × 1 ml |
| 50 mM Magnesium Chloride | Y02016 | 1 ml |
| 10X PCR Buffer, Minus Mg | Y02028 | 2 × 1 ml |
Storage Buffer
20 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 1 mM DTT, stabilizers, 50% (v/v) glycerol
10X PCR Buffer
200 mM Tris-HCl (pH 8.4), 500 mM KCl
Unit Definition
One unit incorporates 10 nmol of deoxyribonucleotide into acid-precipitable material in 30 min at 74°C.
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-fre environment. Use of aerosolresistant 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 fro the reaction assembly area.
General Recommendations and Guidelines
Buffer Selection
PCRx Enhancer Solution
Primer Design
General Recommendations and Guidelines
Buffer Selection
- TaqPCRx DNA polymerase is supplied with two different buffers to provide optimal reaction conditions for the widest variety of DNA templates. The standard 10X PCR Buffer and 50 mM MgCl2 are recommended for PCR of routine templates (30–50% GC content).
- Conventional PCR buffer components in established protocols can be directly substituted with the 10X PCRx Amplification Buffer and 50 mM MgSO4 resulting in more robust amplification and improved product yield in some cases. However, optimal reaction conditions vary and may need to be evaluated by the customer. Use of 10X PCRx Enhancer Solution for PCR of GCrich templates results in wider reaction optima and significantly improves the probability of successful PCR amplification. Note: Targets with GC content ≤45% may require higher Mg++ concentrations (≥2 mM) or use of the standard PCR buffer for optimal amplification. In addition, primer sets that generate specific product using standard PCR buffer may not benefit from using PCRx Amplification Buffer.
PCRx Enhancer Solution
- Optimal concentration of 10X PCRx Enhancer Solution will vary depending on GC content, Mg++ concentration and annealing temperature. Many GC-rich templates (up to 80%) may benefit by simply using the optimized 10X PCRx Amplification Buffer without added PCRx Enhancer solution. For targets with 45 to 60% GC, we recommend testing 10X PCRx Enhancer Solution at 0X, 0.5X, and 1X final concentration. Targets with higher GC content (65 to 90%), or simple repeat sequences, may require up to 4X concentration.
- 10X PCRx Enhancer Solution lowers DNA melting temperature (Tm). Consequently, the maximum primer annealing temperature is lowered approximately 2°C per 1X PCRx Enhancer Solution concentration; however, effective annealing temperatures are widened over a much broader range. While no single thermal cycling protocol is optimal for every template, we recommend starting with an annealing temperature of 55 to 60°C and varying the amount of 10X PCRx Enhancer Solution.
- Addition of 10X PCRx Enhancer Solution extends the range of effective Mg++ concentration (1 to 3 mM). For most target sequences, best results are obtained using 1.5 mM MgSO4.
- Best results with PCRx reagents have been obtained using 2.5 units of Taq DNA Polymerase in a 50-μl amplification reaction. For long templates (up to 10 kb) or six-fold higher fidelity, we recommend using PLATINUM® Taq DNA Polymerase High Fidelity (Cat. Nos. 11304-011/-029).
Primer Design
- Effective primer design is the single most important parameter in PCR. PCR primers should be designed to: form stable, highly specific duplexes with the target sequence, minimize potential for secondary structure and dimer formation, minimize 3′- terminal complementarily (primer dimer), contain high stability (i.e., GC clamps) in the central and/or 5′- region, contain low stability in 3′-region (no more than two C or G in last five 3′- bases) to maximize specificity of priming, and have an equivalent/balanced Tm (DNA melting temperature).
- Primers for high GC content templates should have a GC content similar to that of the target sequence and a Tm of at least 70°C as calculated using nearest neighbor algorithms (2). For optimum PCR efficiency, the difference between PCR product Tm and primer Tm should be less than 25°C. Primer Tm can be adjusted by increasing primer length at the 5′-end. Ideally, primers should be 20 to 25 bases long; however, primers up to 30 bases are effective for amplification of GC-rich sequences.
Basic PCR Protocol
The following basic protocol serves as a general guideline and a starting point for any PCR amplification. Optimal reaction conditions (incubation times and temperatures, concentration of Taq DNA Polymerase, primers, MgCl2, and template DNA) vary and need to be optimized.
