User inputIn this mode the user must define either a Forward primer region, a Reverse primer region, or both. These are defined by making a selection on the sequence and right-clicking the selection. It is also possible to define a Region to amplify in which case a forward- and a reverse primer region are automatically placed so as to ensure that the designated region will be included in the PCR fragment. If areas are known where primers must not bind (e.g. repeat rich areas), one or more No primers here regions can be defined.
If two regions are defined, it is required that at least a part of the Forward primer region is located upstream of the Reverse primer region.
After exploring the available primers (see Graphical display of primer information) and setting the desired parameter values in the Primer Parameters preference group, the Calculate button will activate the primer design algorithm.