13.2 Manual primer design

It is possible to create PCR primers by adding a primer annotation directly onto a sequence. This is especially useful for cloning applications as generally the primers must bind to a specified set of bases at the beginning and end of the gene to be cloned. To manually add a primer, select the region of sequence where you wish the primer to bind. You will see a selection hint with the length and rough Tm of the selection, and an Add Primer button.

Once satisfied with the position and Tm of the selection then you can click the Add Primer button to open the Add Annotation dialog. The Add Annotation dialog will open with settings appropriate for creation of a new primer as shown in Figure 13.6 . You can then give the primer a name, set the primer direction and, if required, add an extension. Changing the primer binding site position in the Add annotation window will automatically update the primer sequence and characteristics. A 5^′ extension can also be added directly onto a primer in this step by clicking the button next to “Extension”. See section 13.9 for more information on adding 5^′ extensions.