Q5 High-Fidelity 2X Master Mix is a premixed solution containing the necessary reagents for polymerase chain reaction (PCR). Its formulation provides a DNA polymerase with an error rate approximately 280 times lower than that of standard Taq polymerase, making it suitable for applications that require high accuracy, such as cloning. The master mix format simplifies the experimental process by reducing the number of pipetting steps, which in turn minimizes the potential for contamination and user error.
PCR Reaction Setup
Setting up a PCR reaction with this master mix involves combining a few core components on ice. The mix itself is supplied at a 2X concentration and contains the Q5 DNA polymerase, dNTPs, and magnesium chloride (MgCl₂). The user needs to add the DNA template and primers, which are short, single-stranded DNA sequences that define the target region for amplification. Nuclease-free water is used to bring the reaction to its final volume.
A typical reaction is assembled in a total volume of either 25 µL or 50 µL. For a 25 µL reaction, 12.5 µL of the Q5 Master Mix is combined with 1.25 µL of a 10 µM forward primer and 1.25 µL of a 10 µM reverse primer. This achieves a final primer concentration of 0.5 µM for each, which is recommended for most applications. The amount of template DNA added is variable, but generally should not exceed 1,000 ng. The remaining volume is filled with nuclease-free water.
The quantity of template DNA is adjusted based on its complexity. For instance, 1 pg to 10 ng of DNA is usually sufficient for a simple plasmid. For more complex sources like genomic DNA, a higher amount, typically 1 ng to 1 µg, is used. After all components are added, the reaction tube should be mixed gently and spun briefly in a microcentrifuge to collect the contents at the bottom.
Thermocycling Conditions
Once assembled, the reaction tubes are transferred to a thermocycler for DNA amplification. The cycling process begins with an initial denaturation step, typically at 98°C for 30 seconds. This step separates the double-stranded template DNA into single strands, making them accessible to the primers.
Following initial denaturation, the reaction undergoes 25 to 35 cycles of amplification. Each cycle consists of three distinct steps: denaturation, annealing, and extension. The denaturation step is at 98°C for 5-10 seconds to re-separate the DNA strands. Next, the annealing step’s lower temperature allows primers to bind to the template DNA. Finally, the extension step is performed at 72°C for the Q5 polymerase to synthesize new DNA.
After the cycles are complete, a final extension step at 72°C for 2 minutes ensures any remaining single-stranded DNA is fully extended. The protocol concludes with a hold step, where the temperature is lowered to 4-10°C to preserve the amplified DNA.
Important Protocol Parameters
Optimizing two parameters is often necessary for specific experiments: the annealing temperature (Ta) and the extension time. The annealing temperature is a primary variable to adjust, as an incorrect Ta can lead to poor or no amplification. The unique buffer composition of the Q5 mix means that traditional methods for calculating melting temperature (Tm) are not accurate. It is highly recommended to use the NEB Tm Calculator. This tool calculates the specific Ta for your primers based on their sequence and the Q5 buffer system.
The NEB Tm Calculator will provide a recommended annealing temperature, which is typically 3°C above the calculated Tm of the lower-Tm primer. The annealing step itself should be run for 10-30 seconds. Following this guideline ensures specific primer binding and minimizes the amplification of non-target products.
The second parameter, extension time, depends on the length of the amplicon. The Q5 polymerase is highly processive, meaning it can synthesize DNA quickly. The general recommendation for extension time is 20-30 seconds per kilobase (kb) of amplicon length. For example, amplifying a 2 kb fragment would require an extension time of 40-60 seconds at 72°C. For amplicons larger than 6 kb, increasing the extension time to 40-50 seconds per kb can be beneficial.