Although Design Primers (Primer3) allows you to quickly design primers it also allows knowledgeable users to use the full power of the Primer3 algorithm.
Primer vs Primer, 3' end vs 3' end and Primer vs Product control the maximal accepted binding/mis-priming between the primers and product. The units are defined internally by Primer3. Raise the values to reduce the stringency of the filtering - this may be required if you are scanning a short sequence and Primer3 cannot find suitable primers. For Primer vs Product set the value to "-1" to turn off filtering for that parameter. Select the Allow ambiguous residues checkbox if you have ambiguous residues in your sequence. In that case, you may want to limit the number of ambiguous residues in the primers so choose the maximum number you are willing to accept and put that in the Allowed Ns in primers edit box.
Set these to match the reaction conditions you use in the lab. These will affect the reported Tm of the primer binding to the template. Reaction Conditions are shared by all primer design tools.
Use this tab if you want the Hybridization Primer scan to use different basic parameters than used for the external primers. Please note this are independent of the main parameters, and even if you change the ones used for the main primers, then if these are still at the defaults then they will be used.
This controls the number of primer pairs that are reported. The default is 5 but you can choose up to 100. However, note that the Graphic Map output is limited to the top 25 primers.
Choose Only Unique Primers will ensure the list of primers will only contain unique primers. When disabled a primer may appear in more than one unique pair.