![]() For example, mammalian genomic DNA may require longer incubation periods than plasmids and PCR products, based on DNA complexity and size. The time and temperature of this step can vary depending on the nature of the template DNA and salt concentrations of buffer. ![]() The initial denaturation step is commonly performed at 94–98☌ for 1–3 minutes. When using a hot-start DNA polymerase, this step also serves to activate the enzyme, although a separate activation step may be recommended by the enzyme supplier. Furthermore, the high temperature at this step helps inactivate heat-labile proteases or nucleases that may be present in the sample, with minimal impact on thermostable DNA polymerases. Complete denaturation of the input DNA helps ensure efficient amplification of the target sequence during the first amplification cycle. The initial denaturation step is carried out at the beginning of PCR to separate the double-stranded template DNA into single strands so that the primers can bind to the target region and initiate extension. ![]()
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