Theoretically after 30 cycles approximately a billion copies of the target region on the DNA template have been generated (Table 4.1). This PCR product, sometimes referred to as an 'amplicon', is then in sufficient quantity that it can be easily measured by a variety of techniques that will be discussed in more detail in the technology section.
PCR is commonly performed with a sample volume in the range of 5-100 ||L. At such low volumes, evaporation can be a problem and accurate pipetting of the reaction components can become a challenge. On the other hand, larger solution volumes lead to thermal equilibrium issues for the reaction mixture because it takes longer for an external temperature change to be transmitted to the center of a larger solution than a smaller one. Therefore, longer hold times are needed at each temperature, which leads to longer overall thermal cycling times. Most molecular biology protocols for PCR are thus in the 20-50 ||L range.
The sample is pipetted into a variety of reaction tubes designed for use in PCR thermal cyclers. The most common tube in use with 20-50 ||L PCR reactions is a thin-walled 0.2 mL tube. These 0.2 mL tubes can be purchased as individual tubes with and without attached caps or as 'strip-tubes' with 8 or 12 tubes connected together in a row. In higher throughput labs, 96-well or 384-well plates are routinely used for PCR amplification.
PCR has been simplified in recent years by the availability of reagent kits that allow a forensic DNA laboratory to simply add a DNA template to a pre-made PCR mix containing all the necessary components for the amplification reaction. These kits are optimized through extensive research efforts on the part of commercial manufacturers. The kits are typically prepared so that a user adds an aliquot of the kit solution to a particular amount of genomic DNA. The best results with these commercial kits are obtained if the DNA template is added in an amount that corresponds to the concentration range designed for the kit.
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