- Add the following components to a 0.5-ml microcentrifuge tube sitting on ice:
Component Volume Final Concentration 10X PCR Buffer, Minus Mg 10 μl 1 X 10 mM dNTP mixture 2 μl 0.2 mM each 50 mM MgCl2 3 μl 1.5 mM Primer mix (10 μM each) 2–5 μl 0.2–0.5 μM each Template DNA 1-20 μl as required Taq DNA Polymerase (5 U/μl) 0.5 μl 2.5 units Distilled water to 100 μl Not applicable
If desired, a master mix can be prepared for multiple reactions, to minimize reagent loss and to enable
accurate pipetting. - Cap tubes and centrifuge briefly to collect the contents to the bottom.
- Incubate tubes in a thermal cycler at 94°C for 3 min to completely denature the template.
- Perform 25 to 35 cycles of PCR amplification as follows:
Denature 94°C for 45 s
Anneal 55°C for 30 s
Extend 72°C for 1 min 30 s - Incubate for an additional 10 min at 72°C and maintain the reaction at 4°C. The samples can be stored at –20°C until use.
- Analyze the amplification products by agarose gel electrophoresis. Use appropriate molecular weight standards. Visualize by ethidium bromide staining.
"Hot-start" Protocol
In the "Hot-start" method, the addition of Taq DNA Polymerase is withheld until the reaction temperature is at 80°C, so as to ensure high specificity of the products being synthesized. Alternately, Platinum® Taq Antibody (Cat No. 10965-010) can be included in the Basic PCR protocol to provide the benefits offered by an automatic hot-star with the convenience of ambient temperature reaction assembly.
TOP- Add all components as in the Basic PCR Protocol, except for the Taq DNA Polymerase.
- Cap tubes and centrifuge briefly to collect the contents to the bottom.
- Incubate tubes in a thermal cycler at 94°C for 3 min to completely denature the template.
- After denaturation at 94°C, maintain the reaction at 80°C.
- Add 0.5 μl of Taq DNA Polymerase (2.5 U) to each reaction. Be certain to add the enzyme beneath the layer of oil, if necessary.
- Continue with 25 to 35 cycles of denaturation, annealing and extension as in the Basic PCR Protocol.
PCR Optimization Protocol for Problematic/GC-Rich Templates
The following basic protocol serves as a general guideline and a starting point. It is designed to test varying concentrations (0X to 4X) of PCRx Enhancer Solution.
- For optimization, prepare enough master mix for seven reactions by adding the following components to a microcentrifuge tube. Alternately, individual PCRx mixtures can be prepared according to the guidelines given below.
Component Seven Rxn Master Mix One Rxn Final Concentration 10X PCRx Amp. Buffer 35 μl 5 μl 1X 10 mM dNTP mixture 7 μl 1 μl 0.2 mM each 50 mM MgSO4 10.5 μl 1.5 μl 1.5 mM Forward Primer (10 μM ) 7 μl 1 μl 0.2 μM Reverse Primer (10 μM ) 7 μl 1 μl 0.2 μM Template DNA ≥7 ≥1 μl (as required) Taq DNA Polymerase (5 U/μl) 3.5 μl 0.5 μl 2.5 units/rxn - Add the following reagents to six thin-walled microcentrifuge tubes sitting on ice:
Final PCRx EnhancerConcentration Component 0 0.5X 1X 2X 3X 4X Master Mix 30 30 30 30 30 30 10X PCRx Enhancer Solution - 2.5 5 10 15 20 Distilled Water 20 17.5 15 10 5 - - Equilibrate a thermal cycler to 95°C. Transfer the reactions from ice to the thermal cycler and incubate at 95°C for 2 min to completely denature the template.
- Perform 25 to 35 cycles of PCR amplification as follows:
Denature 95°C for 30 to 45 s
Anneal 55°C to 60°C for 30 s
Extend 68°C for 1 min per kb
- Analyze the amplification products by agarose gel electrophoresis. Use appropriate molecular weight standards. Visualize by ethidium bromide staining.
References
- Innis, M.A., Gelfand, D.H., Sninsky, J.J., and White, T.J., eds. (1990) PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego, CA.
- Rychlik, W. and Rhoads, R.E. (1989) Nucl. Acids Res., 17, 8543
MAN0000986 18-Jun-2010
