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ADD STEP
On the right side of the page, there is an option to
Add Step
(Figure 4.6). Designate the
respective sections to add the additional step(s). To do so, click on the arrows at either side of
the screen or click directly on the appropriate stage in the orange section; the editable step will
be bordered in black (Figure 4.7).
Figure 4.6
Figure 4.7
ADD STAGE
Under the “Add Stage” icon, there are three different stage options to select. Each stage may
consist of one or more steps.
Figure 4.8
Holding Stage
The default protocol comes with a holding stage that contains the initial denaturation step.
Additional steps may be added to a holding stage if needed. Generally, in both two-step and
three-step PCR protocols, a holding stage exists at the very beginning since it houses the initial
denaturation step.
In some cases, depending on whether you plan to run downstream applications such as a post-
PCR agarose gel, an additional holding stage may be added at the very end of the amplification
cycle for the final extension step.
Cycling Stage
The cycling stage is akin to a holding stage, except that it repeats for a specified number of
times. Denaturation, annealing and extension occurs during this stage so the number of DNA
copies doubles with each completed cycle.
