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Taq DNA Polymerase Guidelines for PCR Optimization |  | | Taq DNA Polymerase Guidelines for PCR Optimization |
Introduction
Taq DNA Polymerase is the enzyme most widely used in the Polymerase Chain Reaction (PCR). The following guidelines will help ensure the success of PCR using New England Biolabs' Taq DNA Polymerase. These guidelines cover routine PCR reactions. PCR of templates with high GC content, high secondary structure, low template concentrations or which produce products greater than 5 kb may require adaptations of these conditions.
Buffers
ThermoPol Reaction Buffer
ThermoPol II (Mg-free) Reaction Buffer
ThermoPol DF (Detergent-free) Reaction Buffer
Standard Taq Reaction Buffer
Protocol
- DNA Template: Using high quality, purified DNA templates greatly enhances the success of PCR reactions. It is also critical that contamination from previous related PCR reactions be avoided.
For typical detection, approximately 104 copies of the target DNA are required to detect a product in 25-30 cycles of PCR. Typically, this means 0.1-1 ng/ml final concentration of plasmid or viral templates, and 1-10 µg/ml of genomic templates. In general, higher DNA concentrations decrease the specificity of the reaction (i.e., extra bands are seen), particularly when a large number of cycles are employed. High DNA concentrations can be advantageous when fewer cycles are desired, for example to increase the fidelity of the reaction. - Primers: Oligonucleotide primers are generally 20-30 nucleotides in length, and ideally have a GC content of 40-60%, with GC residues spaced evenly within the primer. Calculated melting temperatures (Tm) for the two primers should generally be from 42-65°C, and within 5°C of each other. Secondary structure, such as hairpins, within each primer, and potential dimerization between the primer and its primer pair must be avoided to maximize PCR yields. A variety of computer programs can guide the selection of primers.
The final concentration of each primer should be 0.1-0.5 µM. Higher concentrations increase the possibility of secondary priming, which potentially creates spurious amplification products. - Magnesium Concentration: A magnesium concentration of 1.5-2.0 mM is optimal for most PCR products generated with Taq DNA Polymerase. The optimal concentration can depend on the template employed, and also on the buffer, DNA and dNTPs added to reaction as each has the potential to chelate magnesium. If the concentration is too low, no amplification product will be seen. Too high a concentration may result in undesired amplification products. Very occasionally a higher level may be optimal. In these situations, final levels upto 4 mM can be tested in 0.5 mM increments.
- Deoxynucleotides: The concentration of dNTPs is typically 200 µM of each nucleotide. Concentrations of 100-500 µM may be useful for specific applications. Lower concentrations (50-100 µM) may enhance the fidelity of polymerization, but reduce yields. Higher concentrations than this suggested range can increase yields, particularly in long PCR, but can reduce polymerase fidelity.
- Taq DNA Polymerase Concentration: Taq DNA Polymerase is normally present at a concentration of 20 units/ml, but can range from 5-50 units/ml in specialized applications.
- Starting Reactions: Nonspecific primed synthesis during reaction setup, and prior to PCR cycling, has been identified as a source of nonspecific products in some PCR reactions. These undesired products can often be avoided by assembling all reaction components on ice, adding the polymerase last and immediately transfering the reactions to a thermocycler preheated to the denaturation temperature (94°C). Alternatively, a hot start enzyme, such as DYNazyme II hot start (NEB # F-504) can be employed.
- Denaturation Temperature and Duration: An initial denaturation at 94°C is required prior to PCR cycling to fully denature the DNA. Longer incubations and higher temperature incubations can significantly damage the template DNA, and should be avoided unless required due to the high GC content of the template.
During thermocycling, a 15-30 second denaturation at 94°C should be utilized, although this can depend on the thermocycler and tubes used. Consult the product literature accompanying the thermocycler being used for more specific recommendations. - Annealing Temperature and Duration: Annealing temperatures should be chosen to match the Tm values of the primer pair, and are typically between 55-60°C. If extra bands are observed, higher annealing temperatures should be tested. Annealing times of 15-30 seconds are usually adequate.
- Extension Time: Extensions are normally done at 72°C, with the time of the extension determined by the size of the expected amplification product. As a general rule, extension times of one minute per 1000 base pairs should be employed, i.e. 1 minute for a 1 kb product, 3 minutes for a 3 kb product, etc. For products less than 1 kb, an extension time of 45-60 seconds should be used. Products greater than 3 kb, or reactions using more than 30 cycles, may require more extended extension cycles.
- Typical Cycling Conditions: A typical PCR reaction for a 500 bp amplification is shown:
1 cycle: 94°C 2 minutes
25 cycles: 94°C 15 seconds
55°C 15 seconds
72°C 45 seconds
1 cycle: 72°C 5 minutes (to finish replication on all templates)
1 cycle: 4°C indefinite period (storing the sample prior to further analysis)
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