Although all PrimerBank primers are designed to be gene specific, the choice of PCR conditions is important and has a major impact on the success rate. Non-specific primer extension of only a few bases at room temperature by Taq polymerase can lead to non-specific PCR amplifications. Therefore, some variety of hot-start PCR is strongly recommended. AmpliTaq Gold® DNA polymerase has been our preferred enzyme as we found it to be more specific than other hot-start DNA polymerases we tested.
The annealing temperature may affect PCR specificity. To avoid nonspecific PCR products, a high annealing temperature is recommended. In general, the annealing temperature should be at least 55°C or higher. Here we present a standard SYBR® Green real-time PCR protocol using PrimerBank primers. A more detailed and complete protocol is also presented in Chapter 7.
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