Create a Primer

To add a known primer, first open a target sequence, then click menu Primers → Add Primer... to open the "Add Primer" dialog.

1. Paste or type a sequence into the Primer field.
2. Enter an appropriate name into the Primer field.
3. Check the box if the primer is to be phosphorylated (Note primers are not 5'-phosphorylated during standard synthesis procedures).
4. Optional: Add a description for the primer.
5. Optional: Set a color for the primer sequence.
6. The binding location of the primer (provided it matches the target sequence) will be displayed in the lower panel.

Click Add primer to Template to add the new primer to the sequence.

Switch to Sequence view, then select the desired primer binding site on your sequence. When you click the mouse and drag to select, the length and predicted melting temperature of the selected region will be displayed.  

To add a primer based on the selected region click menu Primers → Add Primer.

Specify whether the primer should be based on the Top Strand (5'-3') or Bottom Strand (5'-3') of the selection.

1. Enter a primer name.
2. If required add a description.
3. Details of the new primer are shown above the view of the binding site.

Click Add Primer to Template to add the primer to the target sequence.

Click at the appropriate location within the primer sequence (in most cases at the 5' end) to add to the primer sequence.

"Insertion tools" will appear to assist with addition of Codons, Enzyme sites or short features to the primer sequence .

Manually edit the primer sequence, or use the insertion tools to add sequence to the primer.

1. Place the cursor at the 5' end of the primer.
2. Choose a restriction site from the Enzyme site drop down.
3. Choose whether to add upstream bases to ensure efficient digestion.

Click Insert to add the site to the primer.

1. Any addition will be shown in red.
2. The new restriction site will be annotated with a green line. Hover over the site to see the enzyme name.
3. Information about the primer, including length, number of detected binding sites, number of annealed bases, and recalculated Tm are shown.
4. The new primer extension and it's predicted interaction with the target will be shown in the lower panel.

Click Add Primer to template to add the primer to your target sequence.

The new primer will be displayed on the target sequence.

Hover the pointer over the primer to see a tooltip with details about the primer and binding site.

Click Save to save the target sequence with the new primer.